text:
If the covid-19 appeared once, could it reappear anytime?
Governments are imposing confinement measures on their populations, trusting that if there is no more transmission the virus will die out.
But since this coronavirus appeared a first time, what could prevent it from reappearing any number of times ?

SE Biology is a question and answer site — not a discussion site or a site for floating ideas. It is concerned with the mechanisms of biological processes, not medical or social aspects of biology. For one of these reasons I think  that your question on the coronavirus outbreak is off-topic here. Question of a medical nature might be on-topic at SE Medical Sciences. Otherwise you are advised to consult more appropriate reputable sources for such information, some of which are listed here.

Frame challenge answer: you are jumping to the conclusion that they really hope the virus will "die out". But it's more likely they adopted those measures to slow its progression to a manageable degree. See e.g. this conceptual image from ECDC

Even if there are new outbreaks of COVID-19 in the future, when they happen is not irrelevant, as we could be much better prepared given the current/ongoing research on both (antiviral) treatments and possibly a vaccine as well.
Regarding the "meat" of the question, it's a matter of probabilities, not certainties. It was and is generally assumed that other [bat etc.] coronaviruses will jump hosts to humans in the future.
Besides SARS, another (much) lesser known cross-species jump actually occurred in China in 2016 with SADS-CoV, which killed thousands of pig[let]s, but was not infectious to humans. Annoyingly enough for the Chinese pig farmers, SADS-CoV re-emerged in early 2019 despite the fact that there were no cases reported between May 2017 and January 2019.

I wish the (probably high-rep) users who downvote answers here, would put the effort into closing questions instead, if answering off-topic (??) questions  is the reason for downvoting answers. Otherwise please explain why you are downvoting answers...

Why close a question that, even if poorly worded, is likely to be of interest to many people?

@jamesqf: I guess you meant to ask David that.

No, I meant it as a general comment to anyone who cares to read it.  Of course it's rhetorical, because we can easily guess why certain questions are closed, and lots of interesting comments deleted.

This recent article explores the origin of the current covid-19 by performing comparative genomic analyses: https://www.nature.com/articles/s41591-020-0820-9
One of the main point of the article is that to be able to answer your question one would need to know how the virus started to emerge in humans in the first place.
The authors describe 3 main hypotheses:

Natural selection in an animal host before zoonotic transfer
Natural selection in humans following zoonotic transfer
Selection during passage

The first two hypotheses are probably more likely to be an explanation the 3rd being very unlikely (I leave the details to the article).
Here is a scheme coming from a different article (Source: https://www.nature.com/articles/nm.3985.pdf?origin=ppub) explaining graphically hypotheses 1 and 2 as emergence paradigms:

Now your answer to your question is in the conclusion of the first mentioned article:
"In the midst of the global COVID-19 public-health emergency, it is reasonable to wonder why the origins of the pandemic matter. Detailed understanding of how an animal virus jumped species boundaries to infect humans so productively will help in the prevention of future zoonotic events. For example, if SARS-CoV-2 pre-adapted in another animal species, then there is the risk of future re-emergence events. In contrast, if the adaptive process occurred in humans, then even if repeated zoonotic transfers occur, they are unlikely to take off without the same series of mutations. "
Essentially if it is hypothesis #1, it could be likely. If it is hypothesis #2 less likely. I don't think there is enough evidence yet to prove or disprove any of the two.


text:
How has the theory of evolution changed over time?
I learned at school and in the documentaries like everybody that (to summarize) molluscs evolved into fishes then reptiles then mammals then humans.
Does the theory still make the same claim ? or does neo darwinism only defends speciation where for example all camelidae are related and all felidae are related, but both aren't related.
If not, why the famous evolution trees never identify any of the supposed common ancestors ?

Comments are not for extended discussion; this conversation has been moved to chat.

How has the theory of evolution changed over time?

It improved quite a bit. To put things in perspective; today most of evolutionary biology is about evolutionary genetics while at Darwin time (On the Origin Of Species was published in 1859), we really had no idea about DNA (the basic structure of DNA was only discovered in the 1950s).
A number of the original claims were found to be wrong though (or off-topic). We've added much more than we've removed though!

I learned at school and in the documentaries like everybody that (to summarize) molluscs evolved into fishes then reptiles then mammals then humans.
  Does the theory still make the same claim ?

This claim is not correct (and has never been) or at least is very misleading. I hope your teacher did not really tell you that!

or does neo darwinism only defends speciation where for example all camelidae are related and all felidae are related, but both aren't related

Nope, this would be even more wrong!
Among all extant species today, none are ancestors of others. They all share a common ancestor though. The common ancestor between a lynx and cat lived more recently than the common ancestor between a lynx and an oak tree.
Also, the claim put an emphasis on humans, which sounds like you viewing some kind of goal to evolution or eventually some kind of 'improvement' where humans would be the best or something like that. That notion of good or better is not just wrong, it is off-topic in any scientific discussion.
Some of the clades you mention contain other mentioned clade (for example, humans are mammals), however 1) fish is not a clade (not monophyletic, see this post for an intro to phylogeny) 2) ancestors of mammals have never been mollusc 3) note that the term reptile you use can take two different definitions; a popular meaning and a rigorous scientific meaning (which we use called Reptilia) (have a look at the post If dinosaurs could have feathers, would they still be reptiles?). In any case, no ancestors of mammals were reptiles (although mammals are Reptiliomorphia).

If not, why the famous evolution trees never identify any of the supposed common ancestors ?

I don't really understand this question.
We are able to date pretty well the common ancestors of most lineage of interest through molecular tools. We have a lot of fossil records which, when dated, match our finding with molecular tools. Note that the one most recent common ancestor between two lineages is a single individual (although recombination makes this point more complicated but I won't talk about that here). Of course, we probably never have a fossil of this single individual, but we often have fossils of individuals that are quite closely related to this individual.

You seem to have an extremely biased view of the theory of evolution. I am sorry if your teacher taught you so wrong. The best for you is to simply start with an intro course such as Evo101 for example. Evo101 is short, very introductory and easy to read.

There's also a problem with "famous evolution trees", in that we don't know what the OP considers "famous".  Lots do show common ancestors, though it's hard to show millions of species on a piece of paper.  Here's one site (of many) that attempts to do it: http://www.onezoom.org/  And another: http://www.sciencemag.org/sites/default/files/styles/article_main_large/public/images/sn-treeoflife.jpg?itok=AXS5irW-

@user2534 Comments are not for extended discussion. Asking for evidence of speciation has nothing to do with the main point of the post to start with. You should have a look at this post and probably this one too.

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text:
Nomenclature of genes and proteins
With respect to this paper:
Identification of Host Proteins Required
for HIV Infection Through a Functional
Genomic Screen
In the abstract, I found names such as Rab6 and Vps35 and TNPO3. So Rab6 and Vps35 are proteins and TNPO3 is a gene? So the strings written in full caps are the genes and those strings whose just first letter is capital and the rest of the letters are small is a protein?

Thats a really tough one due to the scope of things.. here's a resource as to gene nomenclature, and here's another one for proteins.

Genes, conventionally, are always written in italic (e.g. SHH) which is not the case for proteins (in the former example SHH). That should allow you to tell apart gene and protein symbols (SHH vs SHH).
The capitalization of gene/protein names is a bit more in the grey area. Usually one will use capitalized gene and protein symbols for human and primates (e.g. SHH and SHH) and use non-capitalized symbols for mice and rat genes (e.g. Shh but still SHH for the protein). This is dependent on the organism and journals usually have specific guidelines on how to exactly write gene and protein symbols.
In your case the article refers to Rab6, Vps53 (not 35) and TNPO3 as proteins, if not clearly stated differently, such as the Rab6 siRNA, not only in the abstract alone but over the entire article. They use italic names for genes as shown in one of the sentence:

... and 48 hours later staining for p24, produced from the HIV gag gene...

Just as a footnote, full gene names (e.g. the sonic hedgehog gene) are not italicized.

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text:
what does Pro→Glu substitution mean?
Considering the paper:
A single amino acid in E-cadherin responsible for
host specificity towards the human pathogen
Listeria monocytogenes
in the abstract portion, what does Pro→Glu mean? Does it mean that, in place of Glu(glucose), pro-16 is placed?

Proline is replaced by Glutamic acid. Pro16 indicated a position on the protein.

You are looking at a protein here, which is mutated in the higher host specificity, so glucose is not involved here. What is written here are the amino acids in the three-letter-code, the notation Pro→Glu means that Proline (Pro) is replaced by Glutamic acid (Glu).
The notation Pro16 means the proline on amino acid position 16 in the protein. You often find this replacements noted as Pro16Glu or P16E (in the 1-letter-code)

oops.. I shall remove my answer


text:
Doubts regarding definition of upstream/downstream genes and cognate protein
With respect to the research paper, there are a few things I didn't understand:
1. What is upstream and downstream gene
2. This paper identifies proteins that help in secretion, but does not identify the originally secreted protein. Am I correct?
3. Does cognate protein here mean homologous protein(from common parents)?

Upstream means towards 5' direction from the reference point (conversely for downstream). Reference point can be a single position such as transcription start site (TSS) or a bigger segment such as a gene. When we say upstream of a gene, it means some region of DNA that is towards 5' direction from the TSS of the reference gene. Downstream of a gene refers to the region 3' to the transcription termination site of the reference gene.
Similar terminology is applicable to RNA but obviously, in this case your reference cannot be a gene (it can be an exon, for instance). I have not seen this terminology used in case of proteins.

This paper is about identification of the proteins that aid in secretion (TEC) of certain proteins (effectors). They identified both the proteins that help in secretion and the proteins that are secreted

Here, we report a class of T6SS effector chaperone (TEC) proteins that
  are required for effector delivery through binding to VgrG and
  effector proteins. The TEC proteins share a highly conserved domain
  (DUF4123) and are genetically encoded upstream of their cognate
  effector genes.  [....]
  We validated this approach by verifying a predicted effector TseC in
  Aeromonas hydrophila. We show that TseC is a T6SS-secreted antibacterial effector and that the downstream gene tsiC encodes the
  cognate immunity protein. Further, we demonstrate that TseC secretion
  requires its cognate TEC protein and an associated VgrG protein.

Cognate refers to the correct binding partner which will result in a functional complex. It basically refers to biomolecular partners with a known and validated relationship. This relationship need not be synergistic. For example:

RNA interference (RNAi) is an evolutionarily conserved process through
  which double-stranded RNA (dsRNA) induces the silencing of cognate
  genes. (Source)

my question is, are they identifying protein that gets secreted or are they identifying protein that help in the secretion?

@Rishika both.. they identify a relationship between them. It is clearly mentioned in the abstract. I edited the answer to highlight this.


text:
Sequencing two strands of dna
My background is not genetics. 2.I am not interested in knowing how dna sequencing or genotyping is done. 3. I am interested just in the nature of the results as described here.
Now coming to the question: Humans have two sets of chromosome in each cell. So if I say sequencing chromosome1, what does that actually mean? Which of the two chromosome's gets sequenced? Of genotyping-do comparison take place between two+two=four chromosome1's? Or is my entire undertanding flawed?

What is your own research about this question? To avoid closure as "homework question" (which is also used as a synonym for low effort questions), please add this information using the edit function.

From your question I gather that you do not have a basic understanding of sequencing....The answer can get pretty long....It would be advantageous to know your background and as Chris mentioned to know your research on the matter.

No I don't.My background is electronics engineering. But I am keenly interested in this topic and is eagerly waiting for the answer. Please do explain in simple words how it is done and more importantly about the two strands getting sequenced.

http://www.genomenewsnetwork.org/resources/whats_a_genome/Chp2_2.shtml. I have a rough idea that dna is cut into pieces ,stained etc. What I am concerned about is not the process,but the nature of the output. In what order will the output be presented? Or is it completely random?

I would say your understanding here isn't necessarily flawed - rather, there's a little trick being played on you.
The figure depicts typical results of Sanger sequencing. This type of sequencing always requires a so-called primer, a short sequence of around 20 bases which are known. The code following those 20 bases will be sequenced. Using this technique alone, you can't practically sequence a whole chromosome - it's usually only good for about 700-1200 bases after the primer.
The bands in the graphic on the left and the peaks on the right side correspond to the base at position "primer + n" (n being the number of bands/peaks starting to count at the bottom of the graphic).
Now consider that in practice, this technique is performed on a liquid sample containing of course a huge number of DNA molecules. In an ideal scenario, every single molecule is identical. In that case, the sequence behind the 20 primer bases is identical and you will get clear bands or peaks as shown in the graphic.
When you take a sample from an organism like a human, there will be a variety of DNA molecules in the mix.
What gets sequenced? Simple, everything that sits behind the primer you're using to sequence. If different molecules have different sequences there, the result will be bands in different columns at the same position on the gel (left side of the graphic) or two different-coloured peaks at the same location in the chromatography (right side of the graphic).
Example: Genotyping
We consider the XYZ gene. There are two variants of this gene in humans, and the only difference is at position 167 of the gene, where one variant has an A and the other a G. Thanks to the human genome project, I know the full sequence of the XYZ gene, so I design my primer to span positions 50-70 of XYZ. Therefore, my sequencing result should yield the XYZ gene, starting somewhere around position 100 (even the highest quality sequencing reactions miss out a few bases after the primer). If I take a sample from a human who has the same XYZ variant on both sister chromosomes, I will only get a single sequencing result - either with A or G at position ~67 of my sequencing. If my sample came from a human who has one variant on one chromosome and the other variant on the other, my sequencing will be ambiguous at position ~67, and upon closer inspection I sequenced two variants from this sample: one with an A and one with a G at this ambiguous position. This way I can determine a human's genotype for this gene: homozygous XYZA/A, XYZB/B or heterozygous XYZA/B.
PS: Don't let the double-stranded nature of DNA confuse your thinking here, Sanger sequencing always only sequences one strand (the one that you designed the primer for)! So two sister chromosomes = two double strands = four single strands, of which two will be sequenced because the other two contain the complementary sequences.
PPS: The way this works can cause huge problems if the primer isn't designed properly. With 20 bases, it's possible that the same 20 bases appear elsewhere in the genome, and suddenly the sequences following the primer aren't only different in one or two positions, but everywhere.

I am interested just in the nature of the results as described here.

By aligning this sequence to all nucleotides of all organisms, we would realize that the given (short) sequence occurs in various organisms besides humans. Therefore it might come from a species with only one chromosome see NCBI Blast !
For the sake of the example, let's assume that the species had multiple instances of each chromosome (e.g. one by human mother, and one by human father), and look at "the results as described here". As no detail is given on the protocol, let's further assume the most common scenario - namely that there was no experimental distinction or separation of the individual instances of chromosomes.
Looking at the results, we would realize that is not "next generation sequencing", but Sanger-sequencing.
Now we look at the provided scheme again (note: this is not real experimental data): clearly the bands on the left side only occur for one letter/base, and at a given position of the sequence, there only is one very clear peak on the right hand side (as opposed to the possibility, to have multiple peaks corresponding to different bases).
We would have to conclude that every instance of the chromosome has an absolutely identical sequence. Thus we would conclude that it would not be important to distinguish individual instances.
But wait - aren't maternal and paternal chromosomes different? Absolutely, the given example however only looks at 25 bases rather than the full chromosome - and it would quite possible that they are absolutely identical. (note: there are various experimental techniques to select only specific regions of DNA or RNA for analysis).

That's a picture of Sanger sequencing.  If that sequencing had been done in a region where a diploid organism was heterozygous for a simple SNP, the trace file would instead of having single clean peaks, would at the point of the SNP have two half sized peaks of two colors for the two letters present at that site.  If the difference between the two alleles was more involve than a single nucleotide polymorphism, the trace file might look quite different.

So if I say sequencing chromosome1, what does that actually mean?

People don't often just sequence a whole chromosome like that, and pretty much no one would do it with Sanger technology anymore.  But if you were sequencing a diploid non-inbred organism, you would expect at points of heterozygosity to get a mix of signals.

Of genotyping-do comparison take place between two+two=four
  chromosome1's?

Diploid organisms have 2 copies of a given chromosome, not 4.  Each chromosome has two strands, but the information on complementary strands is identical.

UPDATE

Before coming to sequencing you have to consider that you are not sequencing a single chromosome from a single cell. You are sequencing a DNA sample corresponding to a population of cells, containing all the chromosomes. I do not know of a procedure to target a single chromosome, targeted procedures for specific regions on the genome sorrounding genes are in existence.

Or is my entire undertanding flawed?

Yes, I would say lacking not flawed.
That said,

So if I say sequencing chromosome1, what does that actually mean?

Hypothetically that would mean you are sequencing DNA from chromosome 1 corresponding to a population of cells. Meaning you are retrieving the sequence of chromosome 1 for usage in downstream analysis by polymerizing its complementary DNA sequence by taking advantage of the procedure of DNA replication.

Which of the two chromosome's gets sequenced?

There is no way to differentiate between two chromosomes in mainstream sequencing experiments. The cell does not have an idea about which chromosome is which and neither do you, so both chromosomes get sequenced at the same time. Please note, I said no way to differentiate between two chromosomes in mainstream sequencing experiments

do comparison take place between two+two=four chromosome1's?

This has two parts. What do you mean by four?
If you are implying that chromosome 1 has two strands and that equates to 4 then no, the two strands together make one chromosome. Your cell has 2n or two chromosome 1's. If you are sequencing a population of cells (let's say 1000), you have two thousand chromosome 1's which are being sequenced all of which are present in pairs across 1000 cells.
Do comparisons take place?
In mainstream experiments we do not compare. That said, methods for differentiating between the two chromosome 1's exist. The oldest I could find is an article by M Nagano on allele specific sequencing. In this case, since your parents equally contribute towards your DNA, each of them carry some mutations specific for their DNA. Using this feature we can differentiate between the paternal and maternal chromosome 1.
There's also single cell sequencing, in this case you would be sequencing the two chromosome 1's from a single cell, theoretically you can then also do allele specific sequencing on a single cell allowing you to differentiate between the two chromosome 1's present in the cell.
Finally, there is no way to differentiate the two strands, after sequencing when you align your data back to the reference genome, you will get to know the strandedness of the data.
Answer to old question

Nowadays the word sequencing is synonymous with High-throughput sequencing, and the article you linked to in the comment relates also to high-throughput sequencing techniques (I got this from a cursory glance).

While sequencing dna which of the two strands get sequenced?

Both get sequenced.

In what order is the results provided

Random

while sequencing how are these two strands sequenced?

If it is single end sequencing, we do not know which strand was sequenced first. (There are ways, but for simplicity's sake let's not go there)
If it is paired end sequencing, one read originates from the sense strand and the other read originates from the opposite strand.
You can watch a youtube video here to find out what is the difference

Is it that result of one strand is provide first followed by the other or is it that only one of the two strands get sequenced?

Both are reported.
The question you have asked is very broad, I can go on for a handful of A4 pages and still not finish. Coming to your first question, I said both get sequenced, because we have no way of knowing while sequencing which strand is getting sequenced (there are stranded sequencing techniques, but you should digest what is mentioned/linked here first and later on with some more research come back with a question on stranded sequencing).
After sequencing you get your result in random order, because what you get is not really result but more raw data files which are called FASTQ files. Read up on FASTQ a bit. After sequencing you don't really get a FASTQ but you get a BCL or base call file, which must be converted to FASTQ. These FASTQ must then be either filtered or not based on quality then aligned to a reference genome. This is where you get to know which read came from which strand.
You should read up more on single end sequencing and paired end sequencing to better understand how DNA is sequenced. Also check out this video. This is the most simplified version which I could find. It should make you curious without bombarding you with too much information.
Last of all, in a single end sequencing you have no way of knowing without alignment which strand got sequenced, but in paired end (where a DNA fragment is sequenced from both ends, generating two paired reads) generally, one mate aligns to the sense strand while the other aligns to the antisense strand.

I do not know of a procedure to target a single chromosome Check out single cell sequencing. I'm more familiar with it in terms of RNA-Seq, but one can perform single-cell genomic sequencing as well.

I meant that I do not know of any procedure till date, which isolates an entire chromosome for sequencing...there are targeted protocols for sure...but targeting an entire chromosome? I'm yet to encounter a procedure which employs isolation of a chromosome before sequencing.

OK, I understand. I doubt such a thing is possible in somatic cells, as there is no way to differentiate between maternal and paternal chromosomes. However, I don't think there is a scientific need to do that. If you want to know where a particular SNP or other mutation comes from, just sequence the subject's parents (if possible).

This poster is asking about Sanger sequencing, not next gen.  There are no bcl files in Sanger sequencing, and it's not clear to me that returning Sanger data in FASTQ format is standard.  It wasn't in the years that I worked in Sanger sequencing.


text:
How far can a mosquito fly over the sea?
My question is: how far can a mosquito fly over the sea? I am interested to know, because from this, I could possibly deduce on which islands there either might not be mosquitoes or on which it would be achievable to eradicate all mosquitoes until somebody brings another larva.

That won't work because remote islands are known to have been populated by insects and spiders that have been blown by winds to quite high altitudes and then transported over thousands of kilometers. So, even spiders can be found on young volcanic Islands in the middle of the Pacific.You can find insects and spiders right until the edge of the troposphere at about 10 km altitude, the vast majority of them will end up dying, a few lucky ones may end up in some tropical paradise on the other side of the planet.

How much wood could a woodchuck chuck??

Also, mosquitoes can hitch rides on something else that floats or flies across the sea.

they are exhansted in under 5 miles, and they tend to shelter from wind, because it uses their energy until they find a host.

If we ignore the weather effects mentioned by Count Iblis we can do a fairly simple calculation.
The lifespan of a male mosquito is about 10 days whereas females live to about 50 days.
Their flying speed is generally about 1.2mph.
So if we assume that they can fly whilst asleep and maintain a constant velocity for their entire lifespan (obviously ridiculous because of the need to eat etc), their maximum ranges would be:
Male: 1.2 * 24 * 10 = 288 miles.
Female: 1.2 * 24 * 50 = 1440 miles.
It should be noted that these are absolute theoretical maximums. I would suspect real life values to be somewhere in the region of 10%-20% of these. However, once wind is taken into account they can easily spread globally.

Can a mosquito not land on the sea water to take a rest, or mate in the air, or find some blood to suck along the way?

Mosquitos are fragile. They don't like direct sun, it dehydrates them fast. they come out a lot at night and in humidity. they don't like spending energy uselessly, their only food source is blood, so they have limited reserves for flight life. I suspect that they can't fly more than 2-3 days continuously without exhaustion. freshwater mosquitos may struggle with sea mist and seawater landings, it may coat them with too much salt for them to preen and work effectively. Either way, they are not fast flyers and they have low energy reserves.

sorry, that answer is inaccurate, they can only fly 1-2 percent of the given figure, not 10-20%, 2.8 miles not 28. They are thought to travel globally by airplane much more often than from wind!

They fly maximum a few kilometers, normally about 2-3 kilometers, before being tired.
Mosquito species preferring to breed around the house, like the Asian Tiger Mosquito, have limited flight ranges of about 300 feet. Most species have flight ranges of 2-5 km. Certain large pool breeders in the Midwest are often found up to 10 km from known breeding spots. The undisputed champions, though, are the saltmarsh breeders - having been known to migrate up to 160 km in exceptional circumstances, although 30 to 50 km are much more common when hosts are scarce. When caught up in updrafts that direct them into winds high above the ground, mosquitoes can be carried great distances. When it's windy they tend to hide in forest leaves and in shelters because wind fatigues them, If you can feel the wind, then they probably can't navigate in it.
Time (h)        Speed (km/h)        Distance (km)       Glycogen (J)

WT  3.68 (±0.31)......      A   0.68 (±0.03)......  A       2.39 (±0.19)........        -A  2.75 (±0.10)... A
Here is a full study, you may find different figures, and even if some species can cly for 10-20 hours total, they would get less than 10-20km without favorable wind conditions.
Wind can sweep them kilometers high where they can travel 1000ds of kilometers, as long as they don't freeze, land in the sea, exhaust themselves, or land somewhere without food, it's a good tactic for them.
To measure their energy reserves of insects, they can do this:

The glycogen, sugar and lipid contents of individual adults of four mosquito species
(Armigeres subalbatus, Culex quinquefasciatus, Aedes aegypti and A. albopictus) were
estimated following standard methods (VAN HANDEL 1965, 1985a, 1985b). Dead and dried
mosquitoes were individually homogenized in 0.2 ml 2% sodium sulphate and a few drops
of methanol in a 15×155 mm glass tube. This solution was vortexed
vigorously, after which a mixture of methanol and chloroform (1:1) was added to it.
The
tissues were crushed and then stirred. The mixture was centrifuged (
MOHAN S. et al.: Energy reserves in for co-occurring mosquito species 53
USA) at 2000 RPM for 20 min. The supernatant was transferred into a 15×155 mm glass
test tube for the sugar assay, while the precipitate
was left for the glycogen and lipid assays. The test tubes containing sugar were heated to
evaporate the solution in it to 0.1-0.2 ml. Anthrone solution was added (up to the 5 ml
mark), stirred and heated in a water bath for 17 min. Following this the test tubes were
cooled in an ice box and then stirred.
The absorbance of the resultant solution was
measured at 625 nm in a spectrophotometer (SHIMADZU® UV-17000, India) and
compared with glucose standards.
To the precipitate left earlier, 0.5 ml chloroform and methanol were added and the
whole gently shaken. The mixture was centrifuged at 2000 RPM for 10 min using
a centrifuge, yielding a supernatant (containing glycogen) and
precipitate (lipid).
The supernatant containing glycogen was collected in separate test tubes
(15×155 mm, marked up to 10 ml, graduated at 0.1 ml) and then heated well to evaporate
the solution. Next, anthrone solution was added to it (up to the 5 ml mark), stirred and
heated in a water-bath for 17 min. It was then allowed to cool in an icebox and stirred. The
absorbance of the resultant solution was taken at 625 nm using spectrophotometer and compared with glucose standards.
For estimating the lipid content, the precipitate was dissolved in a mixture of methanol:
chloroform (1:1). The test tubes (15×155 mm, marked up to 10 ml, graduated at 0.1 ml)
were put in a water bath for evaporation and 0.2 ml concentrated sulphuric acid added.
Then they were cooled in an icebox and vanilin was added up to the 5 ml mark. The
solution was stirred and a reddish colour allowed to develop within 5 min. The absorbance
was measured at 525 nm using a spectrophotometer (SHIMADZU® UV-17000, India) and
compared with the lipid standard.
*
 All the standard curves for sugar, glycogen and lipids
were prepared with 10, 20, 30, 40, 50 ml concentrations of glucose and lipid solution
(mg/ml) using the same reagents, and the same treatment was applied to the mosquito
tissues. A schematic diagram of the experimental setup is shown in Fig. 1.*

... that's what they do in the lab!!! sugar, alcohol, it's only missing some fruit and spices.
Dragonfly and Locusts are long distance flight champions, they can cover 1000ds of miles overseas, some dragonflies can feed on the wing. Small animals generally have to feed very often to survive, mice, moths, run out of energy quickly.


text:
Do HIV particles die without host cells?
When a person is on antiretroviral therapy (ART) which blocks HIV particles from infecting cells, where do those particles go? Do they die out while floating in the bloodstream? I particularly want to understand if this is the reason viral loads go down once a person starts ART.

What do you qualify as dying? It's questionable whether to call viruses "alive".

I mean infectious by alive. Sorry for the terminology

I've learnt that our circulatory system can clear foreign matters from blood stream. Phagocytes can engulf and breakdown virions. Our liver and spleen is the blood filter system, which sequesters foreign matters all the time. So free floating HIV virions can't stay indefinitely in our blood stream.
However, without an effective adaptive immune response, these mechanisms are still unable to suppress viral replication. That's why HIV patients still have high viral load if untreated.


text:
Why does hair turn white but not skin?
From my understanding melanocyte stem cells (McSCs) stop being produced and we have a finite reserve that gets depleted with time. They're at the origin of melanocytes that create hair and skin pigments. Our hair turns white when we don't have any more McSCs. So why does our skin stay the same color with aging?

Welcome to Biology.SE! Please take the [tour] and then go through the help pages starting with [Ask] questions effectively on this site and [edit] your question accordingly. In particular, please add some more details with references to reliable sources (e.g. You say MeSCs stop being produced and are depleted with age. Where did you read this? At what age do they stop renewing themselves? How long do they typically live?) Thanks!

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Nice question. Will write an answer later, when I have some time.

I thought it was common knowledge among biologists that melanocyte stem cells don't get regenerated. I could throw this link where you can read: "After just a few days, all of the pigment-regenerating stem cells were lost. Once they're gone, you can't regenerate pigment anymore. The damage is permanent."

whether they get depleted or not, the question remains. Hope  @Chris gets some time for a basic answer

Biology includes a huge range of disciplines including many specialties (e.g.ecologists and plant biologists) the practitioners of which have no particular reason to know this kind of information!  ——— I think you may be taking that quotation out of context — I think it is saying you don't make more McSCs once they've been killed. Also note that it is best to reference primary literature rather than secondary sources. Finally, if you can find a good reference, please [edit] that information into your question since comments are ephemeral.

@borilla I haven't forgot it, but there is too much work...

The answer to this question has its reason in the hair cycle. Our hair goes through a cycle of growth. At the end of this cycle, the cells in the hair follicle die and have to be replenished before a new hair cycle starts, also the hair falls out eventually. See the figure from reference 1:

The replenishment process is done by to two cell types: Epithelial stem cells which replenish the hair follice itself and melanocyte stem cells (MCSC) which replenish the melanocytes in the follicle and produce the pigment of the hair.
The MCSC are located in the bulge region of the hair, to be able to successfully replenish the pool of pigment cells the MCSC need to do things: proliferate and also differentiate. Proliferation makes sure that there are enough cells in the bulge region to repeat this process in further hair cycles, while the differentiation generates the cells who actually produce the pigment. When the MCSC are lost, no further pigment producing cells and hence no pigment can be made.
It has now shown that with age this pool of MCSC is not replenished completely anymore, eventually resulting in the loss of the melanocyte stem cells. The reason that our skin does not become gray at some point is that this process doesn't happen there, so pigment cells are not depleted. Reference 1 summarizes this topic nicely, while reference 2 is the original paper.
References

Melanocyte Stem Cell Maintenance and Hair Graying
Mechanisms of Hair Graying: Incomplete Melanocyte Stem Cell
Maintenance in the Niche

If I understand correctly, after a certain age hair follicles are not replenished with MCSC. But MCSC are still present somewhere in the skin to differentiate into the melanocytes of the basal layer . And these basal layer melanocytes don't last forever so they need to be replaced.
If this is correct then my question is:   where are those MCSC that still create melanocytes for the basal skin layer?

MCSC need to proliferate before they can differentiate into melanocytes. This happens to rise the number of cells present in the bulge region and to avoid the depletion of the stem cell niche. If the would only differentiate, then there are no cells left after a few hair cycles. So actually the stem cell niche is empty when you have no pigment anymore.

humm I still don't understand where the melanocytes in the basal layer of the skin come from after all your hair have turned gray. I read both articles and I can't find an explanation on the net. Maybe I am misunderstanding the whole process...

license: Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/


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"bead" on a string theory of genetics, source?
Thomas Hunt Morgan was a pioneer in genetics and proposed the now false model of genes being "beads" on a string. These beads being indivisible and responsible for a single phenotype, if I understand correctly.
Does anyone know of a good, reputable source of information on this theory that would be sufficient to use as a citation? I would use wikipedia, but it is disallowed.

I found this reference on the Thomas Hunt Morgan wikipedia page. It uses the "Beads on a String" phrase and may be more appropriate material for your homework. I would recommend you read the references on the bottom of that page for more information on his work and hypotheses.

If the Cold Spring Harbor Laboratory is considered a reputable source (it should be), you can check out their Defining the gene page which has an overview of the beads-on-a-string theory, and how it was disproved by Seymour Benzer. You could also try to find Benzer's original paper, although it doesnt seem to be available online.
Related and possibly helpful:

Benzer's page on Cold Spring Harbor website
Evolution of Genes and Genes in Evolution - very interesting paper from 1959 on genes/genetics, look on the fourth page (marked 18) for the bead-on-string theory


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Can anyone identify this?
Found on Cape Cod MA near a fresh water pond with a muddy bottom. Strange looking creature with a suction cup single "foot" with a mouth opening on opposite end, no limbs, mouth ridges inside mouth, spiral rings of tissue up entire body. Any idea what it is? I thought it might be a tunicate of some kind but I can't find a match anywhere.

It is a artificial fishing lure.specifically a soft worm T-tail, there are hundreds of variations.

Really, just wow on this one.

This may be a mimic but what I have is NOT plastic,

Okay, now we may BOTH be right. This thing I have is not plastic but I finally cut in to it an VIOLA! It is a colony of mud worms that probably took up residence in one and the plastic is long gone, the outside is a mud shell, the inside is a mess of worms...............

Not all lures are plastic many are latex


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Protein structure prediction from amino acids sequence
Information given at this resource https://predictioncenter.org/ is close to impossible to digest (as with everything in this field), so if anyone could tell me what is the accuracy we can predict tertiary protein structure now - I would be grateful.
Also would love to hear your thoughts on 'why a cell can make exactly the same protein structure thousands of time using known to us physical laws, but we have to guess it using machine learning'? Why is it difficult?

"using known to us physical laws" — Do you mean that we know the laws of Physics or that we know that the laws of thermodynamics determine that a protein folds to the lowest free energy possible, or that we know how a protein actually progresses to its thermodynamically favoured state? If you tell us what you think we know and what precisely you think the "it" is we need to guess, we may be able to explain why "it" is difficult. At present your question lacks the clarity needed to help you effectively.

@David yes you're right, I had too simple image of the process, after more studying I understand why you're objecting

what is the accuracy we can predict tertiary protein structure

That depends on the protein.  If the primary sequence closely matches the sequence of a protein for which the structure is already resolved, then template-based methods to model 3D structure can be used (a.k.a homology modeling).  These methods tend to be accurate, as assessed by template-modeling score, though crystal structure confirmation is only available for a minority of models (1%, per this 2010 paper).
For proteins without structurally-resolved homologs, ab initio folding is often used, which relies on molecular mechanics assessment of iterative peptide chain folding to find structures that minimize the Gibbs free energy. Popular software for protein molecular mechanical modeling include CHARMM and AMBER.  Ab initio methods are computationally intensive and are more difficult to validate.

'why a cell can make exactly the same protein structure thousands of time using known to us physical laws, but we have to guess it using machine learning'? Why is it difficult?

It's hard to know all the cellular factors present when a particular protein is synthesized and how those factors affect the folding of the protein.  What is the temperature and pH proximal to the ribosome?  Are chaperone proteins involved?  Is the lowest-energy structure the true structure, or does the native structure fall in a local, stable minimum with a functional potential selected by evolution?  A good discussion of that last point can be found on Quora.

Thanks +1! There are many good points for further research in your answer :fire:

The predictioncenter.org runs an open contest every two years - Critical Assessment of Structure Prediction (CASP).  CASP 14 is underway now.
The best programs for ab initio folding are highly augmented molecular dynamics with machine learning and a bunch of predictive algorithms aggregated to create a structure.  Look at David Baker's Rosetta software. More recently Google's DeepMind beat out Rosetta and a gaggle of others.
predictioncenter.org/casp13/zscores_final.cgi
Deepmind is a Deep Belief driven network https://deepmind.com/blog/article/AlphaFold-Using-AI-for-scientific-discovery

We trained a neural network to predict a distribution of distances between every pair of residues in a protein (visualised in Figure 2). These probabilities were then combined into a score that estimates how accurate a proposed protein structure is. We also trained a separate neural network that uses all distances in aggregate to estimate how close the proposed structure is to the right answer.

The problem of protein folding is known as Levinthal's paradox: a typical protein of a few hundreds amino-acids can fold into an astronomically big number of configurations, all of them consistent with the laws of physics (e.g., having the same or nearly the same energy). Yet the actual protein in a cell (nearly) always folds in the same structure and all the known proteins fold in about a thousand well-defined structures (moreover, sometimes proteins with little sequence similarity adopt exactly the same structure).
For those who want to know more about the physics behind, a rather readable introduction is Huang's Lectures on statistical physics of protein folding, whereas the other answers in this thread have already given a rather good review of the methods used in practice  - note that these are not necessarily relying on the machine learning, as the OP suggests, although the ML has been used for this purpose for a few decades already, e.g., see this book.


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Because we don't live as closely to livestock, are we at a greater or lesser risk of plagues?
Give recent events, some people have called for the 'wet markets' in China to be shut down, (for many reasons) but largely because it is likely the source of the recent plague outbreak, due to the close human-animal contact there.
(I am not here to debate if the markets should or should not be closed down. That is something for another question.)
However looking at the (very short and incomplete) history of the smallpox vaccine: Someone noticed that milk maids didn't have the ugly smallpox scars those who had survived a smallpox infection had. They conjectured the maids had caught the mild cow pox off their cows, and had gained an immunity to smallpox as a bonus. Cowpox was then developed in to a vaccine, fast-forward a few years and now smallpox has been eradicated. The 'natural' cow pox was basically an attenuated live vaccine for smallpox.
Now today we have supermarkets and mechanized agriculture and so have far less contact with animals (e.g. back in the day many households would have their own cow and chickens, we rode horses for transport, etc.) Therefore we have less of a chance to develop immunity to 'naturally attenuated' diseases, which they can immediately fend off if the disease does mutate/break the species barrier into a plague worthy foe.
I am not suggesting we should go back to milking our own cow before breakfast, however:
Does our lack of contact with domesticated animals mean we have less 'naturally attenuated' disease exposure and therefore we are at a higher risk of plagues?

Cleared out the comments because they were turning into an argument and drifting away from being nice. For OP: I think it's an interesting question but one that's difficult to answer in the SE format. You've identified what are probably the two key factors: immunity due to 'natural immunization' and exposure to zoonotic diseases. Someone might be able to make an answer by compiling some expert speculation on either side, but I don't think it is likely that a comparison has been/can be made.

The question is based on a false premise.  Although a smaller percentage of the population has contact with domesticated (or indeed, wild) animals than in previous centuries, population growth means that there are more individual humans who have such contact.


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Can anyone identify this mite?
I live in a flat in the UK and have caught such mites a few times on my skin. I think they are responsible for my bites. On one occasion one mite had one dark spot in the middle instead of two.
Does anyone know what it is?
Regards

Welcome to Biology.SE! Please [edit] your post to include an estimated size for these creatures. That information can be important for identification. ——— If possible clear, closeup photos of the creature from multiple angles would also be helpful. ——— Please also take the [tour] and then go through the help pages starting with [Ask] questions effectively on this site. Thanks!

Also related: Tiny white very hard cell bug! Please help to identify!


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How does SDS-PAGE separate based on mass?
In SDS-PAGE, electric field and mass-to-charge ratio are approximated to be constant for all proteins. Also, if $F=qE =ma$, then $\frac{m}{q}a=E$.
Thus, all proteins must migrate with a constant acceleration.
So what is the driving force in SDS-PAGE that resolves proteins of different mass? Is it that bigger proteins have a larger size and thus experience larger friction force from the gel sieves? But even if that was true the larger proteins feeling larger resistance forces should be counteracted by their larger migrating force from increased total charge.
Would appreciate if someone could clear up this confusion. Thank you.

Please define the terms in your equations (and set them on a separate line for clarity).

You are correct that molecule mobility depends on the mass-to-charge ratio, and this means that different sized molecules with the same $\frac{m}{q}$ will have the same acceleration.  However, the velocity of a molecule moving through a gel matrix depends on the point at which the force exerted by the electric field is in equilibrium with the frictional forces acting on the molecule.  Modeling different sized molecules as spheres of different sizes, we can apply Stokes' Law:
$$F = 6\pi \mu Rv$$
where $\mu$ is viscosity of the matrix (constant), $R$ is the radius of our molecule, and $v$ is the flow velocity. Rewriting your equation, we see that
$$F = qE = ma = 6\pi \mu Rv$$
and
$$v = \frac {ma}{6\pi \mu R}$$
For constant $a$ and $\mu$, velocity varies with the ratio of mass to radius
$$v \sim \frac{m}{R}$$
Assuming uniform density ($d = \frac{m}{V}$), the radii of our model spheres scales cubically with their mass ($V = \frac{4}{3}\pi R^3$), meaning that $\frac{m}{R}$ decreases as molecule size increases.
So, larger molecules will reach force equilibrium with the opposing frictional resistance at a lower velocity compared to smaller molecules, thus explaining how different sized molecules with the same $\frac{m}{q}$ will separate on a gel.  One caveat to this explanation is that polypeptides and nucleic acids are poorly modeled by spheres, and that the mass of such molecules scales linearly with length.


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If a ubiquitously ill species existed, would that more or less falsify evolution?
It is true that a lot of members of our, and most other species, suffer from cancer, but it is still not ubiquitous nor does it exist throughout the individual's life.
If a species existed, in which all, or almost all of its members suffered from the same pathology, and that pathology showed up and existed throughout the organism's life, and this pathology has always existed within the species, would that violate natural selection enough to consider the current body of theory of evolution falsified?
To be clear, it is recognized that natural selection is not the only process which occurs during evolution, but it is pretty safe to say that enough of our body of theory on evolution rests with the validity of natural selection that if natural selection were falsified, so would the body as a whole, and we would need to rework our theories.
Consider what is said in Evolutionary dynamics in structured populations.

An evolving population consists of reproducing individuals, which are information carriers. When they reproduce, they pass on information. New mutants arise if this process involves mistakes. Natural selection emerges if mutants reproduce at different rates and compete for limiting resources.

In other words, evolutionary dynamics implies natural selection. A violation of natural selection therefore would make the current theory on evolutionary dynamics less likely. Now, it could be that other processes are so powerful that they completely override natural selection, but to find a species in which natural selection has totally failed to weed out a near universal illness should present a problem for current evolutionary theory.

If the disease/illness does hinder the fitness of the species, natural selection has no pressure to stop it.

Although I think @Remi.b's points about selection not being the same as evolution clearly show that this question is misguided, you could argue that aging is a ubiquitous illness that has some selection benefits. Overall, though, this question sounds like it's intended as a trap.

I am not conflating natural selection with evolution, but if natural selection is falsified, then so would evolution, at least as we view it now, which means that our entire body of theory would have to be reworked.

@vasshu Natural selection is only one process by which evolution occurs. There is also genetic drift, gene flow, and mutation. Even if natural selection is falsified, it would not falsify evolution since there are other ways evolution occurs.

True, but natural selection is a consequence of evolutionary dynamics. To see it fail to function, even in one species, would present a problem.

The question is non-scientific and imprecise. The phrase "natural selection is a consequence of evolutionary dynamics" is either wrong or ill-defined. At best, the implication is backwards, and even then only a partial truth. Natural selection is merely the fact that different entities survive differently. This can contribute to evolution, if e.g. there is a mechanism for heredity. As for the question underneath, the answer is no. Evolution is limited by its substrates and environmental factors. That is why we still have cancer, age-associated diseases, etc... which are indeed near-universal.

The question is non-scientific and imprecise. The phrase "natural selection is a consequence of evolutionary dynamics" is either wrong or ill-defined.

I mean, I paraphrased a scientific paper on the topic, but sure. Okay.
As for cancer, etc, I specifically pointed out that I am not addressing conditions, such as aging, which are not necessarily able to be "weeded out" by natural selection. However, even in those situations, the affliction is not ubiquitous throughout most of the life of the organism. The organism does not start off elderly.

Very short answer
Evolution ≠ Natural Selection
Slightly longer answer
Evolution ≠ Natural Selection
You seem to equate evolution with natural selection which is the main issue. Various evolutionary processes may yield a population where all individuals suffer from a specific disease. It may not be an equilibrium state though but a state that may well last long enough to be frequently observed.
For example a bottleneck can cause a disease to reach an extremely high frequency if there was some deleterious recessive alleles in the parent population. A bottleneck is not a process that is encompass within the term Natural Selection but it is still an evolutionary process.
We actually have examples (incl. examples in humans) of populations where the prevalence of particular genetic disease is very high.
The existence of other evolutionary processes than natural selection does not violate the theory of natural selection
The existence of other evolutionary processes than natural selection does not violate natural selection theory. The theory of natural selection does not state that nothing but natural selection can affect a population's evolution.
If one were to think that the existence of genetic drift (another evolutionary process) would violate natural selection, then this person would also think that  the existence of wind that would affect an apple course falling through the air would violate the theory of gravitation.
What is evolution?
Your question comes from a misrepresentation of what evolution is about. You might want to have a look at a short and intro course to evolutionary biology such as Understanding Evolution by UC Berkeley for example.

I did not mean to equate the two, although our current theory of evolution is heavily dependent on natural selection. As for my understanding, I have a fair amount of bio behind me, including evolutionary biology and bio-archaeology.

You got close with the vague reference to diseases w/ high prevalence, but this is not the same as ubiquitous existence and expressed throughout the individual's life.

Can you please expand on diseases w/ high prevalence [..] is not the same as ubiquitous existence and expressed throughout the individual's life? I don't understand it. Do you mean high prevalence is not the same as prevalence of 1? You can have a prevalence of 1 and that would not change anything. Fixed deleterious alleles are really not uncommon.

Cancer, for instance, occurs in many individuals, but it is not something that almost every individual lives with for its entire life. It occurs within a fraction of the population for a fraction of the organism's life.

Evolution doesn't ensure a good quality of life, nor does it ensure a longer lifetime of a species's organism.. It only aims for the continued sustenance of the species in a changing environment.
What does being ill really mean?

Does it mean that the lifespan becomes smaller? No, an insect might have a smaller lifespan while being a very rapidly evolving life form.
Does it mean that the species would be pain-free while living? No, even humans are designed to endure chronic pain.

All the things that contribute to not being "ill", are really just things which contribute to being able to have a faster and continuous sustenance. Long life means more chances of offsprings, being pain-free gives more time and ability to mate.
Hope this answers your question! First post. This thread made me join this exchange!

Evolution [..] defines continuous sustenance That is a little unclear but sounds pretty wrong. Selection (and other evolutionary processes) does not necessarily minimize the probability of extinction.

You are right, I should change that to "Aims to" rather than "Is defined by".

Even "aims" would be wrong. Evolution is not conscious being attempting to do anything. You could say "tends" to avoid the agent view of evolution but even then, it would be misleading. Evolution does not necessarily tend to minimize extinction probability nor does it tend to increase individual's lifespan (whatever you mean by sustenance). A genotype decreasing the group mean absolute fitness to its own benefit will be selected for.

Well, I think it is agreeable that Evolution(Even though it is a phenomenon) can be attributed to doing something. For example: The Cold temperatures in the high altitudes leads to a higher chance of rain.

Evolution refers to the change of allele frequency (and phenotypic traits) in a population over time. It does not do anything (sorry that sounds like I'm being a pain in the neck with definitions but it may be important for our discussion). There are particular evolutionary processes that cause those changes, such as natural selection and genetic drift for example. But genetic drift and natural selection do not necessarily lead a species to increase its probability of survival (or whatever is meant by sustenance in your answer).

The mechanics of evolution imply natural selection.

Welcome to the site and thanks for your answer :) It's probably better to say evolution due to natural selection tends to favor continuation of the species. But, this may not always be true; evolution can be very short-sighted [e.g. the giraffe recurrent laryngeal], and allow a trait that harms the population later e.g. due to environment change (say, allowing a koala's diet followed by a disease that wipes out eucalyptus trees). Further, as Remi noted, a trait that harms the group but helps the individual can easily be selected for.


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Is the backward positioning of photoreceptors bad design?
(Several questions have been asked about this topic but most are quite old and there has been at least one study since then that has attempted to answer this in a new way)
Since photoreceptors are placed at the back of nerve cells in vertebrates, these nerves have to then bundle up before passing to the brain, leading to a blind spot. This video (from a usually reliable channel) from 2:30-4:00 says that its bad design and is just something the initial eyes in vertebrates fixed on, and was too difficult to correct later.
But this report of a study from 2015 says that this arrangement helps vertebrates see colours more easily during daytime, and is not a bad design.
Even if this design does help in seeing colours better, the same effect could have been achieved through other means without having to create a blind spot, no?
Can this be called a bad design or not?


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What kind of snake is this? Seen on Koh Lanta, Thailand
We had this snake in our bungalow on Koh Lanta, Thailand. From the reaction of the employees at the resort I can tell that it is venomous, but that's all I know. I am curious, does anybody know what species it is?
Thanks and cheers, Philip!

If the resort is near a water body then it is highly likely that it is a keelback snake.
For determining whether it is poisonous or not it is extremely important to look at the scale pattern on its ventral side(belly). Unfortunately that is not visible here.


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PCR products with no band in gel
I am a PhD student and stuck with the no band in the gel  results.
I am using the following

1 microliter  Primer R (20mM)
1 microliter  Primer f (20mM)
1 microliter  dNTP  (10mM)
1 microliter  DNA Template ( 448 ng)
1 microliter TAQ polymerase
5 microliter Buffer (10x)  added MgCl2  by 1:1
30 microliter PCR grade water

Program:

95 for 4 minute
95 for 45 sec
55-65 for 1.20 minute
72 for 1 minute
Gradient 35 cycle
72  for 4 minutes

Gel:
110 volt, 200 mPA for 30 - 45 minute
I get PCR products as in the images. There are no bands  while there are intense bands at the bottom and some of the wells.
My question is,  is that at the bottom products??
what can be done to make them real? If not then why there are differences between the two lane of wells. Each 12 well are representing a one type of DNA template (I used three different organisms DNA).

First: Your PCR is working in principle, the bands at the bottom are beautiful primer dimer bands which indicate this. Then: What is the source of your DNA and has it ever been user successfully for PCR? How is it prepared?

Hi Chris, thanks for your reply.  The DNA are from LLactobacillus and Lactococcus  bacteria.  The primer has been designed as degenerated based on both of them.  Up to now I couldnt get the PCR work .

Ladder looks good...

Can you post the sequence you intend to amplify and the sequences of the primers you are using? Also, do you have any positive control for the Taq?

Do you use genomic DNA? Then you can try to enhance the outcome by using either less template or add additional MgCl2. You can also try different PCR methods (i.e. spiked PCR or Touchdown).

My first guess would be that you're using too much template DNA for these conditions. Your wells light up quite a bit, 448 ng is also on the high side.
Try lower concentrations of template DNA (1-100 ng), perhaps only with a 55 °C  annealing step. At the moment you don't have any product at all, so I wouldn't worry about non-specificity yet (and 10 °C is also quite a small range to fix that).
Other things that stand out:

I assume it's 20 uM primer, not mM
You're adding too much buffer
It looks like you're making each reaction individually (looking at the template amounts in the first 12), I suggest making a master mix and aliquot that out.
You have quite some space to run your gel longer, that might be very useful if you do get some product.

You should have a bit more information as well. What's the size of the products that you expect? Do you use high GC organisms? Did these primers work
before for other people? Did the genomic DNA work before for other people?
NEB has a lot of useful information about PCR and other DNA work on their website, check it out: https://www.neb.com/tools-and-resources/usage-guidelines/guidelines-for-pcr-optimization-with-taq-dna-polymerase

Use control primers targeting something which for sure gives a product. If you have none, ask your colleagues or take some out of literature. Make sure your product is small enough to be amplified in the time given and check for the correct band with a restriction enzyme.

I'm not sure about the scale of your ladder but regardless the DNA causing the banding is very small. They are most likely primer dimers. I would check the primer sequences to make sure there isn't a lot of complementarity. You can also try manipulating reaction conditions like concentration of magnesium chloride or the primers themselves etc. I would run an optimization set of PCRs with variable reaction conditions.

You can try a touchdown PCR. It is often helpful when no band is present.


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Does gray/white hair turn gray/white from a normal hair? or does a gray/white hair actually grow gray/white originally?
I am really curious if hair actually tuns white/gray from default color, or does it have a defect as it is made and grows out of your head a different color?

It can actually be for both reasons and it more or less depends on the age what happens. The pigment of the hair is produced by pigment cells (the melanocytes) in the hair bulge and is then integrated into the growing hair (image from here).

The pigment melanin produced by these cells can be destroyed by reactive oxigen species which then leads to grey hair which can still be pigmented at the tip.
The other possibility is connected to the hair cycle. After growing for a while the growth stops, the hair falls out and the hair follicle goes into a resting state. In this phase the pigment cells die. When the hair follicle starts a new hair cycle, the pigment cell population in the hair bulb is renewed from the melanocyte stem cells in the hair bulge. Over time the number of the melanocyte stem cells gets lower until there are none left eventually. At this point new hairs will grow without pigment and be grey/white.
There are two interesting publications about this topic:

Oxidative Stress in Ageing of Hair
Melanocyte Stem Cell Maintenance and Hair Graying

wow, this makes a lot of sense as to why old people lose hair.


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How are the dominance and additive effects reflected in the mean and variance of the offsprings of two individuals with known phenotype?
If I estimated the genetic additive σ2A, genetic dominance σ2D and environmental effect σ2E of a trait in a population, what will be the predicted value of the offsprings of two individuals with known phenotypes?
My understanding: given that the two individuals are not close siblings, and that the mean of the two parents is μparents, the offspring will have a mean of:
μoffspring = μparents + μparents x σ2D
and the variance will be the environmental variance,  σ2E
How the additive effect  σ2A plays a role in a single mating ? (assuming thousands of offsprings (like in some plants) )?

I was starting to make complicated calculations but actually to answer this question one does not need much quantitative genetics. The expected should just be the average between the parent phenotypes $\mu_{offspring} = \frac{1}{2}\left(\mu_{mother} + \mu_{father}\right)$, isn't it? Note btw that the covariance between any parent and the offspring will be $\frac{r \sigma^2_A}{\sigma^2_P}$ and the correlation between the mid-parent and the offspring should be $\frac{1}{\sqrt{2}}\frac{\sigma^2_A}{\sigma^2_P}$, where $\sigma^2_P = \sigma^2_A+\sigma^2_D+\sigma^2_E$

Is the question for an advanced class in quantitative genetics on just an introductory class in evolutionary biology or genetics? If the question is only about the expected offspring phenotype, then I think it is as easy as $\mu_{offspring} = \frac{1}{2}\left(\mu_{mother} + \mu_{father}\right)$.

I see, but how the dominance effect changes the mean? and what will be the variance of the offspring population - is it just the environmental related variance?

The offspring population will have a variance that will be much greater than just the environmental variance. If the trait of interest in not fitness, then the expected variance in the offspring population will be the same if you assume infinite population size and no mutation.

However, you are not asking this question in your post. That gets me confused as to what the question is. Can please highlight (in bold or something) the one single question of your post?

I see you edited the question. Thanks. What do you mean "plays a role" in your highlighted question? Also, I don't quite see the parallel between the question in the title and the one highlighted in the post. I don't want to be pedantic, I am just trying to make sure I really understand what you're asking.

I would like to simulate a population in which I know the real heritability, dominance effects and environment effect, estimated from sibling ANOVA, parent-offspring regression and comparing different environments.  Although I can understand the role of these effects on a large population, I am not sure how to apply this to the predicted parameters of each individual offspring of two individual parents. What will be the right formula to estimate the phenotype of  each offspring in the simulation?

I don't understand. What do you mean by  in which I know the real heritability. Do you mean you want to force heritability to remain constant? However, in the same time you say, you want to estimate it as if you don't want heritability to be constant. By simulate, I suppose you refer to numerical simulation of course, not analytical simulation/modelling.

Let say I have large phenotypic data, spanning many parents, children and environments. I estimated the broad sense Heritability from siblings ANOVA, the narrow sense heritability from parent-offs.  regression (~σ2A). and the environment effect from comparing individuals across environments. So now, given a random parent values, The predicted children mean will be the parent mean + some X value, associated with the dominance effect, and variance coming from the enviro. effect.  how to calculate this "X" factor ? This what puzzles me. - what will be the mean and var. of the population?

oohh...I think I got your question. when you say "children mean" and "parent mean", you imply "mean of the children population" and "mean of the parent population" (assuming discrete generations). I wrote an answer. let me know if it is what you expected.

What is the expected phenotypic trait of an offspring given the phenotypic trait of its parents?

The expected trait of the offspring is equal to the mean of the parents traits.
For this I assumed we don't have more information about the genetic of the trait (typically, without knowing the directionality of dominance and number).

What is the expected phenotypic trait of the offspring population given the phenotypic trait of the parent population?

As a consequence of the above, the expected mean phenotypic trait of the offspring population is equal to the mean of the parent population (in absence of selection).

What is the expected variance in the offspring population?

No drift, no selection
In absence of drift and selection (and environmental changes), the variance of the offspring population equals the variance of the parent population.
Directional selection only
In presence of directional selection, please have a look at How does Natural Selection shape Genetic Variation? and ask follow-up question if needed.
Drift only
In presence of drift only, the heterozygosity is reduced by a factor $\frac{1}{2N}$, where $N$ is the population size. The loss of genetic variance is then dependent on (1) the frequency, (2) the dominance and (3) the effect size of alleles at loci causing this genetic variance. There is therefore no simple answer. The only generalization that can be done is that (1) the expected variance of the offspring population is lower than the variance of the parent population and (2) the smaller the population size, the higher is the expected loss of genetic variance
General Note
My feeling about quantitative genetics (but I am not really an expert in that field) is that it is often frustrating to see that generalization is often impossible and general solution to recursive equations often don't exist implying that predictions to more than one generation must be calculated through iteration.
I suspect that an expert in quantitative genetics would be able to say much more than me in this post.
The book Introduction to Quantitative Genetics (by Falconer and Mackay) is probably still the best book to increase your knowledge in quantitative genetics.


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Bacteria in our colons
During my education in Iran, until now, which I must say that I'm graduating high school this year and will go to college next year, our biology book has said that there's some bacteria in our colons and those little cells live there and use the celloluse of our food and because mostly they are anaerobic they produce methane or hydrogen and hydrogen sulfide and so on and sometimes they are the reason of the smell of our farts. Also they produce little amounts of B and K vitamins which is good for us and also, by using cellulose they make the rejection of feces better and easier. Anyway I wonder that where do these bacterias come from? I mean are they with us since birth? Then it means they come from our mother's body but, wait how our mothers have them? But maybe they are not there since birth and they come to colon from the food we eat during our life, but why don't they die in stomach. Even if we think that they go there by eating food, passing stomach easily, what if we don't eat the "food that can have those bacteria", then could it be possible to some humans don't have those bacteria? But they can't have a good release of feces.

Please read about your microbiota. The organisms both come from your mother and are acquired from the environment, including via food consumption.

It has been demonstrated that there are common patterns of microbiome composition evolution during life. In general, the diversity of microbiota composition of fecal samples is significantly higher in adults than in children, although interpersonal differences are higher in children than in adults. Much of the maturation of microbiota into an adult-like configuration happens during the three first years of life.
As the microbiome composition changes, so does the composition of bacterial proteins produced in the gut. In adult microbiomes, a high prevalence of enzymes involved in fermentation, methanogenesis and the metabolism of arginine, glutamate, aspartate and lysine have been found. In contrast, in infant microbiomes the dominant enzymes are involved in cysteine metabolism and fermentation pathways

evolution of the vertebrate gut microbiota :
Multi-cellular eukaryotes have existed for at least a quarter of Earth's history, or 1.2 billion years. Thus an already long history of interaction between multi-cellular lifeforms and microbial communities preceded, and likely shaped, the evolution of the vertebrates. The legacy of ancient associations between hosts and their epibiotic microbial communities is evident in the present day effects that the gut microbiota exerts on host biology, ranging from the structure and functions of the gut and the innate and adaptive immune systems, to host energy metabolism. Host responses to microbial colonization are evolutionary conserved among diverse vertebrates including zebra fish, mice and humans
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2664199/

"the human body is made up of about 10 times more microbial cells than human cells . Further, there may be millions more microbial genes than human genes in this human+microbiome system (which is often thought of as a human ‘superorganism’), and it is the ways in which these microbial genes interact with the human host that describe their ultimate role in our health. Scientists now believe that infants are sterile (meaning free of microbes) in the womb and receive their first inoculum of microbes from the mother during natural childbirth. This inoculum goes on to colonize the newborn and initiate a succession of events leading to the development of the child’s own microbiome. The newborn relies on this maternal vaginal microbial inoculum and the additional inoculum of microbes from mother’s breast milk for microbial colonization of all exposed surfaces in and on the infant’s body (e.g., oral, nasal/airways, gut, urogenital, skin).2 This is a dynamic process in which microbial abundances increase from effectively zero at birth to over six orders of magnitude (that’s more than a million times!) within just the first few weeks of life, with wide swings in the microbial membership of these communities until the microbiota largely stabilize in composition and numbers after approximately three years of life."
http://www.actionbioscience.org/genomics/the_human_microbiome.html

you can read about Gastrointestinal Tract Microflora here:
https://www.ncbi.nlm.nih.gov/books/NBK7670/
and normal flora :
https://www.ncbi.nlm.nih.gov/books/NBK7617/

It's true -  you are given a starter colony by your mother during a vaginal birth, covering your skin surface and intestinal tract.
The population of your gut is likely to change throughout life, depending on the environment you provide (food, exercise, antibiotics, etc).
It's also unlikely all bacteria are killed in your stomach when you eat, and only one cell is necessary to start a colony.
we've seen correlation of bacterial populations in the gut with certain disease states. I won't go beyond that since it's not my field.
try checking out an amazing paper my prof gave me - The Gut Brain Axis
have fun with your studies.

Which is a problem for caesarean births.

I wonder that where do these bacterias come from?

I'll answer this question since your post revolves around this question and the rest are follow-ups.
The infant gets its first bacteria (commensal or mutualist) from the mother's vagina as the amniotic membrane ruptures during parturition and gradually the diversity increases from its contact with other organisms and environment.

Nolte (1977) suggested that the body acquires its first microflora of the
  skin, nose, mouth and conjuctiva from the mother’s birth canal during normal
  vaginal delivery. The local environment is also important as a source from
  which neonates acquire bacteria (Mortimer et al., 1966; Nolte, 1977; Harvey,
  1979; Shinebaum, Cooke and Brayson, 1979). The colonisation of the skin
  surface starts at the umbilical stump (Mortimer et al., 1966)
The normal commensal flora of the oral cavity is more complex. The
  mouth is sterile at birth but is rapidly colonised by bacteria derived from
  attendants and the local environment. By the second day of life, aerobes and
  facultative species can be detected, but strict anaerobes do not appear until
  after the eruption of the deciduous teeth (Hurst, 1957; McCarthy, Snyder and
  Parker, 1965).

Reference: THE DEVELOPMENT OF THE BACTERIAL FLORA
IN NORMAL NEONATES
Also (updated)
A research published in 2013 has found that colostrum contain more than 700 species of bacteria! It being the only source of food for an infant also contributes to the infant's normal flora, though the composition of the bacteria colonising an infant's oral cavity is inconsistent with the composition of the colostrum.

The birth canal origin of the bacteria is not the whole story. It appear that most of the bacteria are acquired from the environment, which in naturally born infants does include the birth canal, but also included the mother's skin and dozens of other environmental contacts. The mature population of bacteria in the gut seems to be the same in both natural born and cesarean infants however the cesarean infants take significantly longer to develop this population indicating the birth canal is not the only factor.
It also appears that certain factors in breast milk encouraging the growth of the most desirable bacteria, and may have a stronger effect then form of birth.


text:
Cells scatter light in solution. Do they refract?
For part of an exam question, "why do cells grown in liquid cause turbidity?" I answered that cells refract light. The correct answer was that cells scatter light. Isn't the light scattering ultimately caused by lots of refraction events? Is the grader correct to take off full points?

Cells also reflect and absorb light, but this is really a physics question.


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How to identify genes required for biofilm formation
This is just a homework question:
Q: Let’s assume that there is an unknown set of E. coli genes that are required for biofilm formation. Describe a genetic experiment you could perform to try to identify those genes.
The one idea that I can think of is:

Compare strains that produce biofilm against strains that do not produce biofilm. Sequence genes, find differences, interrupt genes in biofilm producing strains and confirm biofilm ability is impaired.

Is there a better approach than this?

You could do different things: First you could, as you say yourself, sequence the complete genomes of the strains from a biofilm and compare these with some, which do not form biofilms. This may give you an idea which genes are involved when they are present in the "biofilm genome" but not in the other. The proof would be to disrupt these genes either by removing or mutating them or by gene silencing techniques and see what the outcome is.
It is also possible that only a mutation in a gene which exists in the sample and the control genome makes the difference. Then site-directed mutagenesis would help to identify the important genes.
The second possibility is that there is no real difference in the sample and the control genome and the difference for developing a biofilm "simply" lies in the difference of gene expression. Then the isolation of RNA (either total or mRNA only) would help to find involved genes. If the environment makes the difference for the development of a biofilm, then the biofilm and the control cells should have show no (or almost no) difference in their gene expression profiles when they are cultivated under identical conditions. You then need to test the gene expression for different environmental conditions (including those that favor the development of biofilms) to find genes which are involved.
This comparison of the different conditions will then deliver genes which are uniquely or more highly expressed in the biofilm group, a lot of genes which are comparable in expression to the control sample, and some genes which have a lower expression than the control. It is now worth looking at the higher (or unique) and lower expressed genes, find out their function and then start mutating them to check their function for the formation of a biofilm.
Additionally there is the chance that the gene(s) which is necessary for the biofilm formation is located on an extra-chromosomal plasmid. I would also check the plasmids present in the biofilm and the control sample, sequence them (if present) and see which genes they express. The function for the biofilm formation can easily be tested with a simple transformation.

Nice answer! :-)

In the second possibility, the difference isn't genes, but expression of genes, then there must be some environmental trigger, right? So if both strains are cultured under the same conditions, they should both produce or not produce biofilm, right? In that case, you would figure out which environmental conditions triggers the difference and compare RNA expression to see which gene is affected. Is this correct?

@clay Yes, you are right. If it is really the environment which makes the difference, then there should be no (or almost no) difference in the gene expression profiles. Then you need to test different environmental conditions. Good point, I will add this to the answer.

Addition to Chris' answer.
If you do not have a strain that doesn't produce biofilm then you would have to screen for the genes that are involved in biofilm production (this goes for any phenotype).
In earlier times people used to do this by random mutagenesis with mutagens such as EMS (Ethyl Methanesulphonate) or UV. Nowadays it is possible to build a library of targeted mutagens against each gene for e.g. Library of CRISPR-Cas nucleases.


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Why is synthesis of tetanospasmin advantageous to C. tetani?
The tetanospasmin is a neurotoxin synthesised by some strains of C. tetani. It is the factor causing tetanus, but what is its role for the bacteria itself?
I do not believe the main objective of C. tetani is to harm people. I believe that any organism that wastes resources for purposes not related to its reproduction or preservation is being wiped out by the evolution.
As tetanospasmin is encoded by a plasmid gene, it should be pretty easy ro get rid of and there are strains of C. tetani lacking the gene. As protein synthesis costs resourcess, I guess there is some advantage from the neurotoxin synthesis - otherwise it would be a searious handicap, leading to competitive exclusion of tetanospasmin positive strains.

It's an opportunistic and generalist bacterium, existing in soil, digestive systems it's probably everywhere. It's related to Clostridium botulinum which ialso makes one of the most powerful toxins know, and the two may have similar ecology and purpose. I know botulin can in bee honey enough for old honey to be dangerous to babies, which gives an idea of habitats. There's some info on tetani ecology on the web, i don't know precisely. The toxins it produces may be harmful to humans as a side effect of it's more common habitat where it needs to establish an ecosystem and defend itself.

This paper suggests that botulin, from a close cousin bacteria of tetani, has an unkown mode of evolution: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6357194/

@LifeInTheTrees Bee honey doesn't contain the toxin otherwise it would be harmful to adults as well. Bee honey contains spores of clostridium. Clostridium can take over the gastrointestinal flora of babies as long as they haven't set up sufficient beneficial germs. Then the clostridium germs can produce the toxin inside the babie's bodies.


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Topology of closed circular DNA

Why are covalently closed circular plasmid DNAs naturally found in an underwound state ?

Is it because this makes it easier for the DNA replication machinery to access and unwind DNA ? Or is it because underwound state is energetically more favourable than an overwound state ?

DNA is not always negatively supercoiled naturally. It is important to keep in mind that different regions of topologically constrained DNA can have different supercoiling values. For example, the action of unwinding DNA for transcription or replication introduces positive supercoils ahead of the polymerase and negative supercoils behind it.
Additionally, supercoiling is present in DNA normally. Supercoiling is the result of more or less twist in a DNA helix, which is most stable when it has ~10.5 bp/turn (for B-DNA). Supercoiling allows under- or overwound DNA to return to its most stable twist. Negative supercoiling is the result of underwound DNA. Underwound DNA is not thremodynamically favourable and will actually lower the melting point of DNA (the point where strand separation occurs). This can be compensated by introducing supercoils but it is also important for processes such transcription, which require ssDNA. Negative supercoils and ssDNA can be thought of as interchangeable. Negative supercoils are also used to package DNA around histones in eukaryotes and archaea. On the other hand, positive supercoiling is the result of overwound DNA (also not thermodynamically favourable) and will increase the melting point of DNA. Positive supercoiling is often found in thermophiles which live at higher temperatures and need to prevent their DNA from melting excessively. It has also been suggested that positive supercoiling can play a role in regulation of gene expression (by inhibiting promoter melting). Organisms like to keep an approximately constant amount of supercoiling in their DNA for the above reasons; this is called the superhelical density and is characteristic of different classes of organisms.


text:
Basic question about evolution
I want to understand a mechanism how at the first times when the life began on earth evolution helped the new species to arise through selection while the same organism also continued to live as not evolved. If natural selection favors survival of the fittest, than why after a living evolves to a new fittest form, the older form still exists while it should be counted as unfit for survival and extinct, I am not sure but bacteria may be an example perhaps and I am not asking about the situations where the unfit form has extinct but both organisms continued to live.

Related question.

Thanks for the answer and showing the related question, I specifically didn't want to give an example like apes to humans evolution but the evolution that happened during the times when the life has begun. Could it be that life has started in different locations and in one habitat mutations that were favorable to survive through selection were not necessarily the fittest in another habitat and the same species was already fit to survive and mutations that lead to new species were not the fittest in that location?

@Charles: And some people find they aren't good at baseball, so they invent a new "evolutionary" niche like basketball instead :-)

I want to understand a mechanism how at the first times when the life began on earth evolution helped the new species to arise through selection while the same organism also continued to live as not evolved.

Well, this beginning is not grammatically correct so it is hard to make much comments on it.

If natural selection favors survival of the fittest, [..]

Selection is the process by which fitter individuals increase in frequency in the population. survival of the fittest is a layman phrasing of the process that is natural selection. Natural selection therefore does not favour survival of the fittest, but it is represented by the phrasing survival of the fittest.

[..] th[e]n why after a living evolves to a new fittest form, the older form still exists

It does not. The older form is called an ancestor. The ancestor does not exist today. If there was a speciation events, then the ancestral lineage would have giving rise to more than a single lineage today. Some of these modern lineages may 'look more' like the ancestors than others but it does not mean that any ancestor still exist.
For example, the chimpanzee is not an ancestor of humans. Chimpanzees (and bonobos) and humans share a common ancestor that is about 8 millions old. To consider another example, humans and an oak tree share a common ancestor. This ancestor was neither a human, nor an oak tree. This ancestor lived about 1.6 billions years ago.
If you want to know why I am using term 'lineage' rather than 'species', you can have a look at this post
See below linked Biology.SE posts for more information

the older form still exists while it should be counted as unfit for survival and extinct

Don't forget that different lineages may (and will be at equilibrium due to the competitive exclusion principle) be adapted to different ecological niche. As such, one lineage will not necessarily outcompete the other one.
Source of information on StackExchange
There is a lot you can learn on the subject. You should start with an introduction to phylogenetics such as the one provided in the post

Why do the ancestors of birds still exist?

You can also have a look at

Extinction of species

You should also have a look at

How does Darwinian Evolution work?

Reading your post, it sounds like you may have too a pan-selectionist view of evolution. Related to this point, you might want to read

Why don't mammals have more than 4 limbs?

Source of information external to StackExchange
Understanding Evolution is a good introductory course to evolutionary biology.

Response to comment

Thank you for the very descriptive answer, as you have figured out easily I am very new to evolution. I will read the references you have provided carefully

You're welcome!

is after evolution of a specie through natural selection,

This show again that you have too a pan-selectionist view of evolution.

unfit ancestor extinct and this is explained through phylogeny ?

From wikipedia

Phylogenetics is the study of the evolutionary history and relationships among individuals or groups of organisms (e.g. species, or populations).

A phylogeny is a tree of life of a given taxon. For example here is a phylogeny of placental mammals.
To be able to fully answer your own question, you need to understand the relationships between lineages and therefore you need to have some understanding in phylogenetics.

Thank you for the very descriptive answer, as you have figured out easily I am very new to evolution. I will read the references you have provided carefully, but I think the main answer to my question is after evolution of a specie through natural selection, unfit ancestor extinct and this is explained through phylogeny ?

It's also important to remember that "survival of the fittest" is essentially a circular definition, because organisms prove that they are "fittest" by surviving to reproduce.  Also "fittest" is variable: an orca may be a supremely fit top predator in the ocean, but it's quite unfit to survive in the Mojave Desert.

@bbbandroid See edit to address your comment.

@Remi.b I have just started by watching the video you have mentioned in your post "If you want to know why I am using term 'lineage' rather than 'species', you can have a look at this post" and there is a video in the end of that post named "What Makes a Species a Species?" and i have watched in ending says biological species concept is not an absolute rule

Yep.... any question about it?! You should maybe read the post if this is unclear to you.

Ok i got that point, so i can reshape my initial question, checking below sites link and link when a species diverge into 2 new species, does it mean that the ancestor that diverges extinct ?

In general, you can ask short follow-up questions but not too many of them and not entirely new questions. Your question here is fine. The ancestor became both descendent lineages. So it does not exist anymore as it became something else. The standard mistake would be to think that one of the descendent lineage is the ancestor.


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A question on exclusion of study participants for an Exome genotyping array
I'm reading a paper that used whole exome sequencing on an African
 American and European populations to discover novel low frequency and
rare variants associated with lipid levels & the risk of coronary
heart disease.
They used the Illumina Human exome genotyping array that was designed
based on coding variants discovered from sequencing the exomes of
12,000 individuals. Therefore, they selected their participants from a
study who were not among the 12,000 individuals utilized to design the
array .
My question is: Why would they select individuals who were not among
the 12,000 used to design the array? (I'm thinking because maybe it
could produce false positives associations? bias?)

Can you add a citation for this paper?

@Chris the source is from the paper Association of low-frequency and rare coding-sequence variants with blood lipids and coronary heart disease in 56,000 whites and blacks.http://www.ncbi.nlm.nih.gov/pubmed/?term=Associatoin+of+low+frequency+and+rare+coding+sequence+variants+with+blood+lipds+and+coronary+heart+disease

I'll have a look tomorrow, and I have added the link to your post.

When you design the arrays, you need to have probes on the surface complementary to the sequence you want to detect. Depending on what you want to detect, you need to design these probes with known sequence on a known position.
If you want to detect single nucleotide polymorphisms (SNP), then you need a library of known SNPs on your ChIP, which are basically the position of the SNP and the surrounding sequence.
SNPs can be roughly grouped into two subgroups, common (I call them like this) and rare (frequency in the population lower than 0.1%). The problem with this ChIP based method is that it can only detect SNPs which are already known (or at least are located close to known SNPs with which they in linkage disequilibrium). So if you want to detect rare SNPs in your population sample, you need a big group of people from which you take the SNPs into you ChIP design. If you look at the numbers, one rare variant will only be present in about 12 of the 12.000 persons used in the design.
Your study group is then different from the group of people, which where used to make the ChIP design. Here you take people with a certain background (high lipid levels for example) and then compare, if people which had coronary heart problems have SNPs in common which might be connected to this disease. This can help to identify a mutation in a protein which is the risk factor.

authors: Hášíl Páůďýál