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Patent application title: METHODS AND KITS FOR REDUCING NON-SPECIFIC NUCLEIC ACID AMPLIFICATION
John Richard Nelson (Clifton Park, NY, US)
Gregory Andrew Grossmann (Halfmoon, NY, US)
Gregory Andrew Grossmann (Halfmoon, NY, US)
Robert Scott Duthie (Schenectady, NY, US)
Robert Scott Duthie (Schenectady, NY, US)
Sonali Jagdish Shah (Avon, CT, US)
Ryan Charles Heller (Guilderland, NY, US)
IPC8 Class: AC12P1934FI
USPC Class:
435 912
Class name: Nucleotide polynucleotide (e.g., nucleic acid, oligonucleotide, etc.) acellular exponential or geometric amplification (e.g., pcr, etc.)
Publication date: 2013-08-15
Patent application number: 20130210078
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Methods and kits for efficient amplification of nucleic acids are
provided. The disclosure generally relates to methods and kits for
nucleic acid amplification of target nucleic acids of interest. The
methods described herein promote the synthesis of the target nucleic acid
(i.e., template nucleic acid) by reducing the production of undesirable
primer-dimer structures and chimeric nucleic acid products during the
amplification process by using novel modified primers.Claims:
1. A method for amplifying a nucleic acid comprising: a) providing a
nucleic acid template; b) contacting the nucleic acid template with a
reaction solution comprising a DNA polymerase, deoxyribonucleoside
triphosphates, and a primer with a 3' end and a 5' end, wherein the
primer comprises: (i) a nucleotide analogue that increases the melting
temperature (Tm) of the primer and (ii) a nucleotide analogue that
prevents primer-dimer formation; and c) amplifying the nucleic acid
2. The method of claim 1, wherein amplifying the nucleic acid template is performed under isothermal conditions.
3. The method of claim 1, wherein amplifying the nucleic acid template is performed under high stringency conditions.
4. The method of claim 1, wherein the DNA polymerase is phi29 DNA polymerase.
5. The method of claim 1, wherein the primer is a random primer.
6. The method of claim 1, wherein the primer is thioated.
7. The method of claim 1, wherein one nucleotide analogue in the primer is a locked nucleic acid (LNA) that precedes a nucleotide base, wherein incorporation of the LNA nucleotide base in the primer increases the Tm of the primer.
8. The method of claim 7, wherein the LNA nucleotide base is not located on the 3' end of the primer.
9. The method of claim 1, wherein one nucleotide analogue in the primer is 2-amino-deoxyadenosine (2-amino-dA), wherein incorporation of 2-amino-dA increases the Tm of the primer.
10. The method of claim 9, wherein the primer further comprises the nucleotide analogue 2-thio-deoxythymidine (2-thio-dT), wherein incorporation of the nucleotide analogue 2-amino-dA and the nucleotide analogue 2-thio-dT in the primer prevents primer-dimer formation.
11. The method of claim 10, wherein the primer further comprises an LNA that precedes a nucleotide base.
12. The method of claim 1, wherein the primer is a hexamer.
13. The method of claim 12, wherein the hexamer has a general structure of (atN)(atN)(atN)(atN)(atN)*N, wherein (atN) is the 5' end and *N is the 3' end nucleotide of the hexamer, and wherein "N" represents a deoxyadenosine (dA), deoxycytidine (dC), deoxyguanosine (dG), or deoxythymidine (dT), "+" indicates an LNA that precedes a nucleotide base, (atN) represents a random mixture of 2-amino-dA, dC, dG, and 2-thio-dT, and "*" represents a phosphorothioate linkage.
14. The method of claim 1, wherein amplifying the nucleic acid template comprises a rolling circle amplification (RCA) or a multiple displacement amplification (MDA).
15. The method of claim 1, wherein the method permits amplification of a trace amount of the nucleic acid.
16. A kit for amplifying a nucleic acid comprising: (a) a DNA polymerase; (b) deoxyribonucleoside triphosphates; and (c) a primer with a 3' end and a 5' end that comprises: (i) a nucleotide analogue that increases the melting temperature (Tm) of the primer and (ii) a nucleotide analogue that prevents primer-dimer formation.
17. The kit of claim 16, wherein the DNA polymerase is phi29 DNA polymerase.
18. The kit of claim 16, wherein the primer is a hexamer.
19. The kit of claim 18, wherein the hexamer has the general structure of (atN)(atN)(atN)(atN)(atN)*N, wherein the 5' end of the hexamer is an (atN) and the 3' end of the hexamer is an *N, "N" represents dA, dC, dG, or dT, wherein (atN) represents a random mixture of 2-amino-dA, dC, dG, and 2-thio-dT, and "*" represents a phosphorothioate linkage.
20. The kit of claim 18, wherein the primer comprises the nucleotide analogue 2-amino-dA and the nucleotide analogue 2-thio-dT.
[0001] This application claims the benefit of U.S. Provisional Application No. 61/599,119, filed on Feb. 15, 2012, which is herein incorporated by reference in its entirety.
[0002] The disclosure generally relates to methods and kits for the amplification of target nucleic acids of interest. The methods and compositions described herein promote the amplification of the desired target nucleic acid through the use of novel primers, thereby reducing the production of undesirable amplification products (e.g., primer-dimers and chimeric nucleic acids).
[0003] A variety of techniques are currently available for efficient amplification of nucleic acids even from a few molecules of a starting nucleic acid template. These include polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), multiple displacement amplification (MDA), and rolling circle amplification (RCA). Many of these techniques involve an exponential amplification of the starting nucleic acid template and are able to generate a large number of amplified products quickly. Kits for the amplification of a target nucleic acid are commercially available (e.g., GenomiPhi® (General Electric, Inc.) and RepliG® (Qiagen, Inc.), but improvements to these methods would be advantageous.
[0004] Nucleic acid amplification techniques are often employed in nucleic acid-based assays used for analyte detection, sensing, forensic and diagnostic applications, genome sequencing, whole-genome amplification, and the like. Such applications often require amplification techniques having high specificity, sensitivity, accuracy, and robustness. Most of the currently available techniques for nucleic acid amplification, however, suffer from high background signals, which are generated by non-specific amplification reactions yielding unwanted amplification products. These non-specific amplification reactions hinder effective utilization of many of these techniques in critical nucleic acid-based assays. For example, use of a traditional amplification reaction may produce a false-positive result, thereby leading to an incorrect diagnosis. Such non-specific, background amplification reactions become even more problematic when only trace amounts of the target nucleic acid to be amplified are available (e.g., whole-genome amplification from a single DNA molecule).
[0005] Non-specific, background amplification reactions may be due to, for example, amplification of a contaminating nucleic acid sequence in the sample, primer-dimer formation, or production of chimeric nucleic acids (e.g., resulting from self-hybridization of the desired nucleic acid products). A frequent source of non-specific amplification in a nucleic acid amplification reaction results from various undesirable primer interactions. A primer may hybridize to regions of a nucleic acid in either a target nucleic acid or in a contaminating nucleic acid that share some homology with a portion of the target nucleic acid. If the 3' end of a primer has sufficient homology to an untargeted region, this region may be amplified.
[0006] Non-specific amplification may also result from unintended nucleic acid template-independent primer-primer interactions. Primers may form primer-dimer structures by intra- or inter-strand primer annealing (e.g., intra-molecular or inter-molecular hybridizations), resulting in amplification of unwanted nucleic acids. The resultant spurious primer extension products may be further amplified and may at times predominate, inhibit, or mask the desired amplification of the targeted sequence. Moreover, the amplification products may self-hybridize, allowing the nucleic acid polymerase to generate hybrid products or chimeric products during the amplification reaction.
[0007] For priming DNA synthesis, current MDA formulations often utilize random hexamers with the sequence 5'-NNNNN*N, where "N" represents a deoxyadeno sine (dA), deoxycytidine (dC), deoxyguano sine (dG), or deoxythymidine (dT) and "*" represents a phosphorothioate linkage.
[0008] Constrained-randomized hexamer primers that cannot cross-hybridize via intra- or inter-molecular hybridization (e.g., R6, where R=A/G) have been used for suppressing primer-dimer structure formation during nucleic acid amplification. These constrained-randomized primers, however, impart considerable bias in nucleic acid amplification reaction. Such primers are also of limited use for sequence-non-specific or sequence-non-biased nucleic acid amplification reactions (e.g., whole genome or unknown nucleic acid sequence amplification).
[0009] For priming DNA synthesis, MDA formulations frequently utilize random hexamers with the sequence 5'-NNNNN*N, where "N" represents a deoxyadenosine (dA), deoxycytidine (dC), deoxyguanosine (dG), or deoxythymidine (dT) and "*" represents a phosphorothioate linkage. One solution to minimizing competing non-target nucleic acid (i.e., template DNA) amplification is to modify the oligonucleotide primers in such a way as to inhibit their ability to anneal with one another.
[0010] Previous research to overcome the issues associated with nucleic acid amplification using random hexamer primers described above includes those methods and kits disclosed in U.S. Pat. No. 7,993,839 (issued Aug. 9, 2011). The techniques described in this patent include but are not limited to the use of primers that are hexamers of the general structure 5'-+W+WNNN*S-3', where "+" precedes a locked nucleic acid base (i.e., "an LNA base"; for example, +A=an adenosine LNA molecule), "W" represents a mixture of only dA and dT, and "S" represents a mixture of only dC and dG. The "*" represents a phosphorothioate linkage between the two nucleotides. Since "W" bases are unable to stably pair with "S" bases, the formation of the oligonucleotide duplex is inhibited, which leads to decreased amplification of non-template nucleic acids. These methods and kits may be referred to as "SD GenomiPhi."
[0011] One improvement to the speed and sensitivity of MDA when amplifying trace nucleic acid samples is the incorporation of LNAs into the oligonucleotide primers. LNAs are a class of conformationally restricted nucleotide analogues that serve to increase the speed, efficiency, and stability of base pairing, thereby promoting the hybridization of the modified oligonucleotides to their target sequences in the nucleic acid of interest. For each LNA monomer incorporated into an oligonucleotide primer, the duplex melting temperature (Tm) is increased by 2-8° C. The increase in Tm of the duplex allows the MDA reaction to be performed under more stringent conditions, such as at a higher temperature or with a lower concentration of salt (e.g., 15 mM KCl as opposed to the 75 mM KCl used in traditional amplification reactions with unmodified primers). While the kinetics of amplification using MDA are dramatically increased by incorporation of LNAs into the random primers, one drawback is that the hexamers also anneal to each other more efficiently, leading to amplification of unwanted nucleic acids (e.g., primer-dimers).
[0012] The problems associated with the undesirable amplification of non-target nucleic acids have also been approached from the standpoint of removing contaminating nucleic acids from reagents and reagent solutions used in nucleic acid amplification methods. Kits and methods for generating nucleic acid contaminant-free reagents and reagent solutions for use in nucleic acid amplification are disclosed in U.S. Patent Application Publication No. 2009/0155859. Such methods include processing of polymerase solutions, nucleotide solutions, and primer solutions to render contaminating nucleic acids inert. The methods employ the proofreading activity of the polymerase and/or exonucleases to decontaminate the reagents and reagent solutions. The methods described in U.S. Patent Application Publication No. 2009/0155859 may at times be referred to as "Clean GenomiPhi" or "Clean GPhi."
[0013] Despite these advancements, there remains a need for developing more efficient nucleic acid amplification methods that have lower bias in terms of sequence coverage and produce lower levels of non-specific, background amplification. Development of primers for nucleic acid amplification without sequence bias that also reduce primer-primer interaction and minimize the production of chimeric nucleic acids (e.g., unwanted nucleic acid products resulting from the annealing of the hexamer primers to the target nucleic acid amplification products) is needed in the art.
[0014] The present application provides methods and compositions for amplification of target nucleic acids. Without intending to be limited to a particular mechanism of action, the methods disclosed herein are believed to more efficiently amplify the desired target nucleic acid (e.g., "DNA template" or "nucleic acid template") through the use of modified primers designed to minimize or prevent the production of unwanted primer-dimers and chimeric products observed with other nucleic acid amplification methods and kits. The methods described herein utilize a novel primer design method to avoid the production of spurious nucleic acid amplification products. For example, in one embodiment random hexamers comprising 2-amino-deoxyadenosine (2-amino-dA) and 2-thio-deoxythymidine (2-thio-dT) are produced and used in a nucleic acid amplification reaction (e.g., MDA). The methods and kits described herein may be referred to as "AT GenomiPhi."
[0015] In one aspect of the invention, these modified hexamers are of the general formula: +N+N(atN)(atN)(atN)*N, wherein "+" precedes an LNA base, as described above, and (atN) represents a random mixture of 2-amino-dA, dC, dG, and 2-thio-dT. The hexamers used in another aspect of this disclosure comprise: (atN)(atN)(atN)(atN)(atN)*N, wherein the notations are consistent between these two hexamer designs. As described in greater detail below, the use of these hexamers in nucleic acid amplification techniques addresses the minimizes or eliminates the problems associated with the production of primer-dimer formation and chimeric nucleic acids observed in traditional methods by inhibiting the ability of the random hexamers to anneal with one another, by increasing the melting Tm of the primers, improving the binding efficiency of the hexamer to the target nucleic acid via the addition of LNAs and 2-amino-dA to the primers, and preventing annealing of the target DNA to itself through the incorporation of 2-thio-dT into the random hexamers. Moreover, these primer modifications, which increase their binding strength to the target nucleic acid, permit the utilization of more stringent hybridization buffers that further minimize the likelihood of the production of primer-dimers and chimeric nucleic acid products.
[0016] These and other features, aspects, and advantages of the chemically modified porous membranes will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:
[0017] FIG. 1 depicts the base pairing combination of: 1) 2-amino-deoxyadenosine (2-amino-dA) and 2-thio-deoxythymidine (2-thio-dT) and 2) 2-amino-dA and unmodified T. While the 2-amino-dA/2-thio-dT pairing is very unstable, the 2-amino-dA/T pairing is actually more stable than a standard A/T pairing.
[0018] FIG. 2 depicts the improvements to the standard GenomiPhi® formulation were the addition of the enzymatic cleaning reaction, adjustment of the potassium chloride concentration, and the composition of the random hexamer oligonucleotide. The abbreviations used in this figure are as follows: N=random base; *=phosphorothioate linkage; +, precedes LNA base; W=only the bases A or T; S=only the bases C or G.
[0019] FIG. 3A provides the results of amplification reactions of dilution series of Bacillus subtilis chromosomal DNA or a no nucleic acid control (NTC) that were performed using the standard GenomiPhi® kit or utilizing the "cleaned" GenomiPhi, SD GenomiPhi, or AT GenomiPhi formulations. The threshold time for DNA amplification signal to increase over background for each reaction was plotted out versus the amount of DNA added as input template into the reaction. FIG. 3B provides the results of amplification reactions of Bacillus subtilis chromosomal DNA that were performed using the standard GenomiPhi® kit using the hexamer sequence NNNN*N*N and 75 mM KCl, the "cleaned" GenomiPhi SD, or the GenomiPhi AT formulations, as described herein above. A no template control was also performed (NTC). Details are set forth in Example 1.
[0020] FIG. 4A provides the results of whole Bacillus subtilis genome amplification reactions from the indicated amounts of chromosomal DNA. A no template control was also performed (NTC). Details are set forth in Example 3 below. The legend for this figure is as follows: 1) solid line open square: NNNN*N*N (where * indicates a phosphorothioate linkage), 2) solid line solid triangle: (atN)(atN)(atN)(atN)*(atN)*N (where a mixture of random bases in which: 2-amino-A is substituted for A, 2-thio-T is substituted for T), 3) solid line open triangle: +N+N+N+N+N*, 4) dotted line open diamond +N+N(atN)(atN)(atN)*N, 5) solid line open circle: WWN+N+N*S, and 6) solid line solid circle +W+WNNN*S. FIG. 4B summarizes the data in bar graph format.
[0021] FIG. 5 provides the coverage level determined for each 100 base pair window of the genome and GC content of windows or the amplification reactions in FIG. 2. The coverage level was plotted against the fractional coverage of windows that had that GC content. Details of this example are set forth below in Example 3.
[0022] FIG. 6A provides the results from amplification reactions of E. coli chromosomal DNA performed using the improved AT GenomiPhi method. A no template control was also performed (NTC). Additional details are set forth in Example 4. FIG. 6B summarizes the analysis of performed by PCR. FIG. 6C provides a histogram of coverage levels, the percent of reads mapped, and genome coverage levels. Experimental details are disclosed in Example 4.
[0023] Nucleic acid-based assays involving single molecule DNA amplification or whole-genome amplification demand highly efficient nucleic acid amplification methods that have high yield, high fidelity and have little bias in terms of sequence coverage. A variety of methods that are currently available for use include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), and rolling circle amplification (RCA). Isothermal nucleic acid amplification reactions such as rolling circle amplification (RCA), or multiple displacement amplification (MDA) employing random primers are more adaptable than temperature-dependent nucleic acid amplification reaction (e.g., PCR) for such applications. However, these methods often yield a dominant background signal due to undesired non-specific nucleic acid amplification reactions, especially when the concentration of target nucleic acid template is low (e.g., below 1 ng).
[0024] The methods and kits described herein are intended to efficiently amplify target nucleic acids with the additional advantage of reducing non-specific amplification of non-target nucleic acids (e.g., primer-dimers, chimeric nucleic acid products, etc.) that are observed with other methods of nucleic acid amplification. Without intending to be limited to a particular mechanism of action, the disclosed methods accomplish these goals by the incorporation of nucleotide analogue bases that increase Tm (e.g., the inclusion of each 2-amino-dA base in the hexamer increases the Tm by approximately 3° C.) or prevent undesirable primer-dimer formation. In certain embodiments, 2-amino-deoxyadenosine (2-amino-dA), 2-thio-deoxythymidine (2-thio-dT), or other nucleotide analogues of interest are incorporated into the random hexamers used for amplification of the target nucleic acid. As shown in FIG. 1, nucleotide analogues bases do not stably pair with each other due to steric hindrance (e.g., 2-amino-dA forms only one hydrogen bond with 2-thio-dT as opposed to the three hydrogen bonds observed with the unmodified dA and dT), formation of unwanted nucleic acid duplexes is significantly reduced, thereby leading to the overall inhibition of non-target nucleic acid amplification. In fact, the inclusion of these nucleotide analogue bases actually improves the ability of the primers to hybridize to the target nucleic acid because 2-amino-dA forms three hydrogen bonds with unmodified deoxythymidine (dT) and, similarly, 2-thio-dT forms a normal stable pair with its unmodified partner (i.e., deoxyadenosine (dA)).
[0025] When combined with one or more LNA nucleotides, the methods set forth herein produce even more robust results. For each LNA nucleotide incorporated into an oligonucleotide primer, increases the Tm by 2-8° C. The use of the modified nucleotides analogues bases and LNA nucleotides also permits the use of more stringent hybridization buffers, thereby further decreasing the production of unwanted non-target nucleic acid amplification. Moreover, higher temperatures and lower salt concentrations can also be used in the nucleic acid amplification reaction to facilitate the amplification of specific target nucleic acids of interest.
[0026] To more clearly and concisely describe and point out the subject matter of the claimed invention, the following definitions are provided for specific terms, which are used in the following description and the appended claims. Throughout the specification, exemplification of specific terms should be considered as non-limiting examples.
[0027] The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Approximating language, as used herein throughout the specification and claims, may be applied to modify any quantitative representation that could permissibly vary without resulting in a change in the basic function to which it is related. Accordingly, a value modified by a term such as "about" is not to be limited to the precise value specified. In some instances, the approximating language may correspond to the precision of an instrument for measuring the value. Similarly, "free" may be used in combination with a term, and may include an insubstantial number, or trace amounts while still being considered free of the modified term. Where necessary, ranges have been supplied, and those ranges are inclusive of all sub-ranges there between.
[0028] As used herein, the term "nucleoside" refers to a glycosylamine compound wherein a nucleic acid base (e.g., nucleobase) is linked to a sugar moiety. The nucleic acid base may be a natural nucleobase or a modified or synthetic nucleobase. The nucleic acid base includes, but is not limited to, a purine base (e.g., adenine or guanine), a pyrimidine (e.g., cytosine, uracil, or thymine), or a deazapurine base. The nucleic acid base may be linked to the 1' position, or at an equivalent position of a pentose (e.g., a ribose or a deoxyribose) sugar moiety. The sugar moiety includes, but is not limited to, a natural sugar, a sugar substitute (e.g., a carbocyclic or an acyclic moiety), a substituted sugar, or a modified sugar (e.g., bicyclic furanose unit as in LNA nucleotide). The nucleoside may contain a 2'-hydroxyl, 2'-deoxy, or 2',3'-dideoxy forms of the sugar moiety.
[0029] As used herein the terms "nucleotide" or "nucleotide base" refer to a nucleoside phosphate. It includes, but is not limited to, a natural nucleotide, a synthetic nucleotide, a modified nucleotide, or a surrogate replacement moiety (e.g., inosine). The nucleoside phosphate may be a nucleoside monophosphate, a nucleoside diphosphate or a nucleoside triphosphate. The sugar moiety in the nucleoside phosphate may be a pentose sugar, such as ribose, and the phosphate esterification site may correspond to the hydroxyl group attached to the C-5 position of the pentose sugar of the nucleoside. A nucleotide may be, but is not limited to, a deoxyribonucleoside triphosphate (dNTP) or a ribonucleoside triphosphate (NTP). The nucleotides may be represented using alphabetical letters (letter designation), as described in Table 1. For example, A denotes adenosine (i.e., a nucleotide containing the nucleobase, adenine), C denotes cytosine, G denotes guanosine, and T denotes thymidine. W denotes either A or T/U, and S denotes either G or C. N represents a random nucleotide (i.e., N may be any of A, C, G, or T/U). A plus (+) sign preceding a letter designation denotes that the nucleotide designated by the letter is a LNA nucleotide. For example, +A represents an adenosine LNA nucleotide, and +N represents a locked random nucleotide (a random LNA nucleotide). A star (*) sign preceding a letter designation denotes that the nucleotide designated by the letter is a phosphorothioate modified nucleotide. For example, *N represents a phosphorothioate modified random nucleotide.
[0030] As used herein, the term "nucleotide analogue" refers to modified compounds that are structurally similar to naturally occurring nucleotides. The nucleotide analogue may have an altered phosphorothioate backbone, sugar moiety, nucleobase, or combinations thereof. Generally, nucleotide analogues with altered nucleobases confer, among other things, different base pairing and base stacking proprieties. Nucleotide analogues having altered phosphate-sugar backbone (e.g., PNA, LNA, etc.) often modify, among other things, the chain properties such as secondary structure formation. At times in the instant application, the terms "nucleotide analogue," "nucleotide analogue base," "modified nucleotide base," or "modified base" may be used interchangeably.
[0031] Any nucleotide analogue that minimizes or prevents the amplification of a non-target nucleic acid may be used in the practice of the invention. In addition to those nucleotide analogues described above, a variety of nucleotide analogues with the desired abilities are well known to those of skill in the art. See, for example, U.S. Pat. No. 5,912,340; International Publication No. WO 2010/021702; and Hoshika et al. (2010) Angew. Chem. Int. Ed. 49:5554-5557, all of which are herein incorporated by reference in their entirety. Such nucleotide analogues include but are not limited to "self-avoiding molecular recognition systems" (SAMRS). SAMRS as described by Hoshika et al. are based on 2-thiothymine, 2-aminopurine, hypoxanthine, and N4-ethylcytosine as T*, A*, G*, and C*. When these are introduced individually into a reference DNA duplex, the corresponding SAMRS:standard pairs contributed to duplex stability to the same extent as an A:T pair. The SAMRS:SAMRS pair contributed less to the stability of the reference duplex than the corresponding SAMRS:standard pair. Hoshika et al. (2010) Angew. Chem. Int. Ed. 49:5554-5557.
TABLE-US-00001 TABLE 1 Letter designations of various nucleotides Symbol Letter Nucleotide G G A A T T C C U U R G or A Y T/U or C M A or C K G or T/U S G or C W A or T/U H A or C or T/U B G or T/U or C V G or C or A D G or A or T/U N G or A or T/U or C
[0032] As used herein, the term "LNA (Locked Nucleic Acid) nucleotide" refers to a nucleotide analogue, wherein the sugar moiety of the nucleotide contains a bicyclic furanose unit locked in a ribonucleic acid (RNA)-mimicking sugar conformation. The structural change from a deoxyribonucleotide (or a ribonucleotide) to the LNA nucleotide is limited from a chemical perspective, namely the introduction of an additional linkage between carbon atoms at 2' position and 4' position (e.g., 2'-C, 4'-C-oxymethylene linkage; see, for example, Singh, S. K., et. al., Chem. Comm., 4, 455-456, 1998, or Koshkin, A. A., et. al., Tetrahedron, 54, 3607-3630, 1998.)). The 2' and 4' position of the furanose unit in the LNA nucleotide may be linked by an O-methylene (e.g., oxy-LNA: 2'-O, 4'-C-methylene-β-D-ribofuranosyl nucleotide), an S-methylene (thio-LNA), or a NH-methylene moiety (amino-LNA), and the like. Such linkages restrict the conformational freedom of the furanose ring. LNA oligonucleotides display enhanced hybridization affinity toward complementary single-stranded RNA, and complementary single- or double-stranded DNA. The LNA oligonucleotides may induce A-type (RNA-like) duplex conformations.
[0033] As used herein, the term "oligonucleotide" refers to oligomers of nucleotides or derivatives thereof. The term "nucleic acid" as used herein refers to polymers of nucleotides or derivatives thereof. A "target nucleic acid or "nucleic acid template" means the particular sequence that is intended and desired to be amplified. The term "sequence" as used herein refers to a nucleotide sequence of an oligonucleotide or a nucleic acid. Throughout the specification, whenever an oligonucleotide/nucleic acid is represented by a sequence of letters, the nucleotides are in 5'→3' order from left to right. For example, an oligonucleotide represented by a letter sequence (W)x(N)y(S)z, wherein x=2, y=3 and z=1, represents an oligonucleotide sequence WWNNNS, wherein W is the 5' terminal nucleotide and S is the 3' terminal nucleotide. The oligonucleotides/nucleic acids may be a DNA, an RNA, or their analogues (e.g., a phosphorothioate analogue). The oligonucleotides or nucleic acids may also include modified bases, and/or backbones (e.g., a modified phosphate linkage or modified sugar moiety). Non-limiting examples of synthetic backbones that confer stability and/or other advantages to the nucleic acids may include phosphorothioate linkages, peptide nucleic acid, locked nucleic acid, xylose nucleic acid, or analogues thereof.
[0034] As used herein, the term "terminal nucleotide" refers to a nucleotide that is located at a terminal position of an oligonucleotide sequence. The terminal nucleotide that is located at a 3' terminal position is referred as a 3' terminal nucleotide, and the terminal nucleotide that is located at a 5' terminal position is referred as a 5' terminal nucleotide. The nucleotide adjacent to the terminal nucleotide refers to a nucleotide that is located at a penultimate position from the terminal position.
[0035] As used herein, the term "primer", or "primer sequence" refers to a short linear oligonucleotide that hybridizes to a target nucleic acid sequence (e.g., a DNA template to be amplified) to prime a nucleic acid synthesis reaction. The primer may be an RNA oligonucleotide, a DNA oligonucleotide, or a chimeric sequence. The primer may contain natural, synthetic, or nucleotide analogues (e.g., those that increase Tm). Both the upper and lower limits of the length of the primer are empirically determined. The lower limit on primer length is the minimum length that is required to form a stable duplex upon hybridization with the target nucleic acid under nucleic acid amplification reaction conditions. Very short primers (usually less than 3 nucleotides long) do not form thermodynamically stable duplexes with target nucleic acid under such hybridization conditions. The upper limit is often determined by the possibility of having a duplex formation in a region other than the pre-determined nucleic acid sequence in the target nucleic acid. Generally, suitable primer lengths are in the range of about 3 nucleotides long to about 40 nucleotides long. The "oligonucleotide primers" used in the methods of amplification of a target nucleic acid described herein will be of a length appropriate for a particular set of experimental conditions. The determination of primer length is well within the routine capabilities of those of skill in the art. In certain embodiments described in the instant application the oligonucleotide primer is a hexamer, more particularly a hexamer comprising at least one nucleotide analogue that increases Tm and/or prevents primer-dimer formation. The primers of the present disclosure may further include an LNA nucleotide base.
[0036] As used herein, the term "random primer" or "complete random primer" refers to a mixture of primer sequences, generated by randomizing a nucleotide at any given location in an oligonucleotide sequence in such a way that the given location may consist of any of the possible nucleotides or their analogues (e.g., complete randomization). Thus the random primer is a random mixture of oligonucleotide sequences, consisting of every possible combination of nucleotides within the sequence. For example, a hexamer random primer may be represented by a sequence NNNNNN or (N)6. A hexamer random DNA primer consists of every possible hexamer combinations of 4 DNA nucleotides, A, C, G and T, resulting in a random mixture comprising 46 (4,096) unique hexamer DNA oligonucleotide sequences. Random primers may be effectively used to prime a nucleic acid synthesis reaction when the target nucleic acid's sequence is unknown.
[0037] As described herein, "partially constrained primer" refers to a mixture of primer sequences, generated by completely randomizing some of the nucleotides of an oligonucleotide sequence (i.e., the nucleotide may be any of A, T/U, C, G, or their analogues) while restricting the complete randomization of some other nucleotides (i.e., the randomization of nucleotides at certain locations are to a lesser extent than the possible combinations A, T/U, C, G, or their analogues). For example, a partially constrained DNA hexamer primer represented by WNNNNN, represents a mixture of primer sequences wherein the 5' terminal nucleotide of all the sequences in the mixture is either A or T. Here, the 5' terminal nucleotide is constrained to two possible combinations (A or T) in contrast to the maximum four possible combinations (A, T, G or C) of a completely random DNA primer (NNNNNN). Suitable primer lengths of a partially constrained primer may be in the range of about 3 nucleotides long to about 15 nucleotides long.
[0038] As described herein, the term "partially constrained primer having a terminal mismatch primer-dimer structure" refers to a partially constrained primer sequence, wherein when two individual primer sequences in the partially constrained primer hybridize each other inter-molecularly, with an internal homology of three or more nucleotides, to form a primer-dimer structure having no recessed ends, or a primer-dimer structure having a single-nucleotide base 3' recessed ends, or a primer-dimer structure having a two-nucleotide base 3' recessed ends, there exists a nucleotide mismatch (i.e., nucleotides do not base-pair) at both the 3' terminal nucleotides in the primer-dimer structure. For example, a partially constrained pentamer primer represented by WNNNS provides a terminal mismatch at both the 3' terminal nucleotides when it is inter-molecularly hybridized to form a primer-dimer structure having no recessed ends. In the primer-dimer structure, there exists an internal homology of three nucleotides (i.e., the three random nucleotides in WNNNS may base-pair with each other when the primer-dimer structure having no recessed ends is formed by inter-molecular hybridization). However, this primer example does not provide a terminal mismatch when it is inter-molecularly hybridized to form a primer-dimer structure with single-nucleotide base 3' recessed ends. Similarly, a partially constrained hexamer primer represented by WWNNNS provides a terminal mismatch at both the 3' terminal nucleotides when it is inter-molecularly hybridized to form a primer-dimer structure having no recessed ends. Moreover, this primer example provides a terminal mismatch at both the 3' terminal nucleotides even when it is inter-molecularly hybridized to form a primer-dimer structure having a single-nucleotide base 3' recessed ends. A partially constrained heptamer primer represented by WWWNNNS provides a terminal mismatch at both the 3' terminal nucleotides when it is inter-molecularly hybridized to form a primer-dimer structure having no recessed ends. Further, this primer example provides a terminal mismatch at both the 3' terminal nucleotides when it is inter-molecularly hybridized to form a primer-dimer structure having a single-nucleotide base 3' recessed ends, or to form a primer-dimer structure having a two-nucleotide base 3' recessed ends.
[0039] As used herein, the term "plasmid" refers to an extra-chromosomal nucleic acid that is separate from a chromosomal nucleic acid. A plasmid DNA may be capable of replicating independently of the chromosomal nucleic acid (chromosomal DNA) in a cell. Plasmid DNA is often circular and double-stranded.
[0040] As used herein, the terms "amplification", "nucleic acid amplification", or "amplifying" refer to the production of multiple copies of a nucleic acid template, or the production of multiple nucleic acid sequence copies that are complementary to the nucleic acid template. These terms may be used interchangeably in this application.
[0041] As used herein, the term "target nucleic acid" refers to a nucleic acid that is desired to be amplified in a nucleic acid amplification reaction. For example, the target nucleic acid comprises a nucleic acid template.
[0042] As used herein, the term "DNA polymerase" refers to an enzyme that synthesizes a DNA strand de novo using a nucleic acid strand as a template. DNA polymerase uses an existing DNA or RNA as the template for DNA synthesis and catalyzes the polymerization of deoxyribonucleotides alongside the template strand, which it reads. The newly synthesized DNA strand is complementary to the template strand. DNA polymerase can add free nucleotides only to the 3'-hydroxyl end of the newly forming strand. It synthesizes oligonucleotides via transfer of a nucleoside monophosphate from a deoxyribonucleoside triphosphate (dNTP) to the 3'-hydroxyl group of a growing oligonucleotide chain. This results in elongation of the new strand in a 5'→3' direction. Since DNA polymerase can only add a nucleotide onto a pre-existing 3'-OH group, to begin a DNA synthesis reaction, the DNA polymerase needs a primer to which it can add the first nucleotide. Suitable primers comprise oligonucleotides of RNA or DNA. The DNA polymerases may be a naturally occurring DNA polymerases or a variant of natural enzyme having the above-mentioned activity. For example, it may include a DNA polymerase having a strand displacement activity, a DNA polymerase lacking 5'→3' exonuclease activity, a DNA polymerase having a reverse transcriptase activity, or a DNA polymerase having an endonuclease activity.
[0043] As used herein the term "proofreading DNA polymerase" refers to any DNA polymerase that is capable of correcting its errors while performing DNA synthesis. A proofreading DNA polymerase possesses a 3'→5' exonuclease activity apart from its polymerase activity, and this exonuclease activity is referred as proofreading activity. Proofreading activity of such polymerases correct mistakes in the newly synthesized DNA. During DNA synthesis, when an incorrect base pair is recognized, the proofreading DNA polymerase reverses its direction by one base pair of DNA. The 3'→5' exonuclease activity (proofreading activity) of the enzyme allows the incorrect nucleotide base pair to be excised. Following the nucleotide base excision, the polymerase re-inserts the correct nucleotide base, and continues the DNA synthesis. When free dNTPs are present in the solution or reaction mixture suitable for DNA synthesis, the primary activity of the proofreading DNA polymerase is DNA synthesis. However, when dNTPs are not available for the DNA synthesis reaction, the primary activity of the proofreading DNA polymerase may be its 3'→5' exonuclease activity. Some of the proofreading DNA polymerases may require the presence of a divalent cation for their proofreading activity as well as for their polymerase activity. Suitable divalent cations that can switch on the proofreading activity of the proofreading polymerases include, but are not limited to, magnesium or manganese.
[0044] As used herein, "a strand displacing nucleic acid polymerase" refers to a nucleic acid polymerase that has a strand displacement activity apart from its nucleic acid synthesis activity. That is, a strand displacing nucleic acid polymerase can continue nucleic acid synthesis on the basis of the sequence of a nucleic acid template strand (i.e., reading the template strand) while displacing a complementary strand that had been annealed to the template strand.
[0045] As used herein, the term "complementary," when used to describe a first nucleic acid/oligonucleotide sequence in relation to a second nucleic acid/oligonucleotide sequence, refers to the ability of a oligonucleotide comprising the first nucleic acid/oligonucleotide sequence to hybridize (e.g., to form a duplex structure) under certain hybridization conditions with an oligonucleotide comprising the second nucleic acid/oligonucleotide sequence. Hybridization occurs by base pairing of nucleotides (complementary nucleotides). Base pairing of the nucleotides may occur via Watson-Crick base pairing, non-Watson-Crick base pairing, or base pairing formed by non-natural/modified nucleotides.
[0046] As used herein the term "high stringency hybridization conditions" or "high stringent hybridization conditions" refer to conditions that impart a higher stringency to an nucleic acid hybridization event than the stringency provided by conditions that are generally used for nucleic acid amplification reactions. For example, a high stringency hybridization condition may be accomplished in a nucleic acid amplification reaction by increasing the reaction temperature or by decreasing the salt concentration. Nucleic acid amplification reactions containing short primers are often carried out at about 75 mM salt concentration. In contrast, a nucleic acid amplification reaction performed at about 15 mM salt concentration may represent a high stringency hybridization condition. High stringency hybridization condition may be provided in an in-vitro isothermal nucleic acid amplification reaction by increasing the temperature from about 30° C., which is often used. For example, the isothermal nucleic acid amplification reaction may be performed at about 35° C. to about 45° C. to provide a high stringency hybridization condition.
[0047] As used herein, the term "rolling circle amplification (RCA)" refers to a nucleic acid amplification reaction that amplifies a circular nucleic acid template (e.g., single stranded DNA circles) via a rolling circle mechanism. Rolling circle amplification reaction may be initiated by the hybridization of a primer to a circular, often single-stranded, nucleic acid template. The nucleic acid polymerase then extends the primer that is hybridized to the circular nucleic acid template by continuously progressing around the circular nucleic acid template to replicate the sequence of the nucleic acid template over and over again (rolling circle mechanism). The rolling circle amplification typically produces concatamers comprising tandem repeat units of the circular nucleic acid template sequence. The rolling circle amplification may be a linear RCA (LRCA), exhibiting linear amplification kinetics (e.g., RCA using a single specific primer), or may be an exponential RCA (ERCA) exhibiting exponential amplification kinetics. Rolling circle amplification may also be performed using multiple primers (multiply primed rolling circle amplification or MPRCA) leading to hyper-branched concatamers. For example, in a double-primed RCA, one primer may be complementary, as in the linear RCA, to the circular nucleic acid template, whereas the other may be complementary to the tandem repeat unit nucleic acid sequences of the RCA product. Consequently, the double-primed RCA may proceed as a chain reaction with exponential (geometric) amplification kinetics featuring a ramifying cascade of multiple-hybridization, primer-extension, and strand-displacement events involving both the primers. This often generates a discrete set of concatemeric, double-stranded nucleic acid amplification products. The rolling circle amplification may be performed in-vitro under isothermal conditions using a suitable nucleic acid polymerase such as Phi29 DNA polymerase.
[0048] As used herein, multiple displacement amplification (MDA) refers to a nucleic acid amplification method, wherein the amplification involves the steps of annealing primers to a denatured nucleic acid followed by a strand displacement nucleic acid synthesis. As nucleic acid is synthesized by strand displacement, a gradually increasing number of priming events occur, forming a network of hyper-branched nucleic acid structures. MDA is highly useful for whole-genome amplification for generating high-molecular weight DNA with limited sequence bias from a small amount of genomic DNA sample. Strand displacing nucleic acid polymerases such as Phi29 DNA polymerase or large fragment of the Bst DNA polymerase may be used in multiple displacement amplification. MDA is often performed under isothermal reaction conditions, and random primers are used in the reaction for achieving amplification with limited sequence bias.
[0049] As used herein the term "reaction mixture" refers to the combination of reagents or reagent solutions, which are used to carry out a chemical analysis or a biological assay. In some embodiments, the reaction mixture comprises all necessary components to carry out a nucleic acid (DNA) synthesis/amplification reaction.
[0050] As used herein, the terms "reagent solution" or "solution suitable for performing a DNA synthesis reaction" or "amplification solution" refer to any or all solutions, which are typically used to perform an amplification reaction or DNA synthesis. They include, but are not limited to, solutions used in isothermal DNA amplification methods, solutions used in PCR amplification reactions, or the like. The solution suitable for DNA synthesis reaction may comprise buffer, salts, and/or nucleotides. It may further comprise primers and/or a DNA template to be amplified.
[0051] In some embodiments, kits for nucleic acid amplification are provided. The kits contain reagents, packaged together, that are required to practice the presently described methods of nucleic acid amplification. In one embodiment, the kit comprises a nucleic acid polymerase, the nucleotide analogues described above (e.g., 2-amino-dA and 2-thio-dT), and LNA bases. The nucleic acid polymerase and the other reagents may be packaged in a single vessel or they may be packaged in separate vessels.
[0052] In one embodiment, the kit comprises a Phi29 DNA polymerase and a partially constrained primer having a terminal mismatch primer-dimer structure, packaged together. The partially constrained primer in the kit may comprise a nucleotide analogue, such as a LNA nucleotide. In some embodiments, the partially constrained primer is a DNA-LNA chimera primer. The partially constrained primer in the kit may be a nuclease-resistant primer, for example, an exonuclease-resistant primer. These exonuclease-resistant primers in the kit may contain one or more phosphorothioate linkages between the nucleotides. In one embodiment, the kit comprises 5'-+W+WNNN*S-3', where "+" precedes a locked nucleic acid base (i.e., "an LNA base"; for example, +A=an adenosine LNA molecule and similarly for the other nucleotides), "W" represents a mixture of only dA and dT, "S" represents a mixture of only dC and dG, the "*" represents a phosphorothioate linkage between the two nucleotides, and a Phi29 DNA polymerase.
[0053] In a further embodiment, the kit comprises random hexamers for performing the GenomiPhi AT methods described herein. Specifically, such kits comprise modified hexamers are of the general formula: +N+N(atN)(atN)(atN)*N, wherein "+" precedes an LNA base, as described above, and (atN) represents a random mixture of 2-amino-dA, dC, dG, and 2-thio-dT. Alternatively, these kits may comprise hexamers represented by the formula (atN)(atN)(atN)(atN)(atN)*N.
[0054] The kit may further comprise reagents or reagent solutions required for performing a nucleic acid amplification reaction. It may further include an instruction manual detailing the specific components included in the kit, or the methods for using them in nucleic acid amplification reactions, or both.
[0055] The following examples are offered by way of illustration and not by way of limitation:
Example 1
Improvement in MDA Speed and Sensitivity
[0056] Amplification reactions were performed using the standard GenomiPhi® kit with the hexamer sequence NNNN*N*N and 75 mM KCl or utilizing the "cleaned" GenomiPhi (see, for example, U.S. Patent Application Publication No. 2009/0155859), GenomiPhi SD (e.g., U.S. Pat. No. 7,993,839), or GenomiPhi AT formulations. Real-time amplification was performed by adding a small amount of SYBR green I to the amplification mixture and monitoring fluorescence increase over time in a Tecan plate reader using a dilution series of Bacillus subtilis chromosomal DNA. A no template control was also analyzed (NTC).
[0057] The threshold time for DNA amplification signal to increase over background for each reaction was plotted out versus the amount of DNA added as input template into the reaction. Reactions containing primers with LNA and amino-A/thio-T showed an approximately 10 fold increase in amplification kinetics. The results are summarized in FIGS. 3A and 3B.
Example 2
Oligonucleotides Containing 2-Amino-dA and 2-Thio-dT Improve MDA Coverage and Overall Amplification Bias
[0058] Standard GenomiPhi® reactions or MDA reactions with the indicated formulations were performed with 100 pg B. subtilis input DNA. Total amplification in the reactions was approximately 20.000-fold. Amplified DNA was processed into libraries and subjected to Illumina GA whole-genome sequencing with a 51 nucleotide read length. 8-10 million reads for each sample were mapped to the B. subtilis reference genome. FIG. 4A provides a histogram that maps coverage level across the length of the genome. Relative standard deviations were calculated by the following formula: coverage standard deviation/mean coverage*100. The number of coverage gaps and the average lengths of those gaps are also indicated. FIG. 4B provides bar graphs comparing the formulations.
Example 3
Oligonucleotides Containing 2-Amino-dA and 2-Thio-dT Improve MDA Sequence Bias
[0059] For the amplification reactions described in Example 3, the coverage level was determined for each 100 base pair window of the genome. GC content of the windows was plotted against the fractional coverage of windows that had that GC content. The results are presented in FIG. 5 and indicate that the inclusion of amino-A/thio-T and LNA nucleotides in the amplification primer provide both increased representative amplification and decreased amplification bias.
Example 4
Amplification of Single Bacterial Cells Using the GenomiPhi AT Formulation
[0060] Amplification reactions were performed using the improved GenomiPhi AT formulation. Real-time amplification was performed by adding a small amount of SYBR green I to the amplification mixture and monitoring fluorescence increase over time in a Tecan plate reader. E. coli cells were diluted with a buffer containing 10 mM Tris, pH 7.5, 100 mM NaCl, and 0.1 mM EDTA, stained with FM1-43FX dye (Invitrogen), spotted into wells of a 384-well plate, and cells were counted using an inverted Fluorescent microscope (Nikon). Cells were lysed by addition of 1 μl of 0.2 M NaOH, 0.015% Tween-20, incubated for 10 min at room temperature, and neutralized by addition of 0.5 μl of 0.4 M HCl, 0.6 M Tris, pH 7.5. To this mixture was added the amplification reagents and reactions were incubated at 30° C. for the indicated times. A no template control was also analyzed (NTC). The results are summarized in FIG. 6A.
[0061] PCR was performed using 16S ready-made primers (NEB) and Amplitaq Gold reagents (Invitrogen) according to the manufacturer's instructions. DNA was analyzed by 1% agarose gel electrophoresis, stained with SYBR gold (Invitrogen), and visualized by scanning with a Typhoon imager (GE Healthcare). See FIG. 6B.
[0062] Amplified DNA was processed and subjected to Illumina GA whole-genome sequencing as described above. A histogram of coverage levels, the percent of reads mapped, and genome coverage levels are plotted in FIG. 6C.
[0063] All publications, patents, and patent applications mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
[0064] While only certain features of the invention have been illustrated and described herein, many modifications and changes will occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.
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IMO Secretary-General Efthimios E. Mitropoulos met the Honourable Sheik Hasina, Prime Minister of Bangladesh, in London on Friday (28 January), as a follow-up to his visit to Bangladesh earlier this month.
The Secretary-General sought to enlist support and action for the early ratification of the Hong Kong International Convention on the Safe and Environmentally Sound Recycling of Ships, 2009 and the implementation and enforcement of the technical standards contained therein; and offered technical co-operation assistance for capacity building in Bangladesh.
IMO – the International Maritime Organization – is the United Nations specialized agency with responsibility for the safety and security of shipping and the prevention of marine pollution by ships.
Web site: www.imo.org
Photo collection: Flickr: http://www.flickr.com/photos/imo-un/collections
Join us on our social media sites: Facebook: http://www.facebook.com/imohq Twitter: http://twitter.com/imohq you can also find us: @imohq YouTube: http://www.youtube.com/user/IMOHQ
For further information please contact: Lee Adamson, Head, Public Information Services on 020 7587 3153 (firstname.lastname@example.com) Natasha Brown, Media and Communications Officer on 020 7587 3274 (firstname.lastname@example.com).

subset: commoncrawl


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Archived Letter: Steve Jobs Called a Joker
Description:A 1976 letter penned by Silicon Valley ad man Mike Rose reads, in part, 'This joker is going to be calling you.' The 'joker' was a young Steve Jobs. (1:31)
Source: Newsy
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Çeşme is a coastal town and seaside resort on the Aegean, west of Izmir and on the extreme western tip of Turkey. It is a popular holiday destination, boasting crystal clear waters and beautiful sand beaches.
Cesme's main square has restaurants, cafes, tea houses, and nice sunset views ofthe Aegean Sea. Çeşme's small fortress, now nicely restored as the local museum, looms over the square. The Sunday market is great for a visit.
Things to See & Do
* Çeşme Castle
* Old town
* Orthodox church of Ayios Haralambos
* Thermal springs at IIica
* Aya Yorgi Bay - views over the Bay
General Information
Cruise Season - March - Dec
Currency - Turkish Lira (TRY)
Language - Turkish
Land Area - 260 km²
Population - 84,631 approx
Electricity - 2 round pins European style.
Time - UTC plus two hours
International Country Telephone Code - + 90
Port Location - The port at Cesme is centrally located, close to the Tourism Information Office.
Travel Links - There are several ways to get to Çeşme. There are several bus companies that offer services from Izmir to Cesme. Bus services are also available from Istanbul, the route lasts approximately 8 hours.
There is one, possibly two, daily ferry(ies) to Çeşme from the Greek island of Chios, the boat ride takes slightly over an hour.


text:
Cumbrian law firm moves out in landlord dispute
Last updated at 15:17, Wednesday, 13 August 2014
A Cumbrian law firm has closed its Workington office without warning.
Staff at KJ Commons Solicitors moved out of their base on Upper Jane Street in Workington yesterday.
Managing director David Thompson said it was part of the firm's long-term plan to move from the offices but admitted the change had been brought forward because of a dispute with the landlords.
Mr Thompson confirmed there had been an issue with bailiffs but said the firm had not been given notice to quit the building.
He denied rumours that bailiffs had visited the premises yesterday.
He added: "There's no love lost between us and the landlords. We are just trying to protect the jobs of all the staff here and the clients' interests so we have got all hands on deck to get things moved as quickly as possible.
"There have been some difficulties that we have had to face but we are confident that we have a bright future."
Mr Thompson said about 20 people worked at the office.
The firm's award-winning conveyancing team will join with law firm Taylor Rose Law, which was announced last month.
It will move to new premises on Murray Road.
The firm's family and criminal law departments will move to new premises at Haig Enterprise Park in Whitehaven.
All calls to the firm's Workington number are being temporarily rerouted to the Carlisle office.
The company was set up by Kevin Commons in 1987.
After Mr Commons was killed by gunman Derrick Bird in 2010, the firm became a limited company, with other staff joining Marcus Nickson as directors.
Last year Mr Nickson and fellow K J Commons solicitor Jonathan Dees left the firm to set up their own company, Nickson Dees, as clinical negligence consultants.
In January, the firm was again hit by tragedy when criminal law solicitor and director Tim Frost died of cancer. Mr Thompson said: "It's been difficult but we're going to get through it."
The firm's website lists its current directors as David Thompson, Lorraine Thompson and Craig Smith.
WEDNESDAY, AUGUST 13: In an article "Cumbrian law firm moves out in landlord dispute" published in the News & Star last Saturday, August 9, it was incorrectly stated that Ms Helen McNeil was a landlord of the Upper Jane Street, Workington, premises occupied by KJ Commons solicitors.
We are happy to clarify that Ms McNeil is not a landlord of the property and also that KJ Commons & Co was formed solely by the late Kevin Commons in 1987 - and not with a partner as reported. We apologise for the error.
The article above has been amended to reflect these points.
First published at 09:18, Saturday, 09 August 2014
Published by http://www.newsandstar.co.uk
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text:
Happy 2014!
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Think about palindromes/secret codes and think about the name in reverse.
If your really stuck then check out on urban dictionary since someone actually added it.
Wish I'd picked a better name now though...Too lazy to change it.
Mine is my name for everything i use!
my nickname is joker
my music name is joker
my manga character is joker
my RP name has somthing to do with the word joker
my passwords,usernames,e-mails have all to do with joker hehehehehe!
I believe…whatever doesn't kill you simply makes you…stranger.
Jokerman's Twitter Page: www.twitter.com/JokermanUno
Thanx Shin Ryuga!!!!
Think about palindromes/secret codes and think about the name in reverse.
If your really stuck then check out on urban dictionary since someone actually added it.
Wish I'd picked a better name now though...Too lazy to change it.
Naruto Shippuden >> 51 | Gundam 00 >> 24 | FMA >> Ch. 81
I've been using mine since I named my Star Wars RP character Senna Markos back in 2001. But now that the Bleach movie is out in Japan and fan subs are not that far off, people keep asking me if I chose my name after that Senna.
Well it's really easy to explain mine. I choose bcvl1124 as my username because B is my first name in the real life and it means white second the letter C is my second name and it means pure. V and L are my last names in the real life. And last and for most the numbers 1124. They are my favorite numbers because they let me remember my birthday. If you count the months 11 is November and the day is 24. So that concludes why I got bcvl1124 as my username. ^^ Although I have lots of username I use... ^^;;
Sorry that I couldn't tell my real name. ^^;;
0-leaders of the world
3-deck of cards
if you dont understand thats beacuase its personal if you do then you have the same logic as i
I'll do it man.
hmm that is an excellent quesiton to ask the reason i made my username dark1angel was beacause i like staying in the dark never in the light as well i like the #1, and i chose angel because i am related to the angel of life other then being in the light og heaven so that is my main reason into choosing my username of it and also describes me on who i am indeed really.
Hmm.. you believe that there are NO true leaders in this world to guide us.
The two locations being Heaven and Hell, there is only ONE God,
I'd have to say the four fingers being the Cardinal directions. They point you North, South, East, and West. I have a feeling I may be wrong on that one.
And as for the three deck of cards .. I'd have to say you're on your own with that one.
Very creative to choose as a Username. It's unique.


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Allianz beats rivals in snagging customers: survey
PUBLISHED: 03 Apr 2013 10:06:00 | UPDATED: 05 Apr 2013 01:46:32PUBLISHED: 03 Apr 2013 PRINT EDITION: 04 Apr 2013Australia's fourth-largest general insurer is netting one out of 10 new or switched commercial insurance policies, beating rivals CGU and QBE, data from Roy Morgan Research indicates.
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Top Headlines
- 23 for 2015: Projecting the USMNT Gold Cup roster (February 2015 Edition)
- Don Garber: MLS can become 'guinea pig' for use of technology in soccer
- SBI MLS Season Preview: Orlando City
- SBI MLS Season Preview: Real Salt Lake
- Saturday European Rewind: Rooney fires Man United to win; Dortmund crushes Schalke; and more
- The SBI Show: Episode 193 (CONCACAF Champions League, MLS labor talks, and more)
- SBI MLS Season Preview: Portland Timbers
- Soccer Saturday: Your Running Commentary
- Luis Robles: Union still fighting for 'some form of free agency' despite MLS owners' refusal to consider it
- Ashton Gotz on the fast track in recruitment for USMNT
- Americans Abroad: Weekend Preview
- This Weekend's Soccer On TV
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Alejandro Bedoya Americans Abroad Aron Johannsson Charlie Davies Clint Dempsey CONCACAF CONMEBOL David Beckham David Carney David Moyes English Premier League EPL FC Nantes John Anthony Brooks John Brooks Jozy Altidore Juan Agudelo Julian Green Jurgen Klinsmann Landon Donovan Los Angeles Galaxy Major League Soccer Manchester United MLS MLS Expansion NASL NBC NBC Sports Group New England Revolution New York Red Bulls Orlando City Portland Timbers Premier League San Jose Earthquakes soccer Soccer on TV Stoke City Terrence Boyd Tim Howard Tom Sermanni Tottenham U.S. Men's National Team USL PRO USMNT World CupSBI Soccer Blog Roll
Monthly Archives: September 2011
Americans Abroad: Europa League Rewind
By FRANCO PANIZO Jermaine Jones' struggles for playing time at Schalke 04 have been alleviated somewhat, as Jones made his first appearance for the club in nearly two months on Thursday. Having last played for Schalke on Aug. 6, Jones … Continue reading
Posted in Americans Abroad
16 Comments
Johnson's one-time switch to join USMNT approved by FIFA
TSG Hoffenheim midfielder/defender Fabian Johnson won't be with the U.S. men's national team for its October friendlies due to a neck injury, but he will be able to represent the United States in the near future. The 23-year-old Johnson has … Continue reading
Posted in Americans Abroad, U.S. Men's National Team
58 Comments
Klinsmann releases roster for October friendlies
photo by Thomas Eisenhuth/ISIphotos.com By FRANCO PANIZO Jurgen Klinsmann has released the roster for the U.S. men's national team's upcoming friendlies against Honduras and Ecuador, and there are plenty of familiar faces and one brand new face. German-born midfielder/defender Danny … Continue reading
Posted in U.S. Men's National Team
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MLS East Notes: United seeking back-to-back wins, Pappa ends slump and more
By THOMAS FLOYD The last time D.C. United recorded back-to-back league wins, Tom Soehn was the coach, Josh Wicks was in goal, and the attack was led by Ben Olsen, Christian Gomez, Luciano Emilio and Jaime Moreno. That was June … Continue reading
Altidore scores another Europa League goal
Jozy Altidore has done it again. Altidore continued his fine start to the season by scoring the opener in AZ Alkmaar's Europa League match against FC Metalist Kharkiv on Thursday. The U.S. international got on the scoreboard after making a … Continue reading
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Americans Abroad resume hunt for Europa League on Thursday
By FRANCO PANIZO The quest for European glory continues for several Americans Abroad on Thursday, as the second round of Group Stage games in the Europa League gets underway. After drawing last week, Clint Dempsey will look to help … Continue reading
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Client: Cascadia Games
Project: GameDock
Project specifics:- Branding
- Site Design
- Packaging Design
Client location: USA
Link: http://iosgamedock.com
The brief:
As the main backer of the GameDock's Kickstarter campaign, I've been heavily involved with the GameDock from the outset. While working on almost all aspects of the GameDocks visual design (more to come soon), this time I was tasked with giving the GameDock a slick new mobile friendly site that would really show off its great features.
The solution:
We wanted something that would be as rich on your computer screen as it would be on your mobile device, so we opted for a long scrolling design that included large images in a very clear/concise layout. As I'd already been working on the branding and packaging at this point, the site came together very naturally.
I cannot stress how exciting the GameDock is! 1080p output, 2 traditional controllers, a charging dock, Bluetooth connectivity, you name it and its cheap to buy too! So cool. Its shipping in late February, so get your orders in before they're all gone!
Client: Unknown Worlds Entertainment
Project: Natural Selection 2 - Full Site
Project specifics:- Website Design
Client location: USA
Natural Selection 2 – Full Site and Forums
The brief:
After having the pleasure of finishing the microsite for Natural Selection 2 I was due to move onto the full site and forum re-design, so the brief, while simple (follow the same styling throughout), was also a challenge as I had the forums to tackle...but this was going to be more fun than you could imagine! Why? I was informed to go wild with the design and not to worry about the development implications as their forum coders were super talented!
The solution:
As the style for this entire site was set due to recently completing the microsite, it was the fun task of taking that style and applying it to something much more complex. This whole process went smoothly and we soon had the full site finished and looking good. The final step was the big one, the forums. NS2 has a HUGE community, many of which have been loyal to the brand for many, many years. Ensuring the community would be happy was the core goal.
As the team had some great forum coders on hand, the design was allowed a lot of creative freedom, so every element could be totally redesigned from the ground up without any limitations! This was easily the best forum brief I've ever had! Needless to say with with a mixture of an open brief and few limitations, it came together very quickly.
If you've not already seen the game I strongly suggest you check it out, its probably the most technically ambitious Inide game to date and it squares off against any AAA FPS with ease. Its a true masterpiece!
Client: Kerosene Games
Project: Bladeslinger
Project specifics:- Icon Design
- Website Design
- App Store Promotional Art
Client location: USA
Link: http://bit.ly/10cog1Y
Bladeslinger Icon Design
The brief:
I've been working with Kerosene Games on the Bladeslinger promotional materials for some time now and it was great to start work on the icon design, as lets face it, icon design can be rather fun! The brief was fairly open as Kerosene wanted to experiment with a handful of ideas and have them all user tested to see which one's would be the most effective. So I was given a healthy block of time to come up with a selection of concepts.
The solution:
The final icon (the 1st one here) was selected based off the user feedback and we slowly but sure tailored the design down to a fine point. We played around with a vast range of directions from dark blood thirsty zombies, to light and bright ideas too. The final design is something of a half way house between the two extremities and really pop's when viewed on the iPhone's little screen. It was such a luxury to have experimentation time and I'm very, very thankful to Kerosene for allowing me such a luxury! If you haven't already, go grab a copy of the game as its breathtaking!
Client: Little Guy Games
Project: Super Snack Time Microsite
Project specifics:- App Microsite Design
Client location: Canada
Super Snack Time
The brief:
Another fantastic little microsite for the guys over at Little Guy Games! This time, for their fantastic arcade popper, Super Snack Time! As SST is a bright, colourful and energetic game, the brief was clear from the outset. The site had to be just as exciting and portray the explosive fun this game has to offer.
The solution:
As the artwork for the game is sooooo cutesy, it was imperative that it took centre stage. So I initially requested a handful of HD character renders that could be used on the site. The team at Little Guy Games set to work on a simply beautiful set of characters and sent them across. Once I had these and some footage from the game for reference, I started work on creating the site you see now. It was so much fun working with these characters as they really make the design jump off screen. And what makes all these even better is the fact that the game is absolutely ace too, I cant put it down!
Client: BSKC
Project: BSKC Site Design
Project specifics:- Site Design
Client location: UK
Link: http://www.bskc.co.uk
The brief:
After re-designing the BUKC site a couple of years ago, I was asked if I'd like to take on the BSKC site for a similar treatment! I love anything Motorsport related so I gave a definite YES! The brief was to breath new life into the BSKC site and give it a more extreme look. The only caveat was that it wasn't to look 'too' extreme as it had to appeal to teachers as well as students. I like a challenge!
The solution:
The solution turned came about quite quickly as by adopting a lighter design (rather than the black BSKC look) it meant that the site looked a lot brighter and thus less extreme. With this direction in hand the rest of the design slotted together smoothly and the end result worked out really well.
Client: Microsoft Games Studios
Project: Black Tusk Studios
Project specifics:- Website Design
Client location: Canada
Link: http://mgsvancouver.com
Black Tusk Studios
The brief:
The brief on this project was extremely limited, as the site had to portray a message that was HEAVILY protected by an NDA and even I wasn't allowed full access to the information that all this was based on. So needless to say, this was as exciting as it was extremely challenging...but if you know me, you know I love a damn good challenge!
The solution:
As I'd only been given light hints as to the what this site had to portray, it was a case of going away and working almost blindly, and sending the design back for feedback to see if I was hitting the target. Luckily, I was very close first time, so with a handful of amends we actually landed on the final design quite quickly. I'm so intrigued by the game that their working on (tipped to be Microsoft's next big franchise since Halo) as from the tip bits of info I got, it was sounding very, very exciting indeed!
Client: Unknownworlds Entertainment
Project: Natural Selection 2 Promo Site
Project specifics:- Microsite Design
- Animated Flash Effects
Client location: USA
Natural Selection 2 – Promo Site
The brief:
Natural Selection 2 is one of, if not the most accomplished Indie title in existence, so much so you'd be forgiven for thinking it was a AAA console title coming out of a studio with hundreds of staff! So the brief for this one page promo site was to give it the online presence that it deserves. In simple terms it had to kick boat loads of ass!
The solution:
So, as I ALWAYS ramble on about sites only being as good as their content, I drilled this message home to the team at Unknownworlds and begged for the assets to be bone shatteringly awesome...and boy did they deliver!!!! When I received the web art package I almost cried in excitement! I was like a giddy kid at Christmas running around shouting 'ooh, ooh look at these pretty pictures!!!!'. After I'd done this for an hour or two, I collapsed into my chair and realised it was time to actually do something with them and get to work on the design. For 'research' purposes, a copy of the game had to be acquired and played too, (how hard this job can be) so with both the running around in excitement and playing the game out of the way, I was ready to start. The core design was nailed down very quickly with only a handful of small amends needed to get it just where the Unknownworlds team wanted it. We had some kick ass servers to work with on this site too, so making it super visually rich wasn't going to be an issue. Plain and simply, this site got me more excited than any other before it...and that's saying something as I always get excited! Keep your eyes peeled for the upcoming full community site and the new Unknownworlds sites too, their launching soon! NS2 rocks!
Client: Ravenous Games
Project: Ravenous Games Website
Project specifics:- Site Design
Client location: Canada
Link: http://ravenousgames.com
Ravenous Games
The brief:
I'd known Ravenous Games even before they'd hit the AppStore with the amazing League of Evil, so it was great to finally get the chance to work on their company site! The brief was to create a new site design that showed off their AMAZING retro games for iOS. In truth, this was going to be easy as I just LOVE their games!
The solution:
The companies colour palette is ace, black, purple and white, so coupling that with their superb pixel artwork was going to be a blast! Needless to say the site design came together pretty smoothly and was warmly welcomed by both the Ravenous team and their dedicated fans! Keep doing what you do Ravenous, I love you :)
Client: 24 Caret Games
Project: Retro/Grade Playstation Store Icon & Banners
Project specifics:- Playstation Store Icon Design
- Playstation Store Banner Ad's
Client location: USA
The brief:
This was a fun little project to create a Playstation Store icon & ad package for Retro/Grade! The brief was to create a design that would show off Retro/Grade in all its retro greatness and catch the eyes of Playstation owners while browsing the store.
The solution:
As the game is a fantastic showcase of bright explosive colours and light, the icon and banner designs had to reflect exactly that. So with a handful of elements taken from the game's promo art, I set to work creating the package. I quite fancy doing more of these PS Store packages, this was rather good fun!!
Client: Analogue Interactive
Project: Analogue Interactive Branding & Site Overhaul
Project specifics:- Branding
- Homepage Overhaul
- Social Icon Design
Client location: USA
Analogue Interactive
The brief:
Being a hardcore arcade & retro gamer, I already knew about Analogue Interactive way before they got in touch, so I was rather excited to talk about this project! The brief was to rebrand the company and give the existing website an overhaul to match the new brand. The brand had to be ultra clean and ultra sleek to match the breathtaking hardware that Analogue Interactive make.
The solution:
Just by looking at the absolutely stunning hardware I could see exactly what this brand had to portray. It had to be clean and it had to be premium. I set to work on the new brand design, slowly but surely drawing it back further and further until it had become the minimal design you see now. The guys at Analogue Interactive were extremely happy with the new look and we soon set to work rolling it out. As I'm known for doing exchanges with my games company clients, it was obvious from the outset that payment for this job HAD to be in the form of hardware...YUM YUM!!
Client: Giants Software
Project: Farming Simulator
Project specifics:- Homepage Design
- Subpage Design
Client location: Switzerland
Farming Simulator
The brief:
As far as real world simulators go, the Farming Simulator series must be the most well known, so when Giants Software got in touch I was rather excited about working on the new site! The brief was to streamline their existing site, re-structure the content and give the whole design a fresh, light and attractive look that would freshen up the online experience.
The solution:
As soon I put pen to paper I knew what look was going to be the perfect fit for this design, a nice bright layout with splashes of greens and touches of nature dotted around. As the games assets were so rich, and I'd been supplied with all the 3D source files, I was able to set, pose and render out my own scenes. This gave me unlimited possibilities for the site design. It was quite an honour to work on this site as even though simulators aren't everyone's staple gaming diet, they are VERY big and have a very dedicated following. And lets be honest, if a real life simulator comes to mind, you think Farming Simulator, its just one of those genre defining titles. I really enjoyed this!
Client: Kerosene Games
Project: Kerosene Games Website
Project specifics:- Branding
- Website Design
- Branded Twitter Styling
- Branded Facebook Styling
Client location: USA
Link: http://kerosenegames.com
Kerosene Games
The brief:
After initially designing the microsite for Bladeslinger I was invited to tackle the re-branding and site design for Bladeslingers publisher, the awesome Kerosene Games! The direction that Kerosene wanted to take was one that would have a broad stylistic appeal to cover both hardcore and casual gamers/developers alike. As we'd just completed the branding project I was raring to go!
The solution:
As the games that Kerosene publish are some of the most visually striking on the mobile market, we had to ensure that the site design was equally striking, but also subtle enough to allow the stunning games to take centre stage. The colour palette of black, white and red meant that making the right elements pop was pretty easy, and couple that with stunning game art and the balance was easily struck. The end result was a bold page design that both showcased the games and promoted the reasons why Kersoene is the ideal publisher. There's A LOT more coming from Kerosene Games so I strongly suggest you keep an eye on what their up to :) Superb project!
Client: Subatomic Studios
Project: Subatomic Studios Website
Project specifics:- Website Design
Client location: USA
Subatomic Studios
The brief:
As I'd had the pleasure of working with Subatomic back when they stormed the iPhone charts with Fieldrunners, it was great to be working together again on their official company site! I was tasked with creating the new home for their current and upcoming games, one that could be infinitely expandable and take them forward as they grow and expand.
The solution:
As Subatomic Studios are known for their levels of attention to detail, we spent quite some time working on the finer points of the overall look and feel. Once we nailed down the exact direction we collectively tailored the design to hold lots of nice little visual touches to make every page element pop. If you look closely there's lots of different box styles, lighting accents and other lovely touches scattered around the site. It was quite a large site all in all, and once complete it really came together well! With Fieldrunners 2 on the way, I strongly urge you check the site out in anticipation for their next epic app store success!
Client: FatFly Digital/Jimmy White
Project: iWhirlwind - The Official Jimmy White Snooker Coach
Project specifics:- iPhone & iPad App Design
- App Icon Design
Client location: UK
Link: http://bit.ly/Mqsyyx
iWhirlwind – The Official Jimmy White Snooker Coach
The brief:
As most of my projects are games related, it was a pleasant surprise to be invited to design the new official Jimmy White training app! I think just about everyone on earth knows who Jimmy White is, so it was rather exciting to say the least! The brief was pretty simple too, to create a super clean and super simple user interface design for the new coaching app. The app itself is streamlined to be as simple and easy to learn from as possible, so the app design had to be the same.
The solution:
We decided that a great visual direction to take was one that evoked the feeling of a dark, atmospheric snooker hall. We figured that after working through the training that this is exactly where the user would be putting it to practice! We soon settled on the overall look and feel and set to work on fleshing out the various app screens. Additionally to this was the app icon which was a blast to work on! I wanted to ensure that the icon had a good level of depth and could immediately grab the attention of any snooker fan. This was such a pleasure to work on! It's also been great to see the app fly up the sports app charts and hover around the top 10 for quite some time!
Client: Klei Entertainment
Project: Mark of the Ninja Website
Project specifics:- Website Design
Client location: Canada
Mark of the Ninja
The brief:
Ninja's, seriously, ninjas…how cool is that? Couple ninja's with the Klei's general degree of awesomeness and you've got the perfect project recipe right there! For this dev blog I was tasked with creating a dark, mysterious and stealthy looking design that would tie in with the gorgeous visuals of the upcoming game!
The solution:
I decided to plunge the site into the dark depths of the ninja's world. So by taking elements from the game and creating a moonlit, atmospheric canvas, the site became a true extension of the game! As ever, Jeff Agala's amazing artwork just made this a pleasure to work on. I'm SO excited about Mark of the Ninja as it genuinely looks to be an original and very beautiful game. If you love ninja's, take a peek, you'll love it!
Kingdom Rush
Well, I think just about everyone on planet earth knows about Kingdom Rush, so when I got invited to design their family of sites, I got just a little bit giddy!
Famous Couture
I was tasked with creating something contemporary, urban and very much in line with the world of vinyl toys (kid robot etc), but only in this instance, it had to be tied in with the company name Famous Couture, coming from the saying 'Making Jesus Christ Famous'. A tough but fun brief!


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