fragments leaving a 3' overhang. If there is significant homology (20-40 bp) on each end of the DNA insert, it can anneal with a complementary backbone. Afterwards the polymerase can fill in the gaps while ligase fuses the nicks at the end. This method greatly increases the rate of cloning and success rate of cloning into a vector backbone. However, it requires the DNA fragment to have significant homology with the plasmid. For this reason, knowledge of the sequence being cloned must be known beforehand. This is not a requirement with functional cloning. TOPO cloning TOPO Cloning is a cloning method that uses Taq polymerase. This is because Taq leaves a single adenosine overhang on the 3’ end of PCR reaction products. Utilizing this knowledge, backbones with a 5’ thymine overhang can be used for cloning purposes. In this case knowledge of the fragment being cloned must be known to be able to make PCR primers for it and the number of TOPO Cloning compatible vectors is relatively small. However, it provides the advantage that reactions only take about 5 minutes to do. Gateway recombination cloning Gateway recombination cloning is a cloning method in which a DNA fragment is moved from one plasmid backbone to another via a single homologous recombination event. However, for this method to work, the DNA fragment of interest must be flanked by recombination sites. While this method isn't strictly an alternative, it does allow the movement of DNA fragments from one plasmid to another quicker than creating a whole new genomic library. The reason this method may be used in conjunction with functional cloning is to put a library under a different promoter or on a backbone with a different selection marker. This can come in handy if an individual wants to try functional cloning in a wide range of bacteria to try to combat the issue with codon bias. Applications Determining homology in the environment Metagenomics is one of the largest fields that commonly uses functional cloning. Metagenomics studies all the genetic material from a specific environmental sample, such as the gut microbiome or lake water. Functional libraries are created that contain DNA fragments from the environment. As the original bacterium that a DNA sequence originated from cannot be easily detected, creating metagenomic functional libraries possesses advantages. Less than 1% of all bacteria are easily cultured in the lab, leaving a large percentage of