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Team:Johns Hopkins-BAG/Building Block synthesis
From 2009.igem.org
Building Block Synthesis
One of the current protocol of producing a specific sequence of DNA within our undergraduate course of Build-A-Genome involves overlapping, gapped oligonucleotides (70bp) forming the basis of the 750bp Building Block. The Ligation Chain Reaction (LCR) is an alternative procedure that produces a more specific overlap between to two strands that eliminates the gaps between DNA strands altogether. In other words, most overlapping oligonucleotides would now have a region of around 35bp in which they complement each other. By using an un-gapped strands of DNA, the Taq DNA Ligase enzyme would be used to string the DNA backbone together. Like a normal PCR, the cycling process produces large quantities of feasible DNA Building Block. One important theoretical advantage is the extensive overlapping complementary DNA that occurs during the annealing process. It would be more unlikely that oligonucleotides that have been incorrectly sequenced to anneal correctly and end up in the final product. The LCR would also reduce the likelihood of problems involved with loxP sites and other anomalies found in the normal TPCR and FPCR protocols. The LCR should however be emphasized as an alternative to the current methods due to expensive enzymes and reagents involved in the process of phosphorylation of the oligonucleotides for use in the LCR cycling program.
