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Team:Johns Hopkins-BAG/Building Block synthesis
From 2009.igem.org
Building Block Synthesis
One of the current protocols 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 two strands which eliminates the gaps between DNA strands altogether. In other words, most overlapping oligonucleotides will now have a region of around 35bp in which they complement each other. By using ungapped strands of DNA, the Taq DNA Ligase enzyme will 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 of complementary DNA that occurs during the annealing process. With the LCR, it is more likely for oligonucleotides to anneal correctly and result in a final product of the correct sequence. The LCR would also reduce the likelihood of problems involved with loxP sites and other anomalies found in the normal TPCR and FPCR protocols. However, the LCR should be emphasized as an alternative to the current methods due to the expensive enzymes and reagents involved in the process of phosphorylation of the oligonucleotides for use in the LCR cycling program.
