Enhanced Diagnostic Tools
The Chemical ligation method ensures the exact sequence that is requested to be made, and certain modifications and labeling could also be incorporated. With the chemical method, we can make up to 250 bases long. The chemical method also has a very low yield, so the overall cost is higher than the alternative method.
The Enzymatic method (IVT) allows very long sequences (up to 3,000 and higher) to be generated and has a much higher yield. The difference to this method is that the sequence could have three added bases leading the sequence as result generated by the promoter. Long RNA synthesis is generated using bacteriophage polymerase such as T7, T3 or SP6. Depending on the complexity and length of the sequence, if the RNA is generated using polymerase T7 and T3, it could generate an additional GGG and SP6 could generate an additional GAA as a result of promoter sequence. For most molecular biology applications, three extra bases on the 5' end shouldn't interfere with anything considering the much longer entire RNA.
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