In cells, the ribosome translates mRNA into proteins. mRNA in eukaryotic cells has either a 5′ Cap 1 or Cap 2 [m7G(5′)ppp(5′)G] structure which stabilizes the mRNA and enhances translation. A cap can be incorporated in the mRNA transcription by including a mixture of cap analog and NTPs. Greater than 90% of transcribed mRNA will possess a 5′ cap, while the remaining mRNA will possess a 5′ triphosphate. In some cases it is desirable to remove the 5′ triphosphate with a phosphatase treatment.
TriLink’s CleanCap analog enables high capping efficiencies (90%+) resulting in more biologically active mRNA per transcription. It also produces a natural Cap 1 structure. Efficient translation of the mRNA into protein also requires a poly(A) tail. This can be efficiently introduced by including a poly(dT) stretch at the end of the transcription template. This can also be accomplished by an additional post-transcription PCR step that utilizes a primer containing the poly(dT) stretch.