Oligo dT VN is used for anchored cDNA synthesis. But what does "VN" in the end do? Please help. Thanks.
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Oligo dT length is normally 30nt but transcript poly A tail could be longer. Including VN at 3’ end of RT primer enriches for non-poly A region of transcripts and without it RT can be primed in poly A region so cDNA would have A/T homopolymer which in most sequencing applications is undesirable. A side effect of VN inclusion is possible bias in some transcripts conversion to cDNA. -
'N' is any base, 'V' is either A,C or G. This allows for anchoring at the beginning of the Poly-A tail.Comment
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why VN, why not just V for oligo dT anchor?
Why does almost everyone use 3'-VN as the anchor for oligo dT primers rather than using 3'-V?
The former only has a 1/12 chance of matching a given mRNA, vs. 1/3 for the latter. Plus 1/4 of the former will have T as the N, meaning there's a greater potential for annealing and extension within the polyA tail with VN vs V.
Only reason I can think of is legacy.
Thanks!
MikeComment
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I have not done any tests. I guess the assumption is that the oligos will make multiple attempts to hybridize and that a two-base anchor is "stronger" than a single base.Comment
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