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I am reading Tang et al. (2009).

In the method description it is stated that:

A single cell is manually picked under a microscope and lysed. Then mRNAs are reverse-transcribed into cDNAs using a poly(T) primer with anchor sequence (UP1) and unused primers are digested.

What is an anchor sequence in this case?

  1. Tang, F. et al. mRNA-Seq whole-transcriptome analysis of a single cell. Nat. Methods 6, 377–382 (2009).
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As shown in figure 1 of that paper, the anchor sequence is additional sequence that is added to the end of the polyT primer that provides a known and non-target binding site for additional PCR reactions. I gave a presentation about a month ago about the process that Oxford Nanopore Technology use in their strand-switching cDNA kit, which may be useful for understanding the process used by Tang et al.. Here are my strand-switch/PCR images from that presentation:

  1. PolyT primer addition, with mismatched anchor sequence

PolyT primer addition, with mismatched anchor sequence

  1. Strand switch primer addition, with overhanging anchor sequence

Strand switch primer addition, with overhanging anchor sequence

  1. PCR amplification, using primers that are complementary to the anchor sequences

PCR amplification using complementary primers

In the case of the Tang et al. paper, they use a polyA-tailing method for strand-switching, but the process is essentially the same.

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  • $\begingroup$ So is it basically a sequence added after the Poly(T) tail, which does not bind to the Poly(A) tail? If so, the main purpose of it is to provide a binding site for the PCR primers? $\endgroup$
    – gc5
    Apr 6, 2018 at 14:26
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    $\begingroup$ That's basically correct. It's not "added after", it's part of the same sequence. $\endgroup$
    – gringer ♦
    Apr 6, 2018 at 20:25

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