<p>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 href="https://f1000research.com/slides/7-251" rel="nofollow noreferrer">a presentation</a> 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 <em>et al.</em>. Here are my strand-switch/PCR images from that presentation:</p>

<ol>
<li>PolyT primer addition, with mismatched anchor sequence</li>
</ol>

<p><a href="https://i.sstatic.net/rpSO9.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/rpSO9.png" alt="PolyT primer addition, with mismatched anchor sequence"></a></p>

<ol start="2">
<li>Strand switch primer addition, with overhanging anchor sequence</li>
</ol>

<p><a href="https://i.sstatic.net/lK3qh.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/lK3qh.png" alt="Strand switch primer addition, with overhanging anchor sequence"></a></p>

<ol start="3">
<li>PCR amplification, using primers that are complementary to the anchor sequences</li>
</ol>

<p><a href="https://i.sstatic.net/QnIow.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/QnIow.png" alt="PCR amplification using complementary primers"></a></p>

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

CC BY-SA 3.0
2018-04-06T14:26:58.117Z
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?
gc5
CC BY-SA 3.0
2018-04-06T20:25:08.010Z
That's basically correct. It's not "added after", it's part of the same sequence.
gringer
CC BY-SA 3.0
2018-04-05T21:50:24.363Z
2018-04-05T21:50:24.363Z
gringer
<p>I am reading Tang et al. (2009).</p>

<p>In the method description it is stated that:</p>

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

<p>What is an anchor sequence in this case?</p>

<ol>
<li>Tang, F. et al. <a href="https://www.nature.com/articles/nmeth.1315" rel="nofollow noreferrer">mRNA-Seq whole-transcriptome analysis of a single cell</a>. Nat. Methods 6, 377–382 (2009).</li>
</ol>

<scrnaseq><primer>
What is a poly(T) primer anchor sequence?
# What is a poly(T) primer anchor sequence?

(Asked by: username_0 on 2018-04-05)

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](https://www.nature.com/articles/nmeth.1315). Nat. Methods 6, 377–382 (2009).

**Tags:** scrnaseq, primer

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## 1 answers

### Answer by username_1 on 2018-04-05. Score: 4 | Accepted answer

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](https://f1000research.com/slides/7-251) 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](https://i.sstatic.net/rpSO9.png)](https://i.sstatic.net/rpSO9.png)

2. Strand switch primer addition, with overhanging anchor sequence

[![Strand switch primer addition, with overhanging anchor sequence](https://i.sstatic.net/lK3qh.png)](https://i.sstatic.net/lK3qh.png)

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

[![PCR amplification using complementary primers](https://i.sstatic.net/QnIow.png)](https://i.sstatic.net/QnIow.png)

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.

> **Comments:**
> **username_0**: 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?  
> **username_1**: That's basically correct. It's not "added after", it's part of the same sequence.  

---

