<p>Module 3 does not exist during PCR amplification. Here is how NEBNext adaptor and multiplex PCR primers work.</p>

<p>During the 1st PCR cycle, only the P7 primers are used to bind and amplify ligated DNA, generating the complementary strands that contain the binding sites for P5 primers (Universal in this case). During the second cycle of amplification, the P5  primer hybridizes to the DNA strand generated during the first cycle and start to amplify. Please refer to the following figure for more details.  </p>

<p><a href="https://i.sstatic.net/fY6K8.jpg" rel="nofollow noreferrer"><img src="https://i.sstatic.net/fY6K8.jpg" alt=""></a></p>

CC BY-SA 3.0
2018-04-24T03:33:00.627Z
2018-04-24T03:33:00.627Z
Pingfang Liu
<p>The original point of the non-complementary sequence is to allow formation of a Y-adapter after USER treatment, since that's what's needed for the Illumina chemistry to work. In reality the bottom strand may not be there in step 3, since USER has already digested one strand (creating the hairpin in step 2) and size selection has been performed. Regardless, it'll get removed once cluster formation is performed.</p>

CC BY-SA 3.0
2018-04-13T19:22:51.370Z
2018-04-13T19:22:51.370Z
Devon Ryan
<p>I am making some infographics of different library prep types and noticed something weird about NEB's Ultra II adapters. </p>

<p><code>Molecule 1</code> is the desired product of the adapter ligation step. <code>*</code> indicates a phosphorothioate bond and <code>-</code> indicates a normal bond. <code>|</code> indicates complementary sequence through hydrogen bonding.</p>

<p><code>Molecule 2</code> is the product after treatment with the USER enzyme.</p>

<p><code>Molecule 3</code> is the result of melting molecule <code>2</code> and hybridizing the NEBNext Universal PCR Primer for Illumina.</p>

<p>My question is: Why does <code>Molecule 3</code> have a large non-complementary region on the 3' end of the original molecule? Is that just to force <code>Molecule 1</code> to have an open loop conformation so that the USER enzyme can access it better? Or maybe it is something to do with PCR kinetics?</p>

<p>I understand that PCR synthesizes in the <code>5'-&gt;3'</code> direction, so <code>Molecule 3</code> only forms with the original adapter-ligated molecules before PCR.</p>

<p><a href="https://i.sstatic.net/eSLst.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/eSLst.png" alt="enter image description here"></a></p>

<phylogenetics><adapter><pcr><library>
Why do NEB adapters have non-complementary sequence?
# Why do NEB adapters have non-complementary sequence?

(Asked by: username_0 on 2018-04-13)

I am making some infographics of different library prep types and noticed something weird about NEB's Ultra II adapters.

`Molecule 1` is the desired product of the adapter ligation step. `*` indicates a phosphorothioate bond and `-` indicates a normal bond. `|` indicates complementary sequence through hydrogen bonding.

`Molecule 2` is the product after treatment with the USER enzyme.

`Molecule 3` is the result of melting molecule `2` and hybridizing the NEBNext Universal PCR Primer for Illumina.

My question is: Why does `Molecule 3` have a large non-complementary region on the 3' end of the original molecule? Is that just to force `Molecule 1` to have an open loop conformation so that the USER enzyme can access it better? Or maybe it is something to do with PCR kinetics?

I understand that PCR synthesizes in the `5'->3'` direction, so `Molecule 3` only forms with the original adapter-ligated molecules before PCR.

[![enter image description here](https://i.sstatic.net/eSLst.png)](https://i.sstatic.net/eSLst.png)

**Tags:** phylogenetics, adapter, pcr, library

---

## 2 answers

### Answer by username_1 on 2018-04-24. Score: 1 | Accepted answer

Module 3 does not exist during PCR amplification. Here is how NEBNext adaptor and multiplex PCR primers work.

During the 1st PCR cycle, only the P7 primers are used to bind and amplify ligated DNA, generating the complementary strands that contain the binding sites for P5 primers (Universal in this case). During the second cycle of amplification, the P5 primer hybridizes to the DNA strand generated during the first cycle and start to amplify. Please refer to the following figure for more details.

[![](https://i.sstatic.net/fY6K8.jpg)](https://i.sstatic.net/fY6K8.jpg)


---

### Answer by username_2 on 2018-04-13. Score: 1

The original point of the non-complementary sequence is to allow formation of a Y-adapter after USER treatment, since that's what's needed for the Illumina chemistry to work. In reality the bottom strand may not be there in step 3, since USER has already digested one strand (creating the hairpin in step 2) and size selection has been performed. Regardless, it'll get removed once cluster formation is performed.


---

