Steps to design genome-specific primers

以下这六步其实就是特异性引物设计的核心，其实所有软件和方法都是根据这六步开发的。我们不仅要会用，更要知其所以然。
1. Blast the marker sequence against the pseudomolecule andfind all the
homeologsand potentially paralogs: >90% similarity
2. Extract the sequences for all the homeologs and potentially paralogs
3. Multiple Sequence Alignment
4. Find all the variation sites among the homeologs and paralogs
5. Use variation sites or combination of variation sites that are unique to
yourtargets to design primers
6. Blast all the primers against the pseudomolecule v1.0 with word length 7 to see whether they also hit other chromosomes

Common practices of PCR primers

·  Length: 18 - 25 nt

· Melting temperature: around 60 °C

· GC clamp: G or C bases within the last five basesin the 3' end helps promote specific binding, but more than 3 G/C should beavoided

· NO secondary structures

· Avoid template secondary structure or othercomplex regions, such as retros

· Amplicon length: KASP and dCAPS are short (<300 bp), other markers usually < 1 kb

· Primer pair Tm difference < 5 °C

Primer Design Tips

以下中第二点不知有多少小伙伴知道，这个思路很巧妙，用过的都说好！
1. Usethe unique variation site as the primer 3' end
7A CGAGCTTGATGACGAAGAAGGAT
7B CGAGCTTGATGACGAAGAAGGAC

2. Two variation sites in the first 4 nt from the 3' end: we canintroduce 1
mutation in the 3rd nt from 3' end (may need to use touchdown PCR)
7A CGAGCTTGATGACGAAGAAGGAT
7B CGAGCTTGATGACGAAGAAGGAC
     CGAGCTTGATGACGAAGAAGAAT
Nucleotide substitution principle:
A → C；T → C；G → A；C → T

Validate Primer Location Using Chinese Spring Nullitetrasomic (NT) Lines
If we tested our primers target for 7A:
N7AT7D (7D7B7D): Absent
N7BT7D (7A7D7D): Present
N7DT7B (7A7B7B): Present
Are our primers 7A-specific?

Common PCR-based genotyping methods for SNP markers

以下的三种标记种类大家可能熟悉CPAS和KASP，不知有多少小伙伴熟悉dCAPS, 这个和上面第二点所用到的思路是一样的。

1. CAPS (Cleaved Amplified Polymorphic Sequences)

• One SNP allele creates or removes a naturally occurring restriction site

• Codominant

2. dCAPS (Derived CAPS)

• For SNPs that donot create a natural restriction site

• Uses introduced mismatches in one PCR primer to create a restriction  site forone allele

• Codominant

3.  KASP (Kompetitive Allele Specific PCR)

• A homogenous,fluorescence-based genotyping variant of PCR

• Codominant

 为了让大家熟悉这个dCAPs，以下是一个例子：

IWB1998:CGAGCTTGATGACGAAGAAGGAGA[T/C]CGGGCAGACCCACGACGT

EcoRV: GAT'ATC

这里又有一个巧妙的思路：We can add some tails to make dCAPS primer longer to better separate after digestion：GAAGGTGACCAAGTTCATGCTCGAGCTTGATGACGAAGAAGGATA

Primer design software
· Primer3 (http://primer3.ut.ee/)
· Polymarker for KASP in wheat (https://github.com/TGAC/biorubypolyploid tools)
· CAPS Designer (https://solgenomics.net/tools/caps_designer/caps_input.pl)
· dCAPS Finder 2.0 (http://biology4.wustl.edu/dcaps)
· indCAPS (http://indcaps.kieber.cloudapps.unc.edu/)
· GSP (Genome Specific Primers) (https://probes.pw.usda.gov/GSP/)

SNP Primer Design Pipeline

这个就是军利兄自己编写的Python小程序，以下也附上了在github上的源代码和相应说明文件。强烈推荐大家下载应用，说明文件也非常详细，学起来也不难。

1. Apipeline to design KASP/CAPS/dCAPS primers for SNPs in wheat
2. A Python script which incorporates:

· Muscle: Multiple sequence alignment program

(http://www.drive5.com/muscle/)

· Primer3: program for designing PCR primers

(http://primer3.sourceforge.net/)

· blast+: BLAST the wheat genome

(https://blast.ncbi.nlm.nih.gov/Blast.cgi)

3. I have a github repository for this tool
https://github.com/pinbo/SNP_Primer_Pipeline 

下面是Pipeline的工作原理：

1.Blast each SNP sequence against the pseudomolecule and get hits that are

· >90% similarity and

· >90% of length of the best hit AND >50 bp

2.Get 500 bps on each side of the SNP for all the hits (SNP is at 501)
3. Multiple Sequence Alignment of the homeolog sequences with MUSCLE
4. Find all the variation sites that can differ the target from other homeologs
5. Use these sites as forced 3' end in Primer3 and design homeolog specific primers
6. Blast all the primers against the pseudomolecule v1.0 with word length 7 to see whether it also hits other chromosomes

· Criterion of matches: < 2 mismatches in thefirst 4 bps from 3'

引申阅读：小麦生信系列文章

1. 第一篇是为序，介绍了一个常用的生信网站Graingenes

• 小麦生信必备网站和数据库（一）

2. 接下来三篇主要介绍了小麦物理图谱的介绍和应用，其中对小麦基因组数据库的总结介绍是非常基础且重要的知识。另外，也介绍了一些比较基因组学的知识和应用，包括野生二粒小麦，山羊草，拟南芥，和水稻。

• 小麦生信菜鸟成长记(二)--高手请直接看第二部分

• 小麦基因组数据库介绍       

• 小麦生信菜鸟(三)--常见问题        

• 如何获取小麦中一个基因家族的所有成员 

• 如何根据模式作物拟南芥和水稻的基因序列寻找小麦里的直系同源基因     

3. 接下来几篇小编打算介绍三个主题：基因表达，特异性引物设计，以及突变体库。

• 小麦生信菜鸟(四)—基因表达数据库          

• 小麦生信菜鸟归来(五)—系列总结以及特异性引物设计
  

• 小麦生信菜鸟(六)— 特异性引物设计（今天推送）

• 小麦生信菜鸟(七)— 基因表达数据分析（待续）

• 小麦生信菜鸟(八)— 小麦突变体数据库介绍及应用（待续）

4.其它相关推送：

• 野生二粒小麦基因组在science发布

• 野生二粒小麦基因组下载

• Nature在线发表小麦D基因组供体——粗山羊草的基因组研究论文

• 又一篇节节麦基因组文章来袭--聊一聊你的关注点在哪？

• 节节麦基因组数据使用和下载

• 吐血推荐：小麦或植物常用网站