# 麻辣个引物

## 二、引物

1. 扩增特异性
2. 扩增效率。

1. 引物长度

2.GC含量

3.退火温度

DNA一半解链时得温度（为什么定义解链一半时得温度，还不是全部解链或者3/4的DNA解链的温度，这里面是个统计学问题，sam’s note后面会在统计学中提高这个问题）。退火温度需要比解链温度低5℃，如果引物碱基数较少，可以适当提高退火温度，这样可以使PCR的特异性增加；如果碱基数较多，那么可以适当减低退火温度，是DNA双链结合。一对引物的退火温度相差4℃～6℃不会影响PCR的产率，但是理想情况下一对引物的退火温度是一样的，可以在55℃～75℃间变化。 有以下公式可以用于粗略计算引物的退火温度。

    在引物长度小于20bp时：[4(G+C)+2(A+T)]-5℃
在引物长度大于20bp时：62.3℃+0.41℃(%G-C)-500/length-5℃
Tm(oC) = {ΔH/ ΔS + R ln(C)} - 273.15
ΔH (kcal/mole) : H is the Enthalpy. Enthalpy is the amount of heat energy possessed by substances. ΔH is the change in Enthalpy. In the above formula the ΔH is obtained by adding up all the di-nucleotide pairs enthalpy values of each nearest neighbor base pair.

ΔS (kcal/mole) : S is the amount of disorder a system exhibits is called entropy. ΔS is change in Entropy. Here it is obtained by adding up all the di-nucleotide pairs entropy values of each nearest neighbor base pair. An additional salt correction is added as the Nearest Neighbor parameters were obtained from DNA melting studies conducted in 1M Na+ buffer and this is the default condition used for all calculations.

ΔS (salt correction) = ΔS (1M NaCl )+ 0.368 x N x ln([Na+])

Where
N is the number of nucleotide pairs in the primer ( primer length -1).
[Na+] is salt equivalent in mM.

[Na+] calculation:
[Na+] = Monovalent ion concentration +4 x free Mg2+.


4.避免扩增模板的二级结构区域

5. 非特异性扩增

1.引物blast，解析两引物blast的结果，即使一对引物blast出来得位置刚好处在某一个非目的位置，但如果两端引物至少有2处gap或mismatch，我们仍认为这样得一对引物不可能扩增出这个非目的位置。这样的工具有blast-primer。这个工具的缺陷是依赖blast，而blast结果又跟seed、e-value等参数有很大的关系，引物在blast的过程有可能漏掉一些非特异性扩增位置

2.建立引物Index。这样速度会快很多，但建立Index需要完全匹配，一般考虑的时3’最后几个碱基。这样的工具有MFEPrimer，但这个的问题在于最后几个碱基中如果出现一个错配也是有可能扩增出非目的片段，但是这样得非特异性扩增容易被漏掉

3.目标序列blast,再次排除blast以及Index漏掉的假基因的干扰

6.引物末端

The presence of G or C bases within the last five bases from the 3’ end of primers (GC clamp) helps promote specific binding at the 3’ end due to the stronger bonding of G and C bases. More than 3 G’s or C’s should be avoided in the last 5 bases at the 3’ end of the primer.

3’ End Stability: It is the maximum ΔG value of the five bases from the 3’ end. An unstable 3’ end (less negative ΔG) will result in less false priming.

7.引物的二级结构

Hairpins: It is formed by intramolecular interaction within the primer and should be avoided. Optimally a 3’ end hairpin with a ΔG of -2 kcal/mol and an internal hairpin with a ΔG of -3 kcal/mol is tolerated generally.（ΔG = ΔH – TΔS）

Self Dimer: A primer self-dimer is formed by intermolecular interactions between the two (same sense) primers, where the primer is homologous to itself. Generally a large amount of primers are used in PCR compared to the amount of target gene. When primers form intermolecular dimers much more readily than hybridizing to target DNA, they reduce the product yield. Optimally a 3’ end self dimer with a ΔG of -5 kcal/mol and an internal self dimer with a ΔG of -6 kcal/mol is tolerated generally.

Cross Dimer: Primer cross dimers are formed by intermolecular interaction between sense and antisense primers, where they are homologous. Optimally a 3’ end cross dimer with a ΔG of -5 kcal/mol and an internal cross dimer with a ΔG of -6 kcal/mol is tolerated generally.

8.重复碱基

**Repeats: **A repeat is a di-nucleotide occurring many times consecutively and should be avoided because they can misprime. For example: ATATATAT. A maximum number of di-nucleotide repeats acceptable in an oligo is 4 di-nucleotides.

Runs: Primers with long runs of a single base should generally be avoided as they can misprime. For example, AGCGGGGGATGGGG has runs of base ‘G’ of value 5 and 4. A maximum number of runs accepted is 4bp.

9.为了下一步操作而产生的不完全匹配

5’端对扩增特异性影响不大，因此，可以被修饰而不影响扩增的特异性。引物5′端修饰包括：加酶切位点；标记生物素、荧光、地高辛、Eu3+等；引入蛋白质结合DNA序列；引入突变位点、插入与缺失突变序列和引入一启动子序列等。额外的碱基或多或少会影响扩增的效率，还加大引物二聚体形成的几率，但是为了下一步的操作就要作出适当的“牺牲”。

10.扩增子长度

For qPCR, the target length is closer to 100 bp and for standard PCR, it is near 500 bp

11. Optimum Annealing Temperature (Ta Opt):

The formula of Rychlik is most respected. Our products use this formula to calculate it and thousands of our customers have reported good results using it for the annealing step of the PCR cycle. It usually results in good PCR product yield with minimum false product production.

Ta Opt = 0.3 x(Tm of primer) + 0.7 x(Tm of product) - 14.9

where Tm of primer is the melting temperature of the less stable primer-template pair Tm of product is the melting temperature of the PCR product.

## 三、特殊目的PCR以及相关引物

1.TaqMan

http://www.biomart.cn/experiment/430/590/597/57994.htm

http://www.premierbiosoft.com/tech_notes/TaqMan.html

http://www6.appliedbiosystems.com/support/tutorials/pdf/taqman_mgb_primersprobes_for_gene_expression.pdf

http://www3.appliedbiosystems.com/cms/groups/mcb_marketing/documents/generaldocuments/cms_042505.pdf

## 四、目前已有引物工具

WASP - Allele-specific primers of SNPs http://bioinfo.biotec.or.th/WASP
github - primer3-py https://github.com/libnano/primer3-py
primer3 - http://bioinfo.ut.ee/primer3-0.4.0/primer3/
Primer3web - http://www.primer3plus.com/web_3.0.0/primer3web_input.htm
primer3 - http://primer3.sourceforge.net/webif.php
MFEprimer-2.0 - http://biocompute.bmi.ac.cn/CZlab/MFEprimer-2.0/index.cgi/
Oligo Calc - http://biotools.nubic.northwestern.edu/OligoCalc.html
idtdna - http://sg.idtdna.com/calc/analyzer
PrimerPlex - http://www.premierbiosoft.com/primerplex/index.html
OLIGO - http://www.oligo.net/index.html
igenetech - http://primerqc.igenetech.com/
PrimerBank - https://pga.mgh.harvard.edu/primerbank/
- - https://www-s.nist.gov/dnaAnalysis/primerToolsPage.do
- - http://www.dnasoftware.com/our-products/visual-omp/