【2.1】麻辣个引物

引物是PCR的关键一步,PCR是分子实验的关键,分子实验室是现代生物学得关键。这么说来,搞好引物,是不是处在现代生物学的最顶端,走上人生巅峰了呢。哈哈

一、PCR原理

二、引物

设计的目的是在两个目标间取得平衡:

  1. 扩增特异性
  2. 扩增效率。

说白了就是要能扩增目的片段,要准确,高效的扩增目的片段。围绕这两个基本的条件,我们在设计引物的时候,要注意哪些呢?

引物说白了,就是一段序列,这个序列由A,T,C,G四个碱基组成,就包含了引物的长度,GC含量,热稳定性(Tm),二级结构….

1. 引物长度

一般引物长度为18~30碱基(最佳为18-22 bp)。引物长度跟特异性和Tm有关。总的说来,每增加一个核苷酸引物特异性提高4倍,这样,大多数应用的最短引物长度为18个核苷酸。引物长度的上限并不很重要,主要与反应效率有关。由于熵的原因,引物越长,它退火结合到靶DNA上形成供DNA聚合酶结合的稳定双链模板的速率越小。

叶兄跟我提过引物的长度最好不要超过36bp,太长,实际并不能提高Tm多少,主要起作用的还是3‘那几个碱基。同时引物过短,小于16bp的时候,往往非特异性比较高,同时这样的序列往往是高GC,Tm很容易算不准。这个时候,已经没有必要通过降低长度来降低Tm了

2.GC含量

一般引物序列中G+C含量一般为40%~60%,一对引物的GC含量和Tm值应该协调。若是引物存在严重的GC倾向或AT倾向则可以在引物5’端加适量的A、T或G、C尾巴。GC含量最大得影响还是Tm,GC含量越高,Tm值相应会越高,解链难度增加。同时根据目前主流算Tm的公式,GC越靠近50%,其值越准。

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。

下面得算法没有验证,是否靠谱未知

	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+.

计算Tm的公式太多了,不同软件也可以对退火温度进行计算,其计算原理会各有不同,因此有时计算出的数值可能会有少量差距。其差值主要体现在根据DNA浓度,Mg离子的矫正。

计算Tm公式的比较:,,

4.避免扩增模板的二级结构区域

选择扩增片段时最好避开模板的二级结构区域。实验表明,待扩区域自由能(△G)小于58.6lkJ/mol时,扩增往往不能成功。若不能避开这一区域时,用7-deaza-2’-脱氧GTP取代dGTP对扩增的成功是有帮助的。

5. 非特异性扩增

非特异扩增容易导致两个问题:1.试剂的浪费;2.分析的干扰(特别是假基因特别容易对基因分型造成困惑)

从计算机层面有两个算法可以去解决:

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

引物3’端是延伸开始的地方,因此要防止错配就从这里开始。3’端不应超过3个连续的G或C,因这样会使引物在G+C富集序列区错误引发。3′端也不能有形成任何二级结构可能,除在特殊的PCR反应中,引物3′端不能发生错配。如扩增编码区域,引物3′端不要终止于密码子的第3位,因密码子的第3位易发生简并,会影响扩增特异性与效率。

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

引物自身不应存在互补序列,否则引物自身会折叠成发夹状结构,这种二级结构会因空间位阻而影响引物与模板的复性结合。若用人工判断,引物自身连续互补碱基(这个属于序列局部blast的结果)不能大于3bp。两引物之间不应该存在互补性,尤应避免3′端的互补重叠以防引物二聚体的形成。

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

不同的需求,扩增子长度不一样

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

工具 介绍 链接
生物软件网 一个汇聚了各种引物工具的网站 http://www.bio-soft.net/primer.html
各种工具的比较 - http://molbiol-tools.ca/PCR.htm
各种工具的比较2 - http://www.hsls.pitt.edu/obrc/index.php?page=pcr_oligos
WASP - Allele-specific primers of SNPs http://bioinfo.biotec.or.th/WASP
github - primer3-py https://github.com/libnano/primer3-py
Bio-Rad PCR Troubleshooting http://www.bio-rad.com/zh-cn/applications-technologies/pcr-troubleshooting
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 primer-blast - https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome
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/
BatchPrimer3 - http://probes.pw.usda.gov/cgi-bin/batchprimer3/batchprimer3.cgi?PRIMER_TYPE=4&CLEAR_FORM=no&LOAD_EXAMPLE=yes
PrimerBank - https://pga.mgh.harvard.edu/primerbank/
- - https://www-s.nist.gov/dnaAnalysis/primerToolsPage.do
- - http://www.dnasoftware.com/our-products/visual-omp/

总结:

在碰到这些情况的时候,我们只能秉着“实践是检验真理的唯一标准”这一原则,要试一试才能知道能否行得通了。

参考资料:

http://www.ebiotrade.com/newsf/2006-4/2006429175034.htm

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

药企,独角兽,苏州。团队长期招人,感兴趣的都可以发邮件聊聊:tiehan@sina.cn
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