PCR enhancers.doc

PCR 增强剂PCR enhancer DMSO and glycerolRecommended by a number of authors to improve amplificationefficiency and specificity of PCR. However, in the multiplex reactionthese enhancers gave conflicting results. For example, 5% DMSOimproved the amplification of some products , decreased theamount of others and some loci were not affected at all. The sameobservations were made with 5% glycerol. Bovine Serum Albumin (BSA):100 g/ml - 1 ug/ul, 0.01% - 0.1% (w/v)The addition of albumin to tissue DNA samples increases theamount of DNA generated by neutralizing many deleterious factorsfound in tissue samples which can inhibit PCR. Concentrations ofup to 0.8 g/L may increase the efficiency of a PCR reaction muchmore than either DMSO or glycerol. Formamide: 1.25% - 10% (v/v).In regions of high G/C content, specificity is reduced and multiplebands are observed when PCR products are separated on anagarose gel. Formamide enhances specificity and yield bychanging the Tm of the primer-template hybridization reaction andlowers enzyme resistance to heat destruction. However, formamidecan also be inhibitory to DNA polymerases so it needs to be testedat various concentrations to determine its optimal concentrationlevels. PEG (polyethylene glycol) 6000: 5% - 15% (w/v) Spermidine:Reduces non-specific reactions between the polymerase and DNA. Tris-HCl: 10 mM - 67 mM, pH 8.2 - 9.0 KCl: 25 mM - 50 mM MgCl2: 1.5 - 5.0 mM Gelatin: 0.01% - 0.1% (w/v) Non-ionic detergents Tween 20: 0.05% (v/v) Triton-X-100: 0.01% (v/v) Tetramethyl ammonium bromide TMANO (trimethylamine N-oxide) Betaine在以前的PCR实验中,实验结果总是不好,而用上其他公司生产的PCR增强因子时,实验结果总体有了很大很大的改变,因此,本人想和大家讨论讨论上述没有提及到的增强因子呢?它的用量与作用机制又如何呢?常见的添加剂有：DMSO，甘油，甲酰胺，硫酸铵，BSA等，它们对PCR反应的影响虽然有所不同，但都可提高PCR的扩增效率。1. DMSO：改善GC含量高的DNA的变性情况，使聚合酶更容易在二级结构处延伸，但是当浓度高时（大于10）时会降低保真性。2. 甘油：提高产量，增加酶的稳定性。3. 甲酰胺：促进某些“引物模板”退火，降低带有二级结构的DNA的变性温度。4. 硫酸铵：增加反应体系的离子强度，改变DNA的变性及退火温度，调节酶活。PCR增强剂常常在模板GC含量70%,或者引物有二级结构的时候应用，使用增强剂可以打开这些结构，增加特异性，降低退火温度，但是会使产率降低，需要增加taq酶的量保证扩增的完成。谢谢您的支持,DMSO，甘油，甲酰胺，硫酸铵，BSA，PEG等书上都有很多介绍。但是还有一些成分并是未知的，现在我的初衷就是想深层揭开这些未知成分。像我用过的PCR MIX中Eppendorf HotMaster Mix 和 百克生物的 PCR enhancer 两种MIXE，虽然两者都能达到增强扩增效率的结果，但是 HotMaster Mix 能使得我的扩增特异性大大的提高，所以我觉得应该更深层的对PCR增强因子加强了解，免得以后花大量时间和金钱在这上面。百克生物的PCR enhancer从性状上看还有颜色反应，所以觉得还有类似指示剂之的物质吧，所以大家一起再深入寻找讨论讨论。这样对以后的新人实验方面有很大的帮助了。PCR AdditivesA variety of PCR additives and enhancing agents have been used to increase the yield, specificity and consistency of PCR reactions. Whilst these additives may have beneficial effects on some amplifications it is impossible to predict which agents will be useful in a particular context and therefore they must be empirically tested for each combination of template and primers. Some of the more popular of these additives are listed in the table below along with references describing their use.AdditiveReferencesDMSO(dimethyl sulfoxide)Amplifications 5: 16Gene 140: 1Nucleic Acids Research 18: 1666Betaine(N,N,N-trimethylglycine= carboxymethyltrimethylammonium)Biochemistry 32: 137BioTechniques 21: 1102Genome Research 6: 633Nucleic Acids Research 25: 3957Proceedings of the National Academy of Sciences of the United States of America70: 298Trends in Biochemical Science 22: 225FormamideNucleic Acids Research 18: 7465Non-ionic detergentse.g. Triton X-100, Tween 20 or Nonidet P-40 (NP-40)Biotechniques 12: 332Nucleic Acids Research 18: 1309TMAC(tetramethylammonium chloride)Nucleic Acids Research 18: 4953Nucleic Acids Research 23: 33437-deaza-2-deoxyguanosine(dC7GTP)Nucleic Acids Research 16: 3360BSA(bovine serum albumin)Applied and environmental microbiology 62:1102-1106BioTechniques 23:504BioTechniques 25:564Nucleic Acids Research 16: 9775T4 gene 32 proteinApplied and Environmental Microbiology 62:1102-1106DMSO at 2-10% may be necessary for amplification of some templates, however 10% DMSO can reduce Taq polymerase activity by up to 50% (Gelfand 1989) so it should not be used routinely. DMSO is thought to reduce secondary structure and is particularly useful for GC rich templates.A number of PCR additives are now comercially available, however the identities of these agents are not usually revealed by their suppliers. Frackman et al.(1998) have demonstrated (using NMR analysis) that the PCR additive provided by QIAGEN in their PCR core kit (Q-Solution) and that provided by CLONTECH in the Advantage-GC cDNA PCR kit is in fact Betaine which is available at a fraction of the cost as a 5M solution from Sigma-Aldrich (cat. # B 0300), but be sure to use Betaine or Betaine (mono)hydrate and not Betaine HCl. Other products suspected of consisting largely of Betaine include the GC-RICH solution enhancer from Roche, TaqMaster enhancer from Eppendorf, GC-melt from Clontech and FailSafe enhancer (formerly MasterAmp PCR Enhancment Technology) from Epicentre (Weissensteiner, pers. comm.). Betaine is generally used at a final concentration of 1.0-1.7M.Formamide is generally used at 1-5% and 10% formamide is reported (Gelfand 1989) to have no effect on the activity of Taq polymerase, however, Sarkar et al. (1990) (see table for ref.) found that 1.25% formamide worked as well as 2.5% and 5%, and no amplification was seen at 10% so it seems prudent not to use concentrations of formamide greater than strictly necessary for optimal amplification.Non-ionic detergents stabilise Taq polymerase and may also supress the formation of secondary structure. 0.1-1% Triton X-100, Tween 20 or NP-40 may increase yield but may also increase non-specific amplification. As little as 0.01% SDS contamination of the template DNA (left-over from the extraction procedure) can inhibit PCR by reducing Taq polymerase activity to as low as 10%, however, inclusion of 0.5% Tween-20 or -40 will effectively neutralise this effect (Gelfand 1989).TMAC is generally used at a final concentration of 15-100mM to eliminate non-specific priming. TMAC has is also used to reduce potential DNA-RNA mismatch (Proceedings of the National Academy of Sciences of the United States of America 82: 1585) and improve the stringency of hybridization reactions (Nucleic Acids Research 16: 4637).The base analogue 7-deaza-2-deoxyguanosine may facilitate amplification of templates with stable secondary structures when used in place of dGTP in a ratio of 3: 1, 7-deaza-2-deoxyguanosine: dGTP.BSA has proven particularly useful when attempting to amplify ancient DNA or templates which contain PCR inhibitors such as melanin.-谢谢bact的好资料.今天的实验还不错,现在得到的这个BUFFER配方与大家分享.这个BUFFER在我的实验中整体效果比较理想,希望此BUFFER能帮到一些人,这BUFFER针对着的是复合引物扩增体系.10 x Reaction MixPH 8.8)120mM Tris-Cl500mM KCl1%(v/v)Triton-X-100100 mM 甜菜碱25 mM Mg22 mM dNTP此MIX与普通未曾优化的原始MIX比较,从扩增效率与特异性比较相差太大,所以有好东西就和大家一起分享,因此希望大家一起并分享更好用的BUFFER配方及新颖的PCR增强剂.10. PCR add