PCRenhancers.docVIP

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 μg/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 μg/μL 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