《高GC含量DNA的PCR》由会员分享，可在线阅读，更多相关《高GC含量DNA的PCR（47页珍藏版）》请在金锄头文库上搜索。
1、PCR Amplification of Highly GCrich Regions 高GC 含量DNA的PCR 扩增 ?1高GC 含量DNA序列扩增困难生物中广泛存在高GC 含量DNA序列, 扩增的DNA有的含70%-80%高GC,常规 PCR条件下,难扩增,原因:易形成复杂的二级结构, 防止DNA模 板变性,DNA聚合酶难以结合.2高GC 含量DNA序列扩增常遇到的问题1.常规PCR. 影响PCR产物合成.多重 PCR. 偏好低GC区扩增导致PCR产物 比例失衡.定量 PCR.富GC区易与内标形成异源双链 核酸分子,导致PCR结果解释错误.DNA测序. DNA聚合酶趋向于在富GC区停留 使
2、测序难以进行.cDNA合成. 合成的是缺GC区的短的cDNA 片段.3高GC 含量DNA序列扩增方法1.模板DNA变性处理 1) NaOH: 2)核苷酸类似物: dITP脱氧黄嘌呤, dGTP(7-脱氮-2-脱氧鸟苷-5-三磷酸) 3)有机添加剂:二甲基亚砜（DMSO）、甲酰胺、甜菜碱(betaine）、甘油、四甲基亚砜、酰胺 、聚已二醇（PEG) 2.使用热启动聚合酶和高Tm引物它们的特异性比普通 Taq polymerases高,如. Tag 加 Pfu, Deep Vent, 4NaOH模板DNA在碱性溶液里不被降解,但一定浓 度的碱性溶液可以破坏DNA高级结构,使 DNA变性,变性的D
3、NA在PCR反应中容易与 引物结合(退火),也容易延伸.模板DNA处理:1ml里含有0.4mol/LNaOH和0.4mmol/LEDTA(乙二胺四乙酸 ) 室温10分钟 3MNaAc调pH 乙醇沉淀 PCR反应5DNA segments with very high GC content have proved difficult to handle in a wide range of molecular analyses. GCrich regions may form rigid, constrained secondary structures that are difficult o
4、r impossible for the DNA polymerases to enter under standard PCR conditions. Why is it difficult for DNA segments with GCrich to be amplified ?6Highly GCrich regions can obstruct PCR dependent analyses in the following situations:1.Standard PCR amplification. Highly GC rich regions prevent template
5、denaturation, and hence product synthesis. 2.Multiplex PCR amplification. The preferential amplification of lowGC content sequences results in misinterpretation of the balance between the two sequences.7多重 PCR (MPCR) 或复合PCR，它是在同一PCR反应体系里加上两对以上引物，同时扩增出多个核酸片段的PCR反应，其反应原理，反应试剂和操作过程与一般PCR相同。所有引物对在统一指定的反
6、应条件下进行扩增。多重PCR Multiplex PCR8electrophoresiselectrophoresisprimersprimers12341234多重多重PCRPCR（复合复合PCRPCR） 用于检测特定基因序列的存在或缺失。用于检测特定基因序列的存在或缺失。9Highly GCrich regions can obstruct PCR dependent analyses in the following situations:3.Quantitative PCR amplification. GC rich targets may form heteroduplexes (
7、异源双链核酸分子) with internal standards, which are designed to be very similar to target sequences, leading to misinterpretation of results.10Highly GCrich regions can obstruct PCR dependent analyses in the following situations:4.DNA sequencing. DNA polymerase tends to stall in GCrich regions, which resul
8、ts in compression and difficulty in interpreting these parts of the sequence. 5.cDNA synthesis. cDNA synthesis of GC rich templates may create shorter fragments that lack the GCrich portions of the sequence. The result is a skewed(歪斜 的) representation of the original mRNA molecules.11How to overcome
9、 these problemsOver the years, different approaches have been used to overcome the problems caused by secondary structure.Hotstart Taq polymerases have been especially designed to function only after an extended initial denaturing step at high temperature. The specificity(特异性) of these polymerases i
10、s higher than that of standard Taq polymerases. 12To overcome the problem of DNA sequence compressions, the template can be denatured using either sodium hydroxide(NaOH) or the nucleotide analogs deoxyinositol triphosphate (dITP脱氧黄嘌呤), and 7deaza GTP(7脱氮 2脱氧鸟苷5三磷酸)could partly substitute the dGTP. H
11、ow to overcome these problems13Organic additives such as dimethylsulfoxide (DMSO), formamide(甲酰胺), betaine(甜菜碱, 三甲铵 乙内酯), glycine(甘氨酸), lowmolecular weight sulfones that are chemically related to DMSO (e.g., tetramethylene sulfoxide四甲基亚砜), and, more recently, lowmolecularweight amides(酰胺) have all p
12、roved successful, to some degree, in solving the problems associated with highly constricted (收缩的)DNA and RNA structures.14BETAINE甜菜碱 扩增增强剂甜菜碱Betaine (N,N,N, -trimethylglycine) 是氨基酸类似物和胆碱的主要代谢物，存在 于肝和肾，调节植物的渗透压。体内可保护 蛋白质，体外可促进蛋白质的折叠. betaine 加到 PCR 混合液里可保护Taq polymerase 免被高温变性伤害, 还能促进 DNA-protein复合物
13、的稳定，保证扩增的效 率.高浓度的betaine能消除AT区和 GC 区熔解 温度的差异，但不改变双链B-型 DNA 的构 型.15BETAINEBetaine (N,N,Ntrimethylglycine) is an amino acid analog and a major metabolite代谢物 of choline (胆碱) metabolism, which is present in liver and kidney cells. Betaine is the major regulator of osmotic(渗透性的) pressure in plants, a rol
14、e that serves to protect proteins in vivo and facilitates refolding of proteins in vitro. 16BETAINEAddition of betaine to a PCR mix protects the Taq polymerase from denaturation during the high temperature steps of the cycles, thereby maintaining the efficiency of the enzyme during amplification. 17
15、BETAINEBetaine is a zwitterion(tsvitrai n 两性离子) at neutral pH and, even at high concentrations (5 M), it has property facilitates the maintenance of stable DNAprotein complexes.18BETAINE High concentrations of betaine eliminate the differences in melting temperature between AT and GC domains, withou
16、t changing the double stranded DNA conformation of the B form. 19LOWMOLECULARWEIGHT SULFOXIDES, AMIDES, AND GLYCINES 低分子量的亚砜、酰胺和甘氨酸 In the search for more potent amplification enhancers for highly GC rich templates, several classes of low molecularweight compounds, sulfoxides(亚砜) and amides(酰胺), have been analyzed. 20LOWMOLECULARWEIGHT SULFOXIDES, AMIDES, AND GLYCINES 低分子量的砜、酰胺和甘氨酸Among the sulfoxides, tetr
