PCR Amplification of Highly GCrich Regions 高高GC 含量含量DNA的的PCR 扩增扩增 ?1高高GC 含量含量DNA序列扩增困难序列扩增困难生物中广泛存在生物中广泛存在高高GC 含量含量DNA序列序列, ,扩扩增的增的DNA有的含有的含70%-80%70%-80%高高GC,常规常规PCRPCR条件下条件下, ,难扩增难扩增,原因原因:易形成复杂的二级结构易形成复杂的二级结构, , 防止防止DNA模板模板变性变性,DNA,DNA聚合酶难以结合聚合酶难以结合. .2高高GC 含量含量DNA序列扩增常遇到的问题序列扩增常遇到的问题1.常规PCR. 影响影响PCR产物合成产物合成.2.多重 PCR. 偏好低偏好低GC区扩增导致区扩增导致PCR产物产物比例失衡比例失衡.3.定量 PCR.富富GC区易与内标形成区易与内标形成异源双链异源双链核酸分子核酸分子,导致导致PCR结果解释错误结果解释错误.4.DNA测序. DNADNA聚合酶趋向于在聚合酶趋向于在富富GC区停留区停留使测序使测序难以进行难以进行. .5.cDNA合成. 合成的是缺合成的是缺GC区的短的区的短的cDNA片段片段.3高高GC 含量含量DNA序列扩增方法序列扩增方法1.模板模板DNA变性处理变性处理 1) NaOH: 2)核苷酸类似物核苷酸类似物: 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使用热启动聚合酶和高Tm引物引物 它们的特异性比普通 Taq polymerases高,如. Tag 加加 Pfu, Deep Vent, 4NaOH模板模板DNA在碱性溶液里不被降解在碱性溶液里不被降解,但一定浓度但一定浓度的碱性溶液可以破坏的碱性溶液可以破坏DNA高级结构高级结构,使使DNA变性变性,变性的变性的DNA在在PCR反应中容易与引物反应中容易与引物结合结合(退火退火),也容易延伸也容易延伸.模板DNA处理:1ml1ml里含有里含有0.4mol/L0.4mol/LNaOH和和0.4mmol/LEDTA0.4mmol/LEDTA( (乙二胺四乙酸乙二胺四乙酸 ) 室温室温10分钟分钟3MNaAc调调pH 乙醇沉乙醇沉淀淀 PCR反应反应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 or 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 PCRdependent analyses in the following situations:1.Standard PCR amplification. Highly GCrich regions prevent template denaturation, and hence product synthesis.2.Multiplex PCR amplification. The preferential amplification of lowGCcontent sequences results in misinterpretation of the balance between the two sequences.7多重多重 PCR (MPCR) 或复合或复合PCR，它是在同一它是在同一PCR反应体系里加上两对以上引物，同时扩增出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多个核酸片段的PCR反应，其反应原理，反应试反应，其反应原理，反应试剂和操作过程与一般剂和操作过程与一般PCR相同。
所有引物对在统相同所有引物对在统一指定的反应条件下进行扩增一指定的反应条件下进行扩增多重多重PCR Multiplex PCR8electrophoresiselectrophoresisprimersprimersprimersprimers12341234多重多重PCRPCR（复合复合复合复合PCRPCRPCRPCR）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 PCRdependent analyses in the following situations:3.Quantitative PCR amplification. GC rich targets may form heteroduplexes (异源双异源双链核酸分子链核酸分子) with internal standards, which are designed to be very similar to target sequences, leading to misinterpretation of results.10Highly GCrich regions can obstruct PCRdependent analyses in the following situations:4.DNA sequencing. DNA polymerase tends to stall in GCrich regions, which results in compression and difficulty in interpreting these parts of the sequence.5.cDNA synthesis. cDNA synthesis of GCrich templates may create shorter fragments that lack the GCrich portions of the sequence. The result is a skewed(歪斜的歪斜的) representation of the original mRNA molecules.11How to overcome 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(特异性特异性) of these polymerases is 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脱氧黄嘌呤脱氧黄嘌呤), and 7deaza GTP(7脱氮脱氮2脱氧脱氧鸟苷鸟苷5三磷酸三磷酸)could partly substitute the dGTP. How to overcome these problems13Organic additives such as dimethylsulfoxide (DMSO), formamide(甲酰胺甲酰胺), betaine(甜菜碱甜菜碱, 三甲铵三甲铵乙内酯乙内酯), glycine(甘氨酸甘氨酸), lowmolecularweight sulfones that are chemically related to DMSO (e.g., tetramethylene sulfoxide四甲基亚砜四甲基亚砜), and, more recently, lowmolecularweight amides(酰胺酰胺) have all proved successful, to some degree, in solving the problems associated with highly constricted (收缩的收缩的)DNA and RNA structures.14BETAINE甜菜碱甜菜碱扩增增强剂扩增增强剂甜菜碱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代谢物代谢物 of choline (胆碱胆碱) metabolism, which is present in liver and kidney cells. Betaine is the major regulator of osmotic(渗透性的渗透性的) pressure in plants, a role 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 hightemperature steps of the cycles, thereby maintaining the efficiency of the enzyme during amplification. 17BETAINEBetaine is a zwitterion(tsvitrai n两两性离子性离子) 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 do。