1、1PCR Amplification of Highly GC-rich Regions 高高GC 含量含量DNA的的PCR 扩增扩增 ?2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, ,扩增的扩增的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. .3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
2、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.4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
3、(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, 5NaOH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变变性性,变性的变性的DNA在在PCR反应中容易与引物结合反应中容易与引物结合(退火退火),也容易延伸也容
4、易延伸.模板DNA处理:1ml1ml里含有里含有0.4mol/L0.4mol/LNaOH和和0.4mmol/LEDTA(0.4mmol/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 乙醇沉淀乙醇沉淀 PCR反应反应6DNA segments with very high GC content have proved difficult to handle in a wide range of molecular analyses. GC-rich regions may form rigid, constrained secondary structure
5、s that are difficult or impossible for the DNA polymerases to enter under standard PCR conditions. Why is it difficult for DNA segments with GC-rich to be amplified ?7Highly GC-rich regions can obstruct PCR-dependent analyses in the following situations:1.Standard PCR amplification. Highly GC-rich r
6、egions prevent template denaturation, and hence product synthesis.2.Multiplex PCR amplification. The preferential amplification of low-GC-content sequences results in misinterpretation of the balance between the two sequences.8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
7、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 PCR91234123410Highly GC-rich regions can obstruct PCR-dependent analyses in the following situations:3.Quantitative PCR amplification. GC -rich targets may form heterod
8、uplexes (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.11Highly GC-rich regions can obstruct PCR-dependent analyses in the following situations:4.DNA sequencing. DNA polymerase tends to stall in GC-rich r
9、egions, which results 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 GC-rich portions of the sequence. The result is a skewed(歪斜的歪斜的) representation of the original mRNA molecu
10、les.12How to overcome these problemsOver the years, different approaches have been used to overcome the problems caused by secondary structure.Hot-start 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
11、性) of these polymerases is higher than that of standard Taq polymerases. 13To 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 7-deaza GTP(7-脱氮脱氮-2-脱氧脱氧鸟苷鸟苷-
12、5-三磷酸三磷酸)could partly substitute the dGTP. How to overcome these problems14Organic additives such as dimethylsulfoxide (DMSO), formamide(甲酰胺甲酰胺), betaine(甜菜碱甜菜碱, 三甲铵乙三甲铵乙内酯内酯), glycine(甘氨酸甘氨酸), low-molecular-weight sulfones that are chemically related to DMSO (e.g., tetramethylene sulfoxide四甲基亚砜四甲基亚
13、砜), and, more recently, low-molecular-weight 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.15BETAINE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
14、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.16BETAINEBetaine (N,N,N-trimethylglycine) 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 ce
15、lls. 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. 17BETAINEAddition of betaine to a PCR mix protects the Taq polymerase from denaturation during the high-temperature steps of the
16、 cycles, thereby maintaining the efficiency of the enzyme during amplification. 18BETAINEBetaine is a zwitterion(tsvitrai n两性离子两性离子) at neutral pH and, even at high concentrations (5 M), it has property facilitates the maintenance of stable DNAprotein complexes.19BETAINE High concentrations of betai
17、ne eliminate the differences in melting temperature between AT and GC domains, without changing the double-stranded DNA conformation of the B form. 20LOW-MOLECULAR-WEIGHT SULFOXIDES, AMIDES, AND GLYCINES低分子量的低分子量的亚砜、酰胺亚砜、酰胺和和甘氨酸甘氨酸In the search for more potent amplification enhancers for highly GC-r
18、ich templates, several classes of low-molecular-weight compounds, sulfoxides(亚亚砜砜) and amides(酰胺酰胺), have been analyzed. 21LOW-MOLECULAR-WEIGHT SULFOXIDES, AMIDES, AND GLYCINES低分子量的低分子量的砜、酰胺砜、酰胺和和甘氨酸甘氨酸Among the sulfoxides, tetramethylene sulfoxide(四甲基亚砜四甲基亚砜) and tetramethylene四亚四亚甲基甲基 sulfolane（环丁
19、砜环丁砜 ） were found to efficiently enhance the amplification of templates with GC contents from 52% to 73%. 22LOW-MOLECULAR-WEIGHT SULFOXIDES, AMIDES, AND GLYCINES低分子量的低分子量的砜、酰胺砜、酰胺和和甘氨酸甘氨酸Acetamide(乙酰胺乙酰胺) , N,N-dimethylglycine, N-monomethylglycine (sarcosine肌氨酸肌氨酸), and trimethylamine N-oxide (三甲胺三甲
20、胺-N-氧氧化物化物TMANO), which are structurally similar to betaine, have been analyzed for their effect on T7 polymerase sequencing of supercoiled DNA. 23Enhencers 扩增增强剂扩增增强剂Betaine proved to be the most efficient facilitator, followed by TMANO, and then by N,N-dimethylglycine, which was intermediate in ac
21、tivity. Sarcosine(肌氨酸肌氨酸) showed only a minor ability to overcome the secondary structures of the DNA template. 24低分子量的低分子量的砜、酰胺砜、酰胺和和甘氨酸甘氨酸扩增增强剂Betaine已经证明是最强的增强剂，其次是 TMANO(三甲胺-N-氧化物，与Betaine结构相似）、乙酰胺， N,N-二甲基甘氨酸。肌氨酸肌氨酸对克服DNA二级结构仅有较小的能力. 25BETAINE APPLICATIONS甜菜碱的甜菜碱的应用应用 DNA Sequencing DNA 测序测序 Mu
22、ltiplex PCR Amplification 多重多重PCR扩增扩增 Reverse Transcription PCR 反转录反转录PCR Quantitative PCR 定量定量PCR Microarray Studies 芯片研究芯片研究26DNA Sequencing DNA 测序测序DNA regions with high melting temperatures, or with the consensus sequence of 嘧啶嘧啶Py-G-C, may cause DNA polymerases to stall, resulting in compressio
23、n of the DNA sequencing ladder and difficulty in the correct interpretation of the sequence . 高高Tm的或有的或有Py-G-C(Pyrimidine 嘧啶嘧啶-鸟嘌呤鸟嘌呤-胞嘧胞嘧啶啶)共有序列的共有序列的DNA能够停止能够停止 DNA 聚合酶聚合酶,导致难以导致难以正确测序正确测序 .27DNA Sequencing DNA 测序测序The addition of 2 M betaine 甜菜碱甜菜碱 to these difficult sequencing reactions can elim
24、inate the problem more efficiently. The efficiency of TMANO, used at 0.25-2.0 M, was similar to that of betaine in these sequencing reactions. 测序反应里加测序反应里加2 M betaine 或或0.25-2.0 M TMANO(三甲胺三甲胺-N-氧化物氧化物)能有效解决这个能有效解决这个问题。问题。28Multiplex PCR Amplification多重多重PCR扩增扩增GC-Rich fragments may be strongly unde
25、rrepresented after standard multiplex PCR amplifications. The addition of 1 M betaine and 5% (vlv) DMSO may equalize the amplification efficiency of different DNA fragments. 正常情况下正常情况下,富富GC区的多重区的多重 PCR扩增受抑制扩增受抑制. 加加 1 M betaine 和和 5% (vlv) DMSO 能够平衡能够平衡不同不同 DNA 片段的扩增效率片段的扩增效率. 29Reverse Transcriptio
26、n PCR反转录反转录PCRAddition of 2 M betaine alone, or accompanied by 0.6 M trehalose(海藻糖海藻糖), can dramatically enhance reverse transcriptase reactions. For example, in the presence of these additives, the yield of 12.5-kb cDNA fragments was increased 9-fold, as compared with reverse transcriptase (RT)-PCR
27、 amplifications without additives. 单加单加 2 M betaine alone, 或同时加或同时加 0.6 M 海藻糖海藻糖能能大大促进反转录反应大大促进反转录反应. 例如加增强剂的比不加的例如加增强剂的比不加的(RT)-PCR会使会使12.5-kb cDNA 量增加量增加 9倍倍.30Quantitative PCR 定量定量PCRInternal standards（内标）（内标） for use in quantitative PCR amplifications are designed to be very similar to the targe
28、t molecule. Target and the internal standard formed heteroduplexes, even after the first amplification cycle. Addition of 2 M betaine, 5% formamide(甲酰胺甲酰胺), and 10% DMSO to the PCR mix reduced the amount of recombinants to below a detectable level.内标与目标分子非常相似内标与目标分子非常相似,两者容易形成异源二聚体两者容易形成异源二聚体,这甚至在第一
29、轮扩增后就可以发生这甚至在第一轮扩增后就可以发生. 加入加入 2 M betaine, 5% 甲酰胺甲酰胺, 和和 10% DMSO 到到 PCR反应液里可以将异源二聚体的量减少到检测线反应液里可以将异源二聚体的量减少到检测线以下以下. 31Microarray Studies 芯片研究芯片研究 In addition to its use in enhancing amplification of GC-rich sequences, betaine proved useful in microarray studies. The addition of 1.5 M betaine to t
30、he DNA solution results in a significant reduction of the nonspecific background signal. betaine 除了能促进富除了能促进富GC序列的扩增序列的扩增, 在芯在芯片研究方面也是有用的片研究方面也是有用的. 加加 1.5 M betaine 到到 DNA 溶液里会大大削减溶液里会大大削减非特异的背景信号非特异的背景信号.32Microarray Studies 芯片研究芯片研究 Betaine reduces the evaporation(蒸发作用蒸发作用) from the DNA samples i
31、n the microtiter plates during the manufacturing of the slides. After the DNA has been applied to the glass slide, the diminished evaporation may provide enough time for the DNA to be distributed uniformly within each spot, thereby inhibiting the “doughnut effect（空（空巢）巢）” that disturbs the results.B
32、etaine 能够减少芯片制作中微量滴定板里能够减少芯片制作中微量滴定板里DNA样本的蒸发作用样本的蒸发作用,保证有充足的时间让保证有充足的时间让DNA均匀地均匀地分布到每一个点分布到每一个点, 从而抑制干扰结果的从而抑制干扰结果的“空壳效空壳效应应”.33The following procedure details the establishment of an amplification procedure for GC -rich sequences. In this example, three amplicons from two different genes containin
33、g GC-rich sequences were used. PROTOCOL 1:ESTABLISHING AN AMPLIFICATION PROCEDURE FORHIGHLY CC-RICH REGIONS草案草案1：高高GC 含量含量DNA序列扩增序列扩增步骤步骤34PROTOCOL 1:ESTABLISHING AN AMPLIFICATION PROCEDURE FORHIGHLY CC-RICH REGIONS 高高GC 含量含量DNA序列扩增序列扩增草案草案35Fragment 1 comprised the initial part of exon 1 from the h
34、uman release factor 3 (GSPT1/hRF3), a 387-bp sequence that contains two trinucleotide repeats: a (GGC) followed by an (AGC), and a total GC content of 75%. The second and third fragments come from the Klotho gene (LocusLink number for the genes Klotho 1: 9365 and GSPTL/hRF3: 2935). Fragment 2 compri
35、sed the 5 end of Klotho exon 1 (375 bp, 81% GC) and fragment 3, the 3 part of Klotho exon 1 (350 bp, 70% GC). 三个扩增子三个扩增子, ,来自两个不同的来自两个不同的富富GC GC 含量含量基因基因片段片段 1: 来自人释放因子来自人释放因子 3 (GSPT1/hRF3),含含 exon 1 起始部起始部分分,长长 387-bp,其中有两个三核苷酸重复其中有两个三核苷酸重复”GGC后后AGC”,总总GC 含量含量 75%. 片段片段 2,3:来自来自Klotho基因基因 (LocusLi
36、nk 号号 Klotho 1: 9365 and GSPTL/hRF3: 2935).片段片段 2含含 Klotho exon 1 的的 5 端端 (375 bp, 81% GC);片段片段 3含含 Klotho exon 1 的的 3 端端 (350 bp, 70% GC). 36MATERIALS 材料材料BUFFERS, SOLUTIONS溶液溶液, AND REAGENTS试剂试剂BetainedNTP solution all four dNTPs, (each containing at 25 mM)Dimethyl sulfoxide (DMSO)Tetramethylene s
37、ulfone (Sulfolane环丁砜环丁砜 )37ENZYMES AND ENZYME BUFFERSTaq polymerasePolymerase buffer (as supplied by enzyme manufacturer)NUCLEIC ACIDS AND OLIGONUCLEOTIDESHuman genomic DNA, 20 ng per amplification reaction模板与引物模板与引物人基因组 DNA38hRF3 (hGSPTL) primers:hRF3f: CCGCCTCTGTCGTCGTCGChRF3r: CCGCGCTGAGGTTCTCCCK
38、b 1 af: CTCGCAGGTAATrATrGCCAGKb lar: GATGGACGCACCCTGKb 1 cf: GTGCAGCCCGTGGTCACKb icr: GACTCAGITCCCACACTTC3940Depending on the chosen conditions, include either DMSO (5%); betaine (1 M); betaine (2 M); betaine and DMSO (1 M and 5%, respectively); betaine and DMSO (2 M and 5%, respectively); tetrameth
39、ylene sulfone (0.4 M); or no enhancers (final concentrations) in the PCR mix.条件设定条件设定: PCR反应液里加反应液里加 DMSO (5%); betaine (1 M); betaine (2 M); betaine 和 DMSO (1 M和5%); betaine 和 DMSO (1.5 M 和5%); PCR反应液里不加增强剂反应液里不加增强剂. 4142The results show that the optimal yield of the PCR product was seen with 5% DM
40、SO and 1.5 M betaine as shown in Figure 4.结果加结果加5% DMSO和和1.5 M betaine 的的PCR产物最好产物最好.43Some advice on solving the problems associated with GC-rich templates (50%GC).解决高于解决高于50%GC模板扩增问题的忠告模板扩增问题的忠告Add 1.5 M betaine and 5% (v/v) DMSO to the PCR amplification.If no effect is seen, increase the betaine
41、concentration to 2.5 M, add 10% (vlv) DMSO, and double the amount of Taq polymerase (DMSO inhibits Taq polymerase activity). PCR反应液里加反应液里加5% DMSO和和1.5 M betaine.如果效果不明显如果效果不明显,增加增加betaine浓度到浓度到 2.5 M, DMSO 到到10% (vlv),同时同时Taq polymerase翻倍翻倍 (DMSO 抑抑制制 Taq polymerase 活性活性).44Many unspecific PCR produ
42、cts.Increase the annealing temperature and add 1.5 M betaine and 5% (vlv) DMSO. If no effect is seen, increase the betaine and DMSO concentrations further.对有许多非特异扩增发生对有许多非特异扩增发生,可以提高退火温度可以提高退火温度,也可以在也可以在PCR反应液里加反应液里加5% DMSO和和1.5 M betaine,如果效果不明显如果效果不明显,再增加再增加betaine和和DMSO浓度浓度.45DISCUSSION 讨论讨论Betai
43、ne as the most successful PCR amplification enhancer of the wide spectrum of additives used to overcome constrained DNA structures, with both RNA and DNA as templates.Betaine是大多数成功的是大多数成功的PCR扩增使用的增强剂扩增使用的增强剂,可可以有效克服以有效克服 DNA和和RNA二级结构二级结构.46We found that highly GC -rich regions are very efficiently a
44、mplified in the presence of 1.52.5 M betaine, and the yield for some fragments is further increased by addition of 510% DMSO. The amplification of a fragment with 81% GC content was only possible in the presence of either 2 M betaine or 12 M betaine and 5% DMSO. 我们已发现，加入我们已发现，加入1.5-2.5 M betaine,富富 GC 区会区会有效扩增有效扩增 ,加入加入 5-10% DMSO 有些片段的扩增产有些片段的扩增产量被进一步提高量被进一步提高. 81% GC区只有在区只有在2 M betaine或或 1-2 M betaine 和和 5% DMSO存在时才可能被扩增存在时才可能被扩增. 47Reading and Writing1. Why is it difficult for DNA segments with GC-rich to be amplified?2. Do a summary about Betaine application in PCR.
