6高GC-含量DNA的PCR汇总课件.ppt
- 【下载声明】
1. 本站全部试题类文档,若标题没写含答案,则无答案;标题注明含答案的文档,主观题也可能无答案。请谨慎下单,一旦售出,不予退换。
2. 本站全部PPT文档均不含视频和音频,PPT中出现的音频或视频标识(或文字)仅表示流程,实际无音频或视频文件。请谨慎下单,一旦售出,不予退换。
3. 本页资料《6高GC-含量DNA的PCR汇总课件.ppt》由用户(三亚风情)主动上传,其收益全归该用户。163文库仅提供信息存储空间,仅对该用户上传内容的表现方式做保护处理,对上传内容本身不做任何修改或编辑。 若此文所含内容侵犯了您的版权或隐私,请立即通知163文库(点击联系客服),我们立即给予删除!
4. 请根据预览情况,自愿下载本文。本站不保证下载资源的准确性、安全性和完整性, 同时也不承担用户因使用这些下载资源对自己和他人造成任何形式的伤害或损失。
5. 本站所有资源如无特殊说明,都需要本地电脑安装OFFICE2007及以上版本和PDF阅读器,压缩文件请下载最新的WinRAR软件解压。
- 配套讲稿:
如PPT文件的首页显示word图标,表示该PPT已包含配套word讲稿。双击word图标可打开word文档。
- 特殊限制:
部分文档作品中含有的国旗、国徽等图片,仅作为作品整体效果示例展示,禁止商用。设计者仅对作品中独创性部分享有著作权。
- 关 键 词:
- GC 含量 DNA PCR 汇总 课件
- 资源描述:
-
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
展开阅读全文