资源描述
1Preparation, analysis and manipulation of nucleic acids and proteins2Hybridization-the base-pairing characteristics of DNA and RNADNA cloning- DNA polymerase, restriction endonucleases and DNA ligasePCR-Thermophilic DNA polymeraseThe methods depend upon, and were developed from, an understanding of the properties of biological macromolecules themselves.3CHAPTER19: Techniques of Molecular Biology by Electrophoresis (电泳分离) by Restriction endonuclease (限制性内切酶切割) by Hybridization (杂交鉴定) by PCR (PCR扩增)5. DNA Cloning and gene expression (DNA克隆和基因表达)6. Genome sequence & analysis (基因组序列和分析) 1. Gel electrophoresis4Topic 1 Nucleic acids-separationseparates DNA and RNA molecules according to size, shape and topological properties.1. DNA and RNA molecules are negatively charged, thus move in the gel matrix (胶支持物) toward the positive pole (正电极). are separated according to . The large DNA molecules move slower than the small molecules. The mobility of is affected by their topological structures. The mobility of the same molecular weight DNA molecule with different shapes is: supercoiled (超螺旋) linear (线性) nicked or relaxed (缺刻或松散) 5Fig 21-1: DNA is separated by gel electrophoresislargemoderate small6Agarose (琼脂糖): (1)a much less resolving power than polyacrylamide, (2)but can separate DNA molecules of up to tens of kb1 kb0.5 kb2 kb3 kb4 kbDNA can be visualized by staining the gel with fluorescent dyes, such as ethidium bromide Gel matrix (胶支持物)7Polyacrylamide(1)has high resolving capability, and can resolve DNA that differ from each other as little as a single base pair/nucleotide.(2)but can only separate DNA over a narrow size range (1 to a few hundred bp).8(1)RNA have a uniform negative charge as DNA does. (2)RNA is single-stranded and have extensive secondary and tertiary structure, which significantly influences their electrophoretic mobility.(3)RNA can be treated with reagent such as glyoxal (乙二醛) to prevent RNA base pairing, so that its mobility correlates with the molecular weight Electrophoresis is also used to separate RNAs910nWhy use endonucleases?-To break large DNA molecules into manageable fragments.Nucleic acids-Restriction digestionare the nucleases that DNA at particular sites by the of specific sequences. RE used in molecular biology typically recognize short (4-8bp) target sequences that are usually palindromic (回文结构), and cut at a defined sequence within those sequences. e.g. EcoRI115.GAATTC.3 .CTTAAG.12the 1st such enzyme foundEscherichia coli Species categoryR13strainHow to name a restriction endonuclease?EcoRIThe random occurrence of the hexameric (The random occurrence of the hexameric (六核六核苷酸的苷酸的) ) sequence: sequence: 1/4096 (4 1/4096 (4-6-6=1/4=1/46 6) )13How to estimate the frequency of the RE in a DNA molecule or genome?What are the frequencies if the recognition What are the frequencies if the recognition sequences are four (tetrameric) and eight sequences are four (tetrameric) and eight (octameric) nucleotides? (octameric) nucleotides? (1) Restriction enzymes differ in the : target sites are different.(2) Restriction enzymes differ in , and thus the frequencies differ.(3) Restriction enzymes differ in : blunt/flush ends (平末端平末端), sticky/staggered ends (粘性末端粘性末端).(4) Restriction enzymes differ in the .1415sticky ends(粘性末端) blunt ends(平末端) Fig 21-4 Recognition sequences and cut sites of various endonucleases16Fig 21-5 Cleavage of an EcoRI site. The 5 protruding ends are said to be “sticky” because they readily anneal through base-pairing to DNA molecules cut with the same enzyme17CHAPTER19: Techniques of Molecular Biology by Electrophoresis (电泳分离) by Restriction endonuclease (限制性内切酶切割)3. Identification by Hybridization (杂交鉴定杂交鉴定) by PCR (PCR扩增)5. DNA Cloning and gene expression (DNA克隆和基因表达)6. Genome sequence & analysis (基因组序列和分析) 3-1. DNA hybridization can be used to identify specific DNA moleculesHybridization: Hybridization: the process of base-pairing between complementary ssDNA or RNA from two different sources.18Probe (Probe (探针探针) )A labeled, defined sequence used to search mixtures of nucleic acids for molecules containing a complementary sequence. 19: put the labels at the ends: put the labels internally Radioactive labeling: display and/or magnify the signals by . Non-radioactive labeling: display and/or magnify the signals by : antibody binding enzyme binding - substrate application (signal release)Labeling of DNA or RNA probes20gelmembraneSouthern and Northern blottingDNA on blotRNA on blot1. Genomic DNA preparation RNA preparation2.Restriction digestion -3.Denature with alkali - 4. Agarose gel electrophoresis 5. DNA blotting/transfer and fixation RNA6. Probe labeling 6. Hybridization (temperature) 7. Signal detection (X-ray film or antibody) 22Blot typeTargetProbeApplicationsSouthern DNA DNA or RNAmapping genomic clonesestimating gene numbers, etcNorthernRNADNA or RNARNA sizes and abundance (gene expression level)WesternProteinAntibodiesprotein size and abundance (gene expression level)Comparison of Southern, Northern and Western bolt hybridization4. Polymerase chain reaction (PCR) amplifies DNAs by repeated rounds of DNA replication in vitro PCR is to used to amplify a DNA sequence using a pair of primers each complementary to one end of the DNA target sequence.2324DenaturationPrimer annealing PolymerizationDenaturation (变性): The target DNA (template) is separated into two stands by heating to 95Primer annealing (退火): The temperature is reduced to around 55 to allow the primers to anneal.Polymerization (elongation, extension) (延伸): The temperature is increased to 72 for optimal polymerization step which uses up dNTPs and required Mg+.251 反转录 PCR (reverse transcription PCR, RT-PCR) 2巢式 PCR3 实时荧光定量 PCR 4 重组 PCR : 用PCR法在DNA片段上进行点突变5多重 PCR 6原位 PCR 7 不对称 PCR : 即在扩增体系中加入不同浓度的引物，而得到 单链 DNA 产物。PCR PCR 的衍生种类的衍生种类27Reverse transcriptase (RT)-PCRAAA(A)n5-CapmRNA(dT)1218 primeranneal5-CapAAA(A)n35Reverse transcriptiondNTP, RT5-CapAAA(A)n5cDNA:mRNA hybridRegularPCR28PCR mutagenesis (诱变)PCR can be used to introduce deletion and point mutations1. Two separate PCR reactions are performed. 2. One PCR amplifying the 5-portion of the insert, and the other amplifying the 3-portion of the insert.3. The point mutation/deletion mutations are located in the primers29SP6 primerT7 primerForward mutagenic primerReverse mutagenic primerFirst PCRRemove primersDenature and annealExtend to full length by DNA polymerase3330Second PCRSP6 primerT7 primerDNA cloningReal-Time PCR -Taqman probe ABI PRISM 7000 and 7700 Sequence Detection SystemReal-time detection: Each cycle produces a fluorescent signal proportional to the amount of PCR product present高分辨熔解（high-resolution melt, HRM）分析技术是近几年来在国外兴起的一种用于突变扫描和基因分型的最新遗传学分析方法。在PCR结束后直接运行高分辨熔解，即可完成对样品基因型的分析。快速、高通量准确、灵敏度高、特异性好重复性好操作简便高分辨率熔解（High-resolution melting，简称HRM） 样品分散（divide）的环节:2011年,利用油包水微滴生成技术 开发了微滴式数字PCR技术(QX100) 2013年 QX200。2012年，RainDance公司, 在高压气体驱动下，将每个标准反应体系分割成包含100万至1000万个皮升级别微滴的反应乳液数字数字PCRPCR（Digital PCR-dPCRDigital PCR-dPCR）与传统定量PCR（qPCR）技术不同，数字PCR采用绝对定量的方式，不依赖于标准曲线和参照样本，直接检测目标序列的拷贝数。极微量核酸样本检测复杂背景下稀有突变检测表达量微小差异鉴定优势明显癌症标志物稀有突变检测、致病微生物鉴定、转基因成分鉴定、NGS测序文库精确定量和结果验证应用前景越来越受到关注34CHAPTER19: Techniques of Molecular Biology by Electrophoresis (电泳分离) by Restriction endonuclease (限制性内切酶切割)3. Identification by Hybridization (杂交鉴定)4. Amplification by PCR (PCR扩增)5. DNA Cloning and gene expression (DNA克隆和基因表达克隆和基因表达)6. Genome sequence & analysis (基因组序列和分析) 5. DNA cloning, analysis and gene expression35Nucleic acids- sequencingThe ability to construct recombinant DNA molecules and maintain them in cells is called DNA cloning.361.Restriction digestion of your insert and vector using the same enzyme.2.Use ligase to join your insert and vector together.3.Transform the ligation products into E.coli. competent cells.4.Grow the cells on a plate containing tetracycline (四环素).cDNA library generationThe mRNAs are firstly reverse transcript into cDNA, and these cDNA, both full length and partial, are cloned to make the cDNA library.3737annealReverse transcriptionRegular PCR38Colony screening 1. Antibiotic screening (抗生素选择)： only the recombinant plasmids grow on the antibiotic-containing plate.2. Blue-white screening (蓝白斑选择): DNA insertion in the vector shuts down the LacZ gene expression, and turns the colony to white. 3. Colony hybridization screening (菌落杂交筛选) from a library.Screening of positive clonesTransfer to nitrocelluloseor nylon membraneDenature DNA(NaOH)Bake onto membraneProbe with 32p-labled DNA complementary to gene of interestExpose to filmSelect positive from master plateKeep master plate Screening by plaque hybridizationColony hybridization-Southern blot 40Analysis of a clone 1. Restriction mapping: digestion of the plasmid prepared from a clone with restriction enzymes to investigate if the interested DNA is inserted the recombinant plasmid.2. Sequencing the cloned DNA to see if the inserted DNA maintains the correct sequence.Analysis of DNA clones1 Kb+ ladder1. Positive clones digested with different restriction enzymesEmpty vectorRestriction mapping2.SequencingTwo ways for sequencing:Two ways for sequencing: 1. DNA molecules (radioactively labeled at 5 1. DNA molecules (radioactively labeled at 5 termini) are subjected to 4 regiments to be broken termini) are subjected to 4 regiments to be broken preferentially at Gs, Cs, Ts, As, separately. preferentially at Gs, Cs, Ts, As, separately. ( (Maxam and Gilbert chemical method, not widely Maxam and Gilbert chemical method, not widely usedused) ) 2. Chain-termination method (2. Chain-termination method (Sangers method, Sangers method, widely usedwidely used) )42Nucleic acids- sequencingMaxam and Gilbert碱基体系化学修饰 试剂化学反应断裂部位GA + GC + TCA Cdimethyl sulphate（硫酸二甲酯）Piperidine formate（哌啶 甲酸）, pH2.0hydrazine（肼，联氨NH2.NH2）hydrazine + NaCl（1.5M）90C, NaOH（1.2M）甲基化脱嘌呤打开嘧啶环打开胞嘧啶环断裂反应GG和AC和TCA和C表表6-1：Maxam-Gilbert化学降解法测序的常用化学化学降解法测序的常用化学试剂：试剂： ddNTPs are chain-terminating nucleotides: the synthesis of a DNA strand stops when a ddNTP is added to the 3 end4445The absence of 3-hydroxyl lead to the inefficiency of the nucleophilic attack on the next incoming substrate molecule.Fig 20-15 DNA sequencing gel46Four separate reactions dNTP+ ddGTP, dNTP+ ddATP dNTP+ ddCTP, dNTP+ ddTTPEach ddNTP carries a fluorescence group, allowing us to “Read” the sequence directly from the gel.“基因组基因组”-生命科学的生命科学的“元素周期表元素周期表”测序与数据分析驱动生命科学发展测序与数据分析驱动生命科学发展化学裂解测序法（Maxam and Gilbert，1977年）第二代测序技术第二代测序技术 第三代测序技术第三代测序技术 第三代测序技术第三代测序技术 测序成本不断下降测序成本不断下降Topic 2: 58CHAPTER19: Techniques of Molecular Biology1. Gel retardation assay2. Nuclease protection assayGel retardation (凝胶阻滞凝胶阻滞) A short labeled nucleic acid is mixed with a cell or nuclear extract expected to contain the binding protein. samples of labeled nucleic acid, with and without being incubated with the extract, are run on a gel. The DNA-protein complexes are shown by the presence of slowly migrating bands. 59DNA bound totwo proteinsDNA-proteincomplexBare DNAA DNA bound with more than one protein to form a larger complex.DNase I footprinting (DNase I 足迹法足迹法) Identify the actual region of sequence with which the protein interacts.615*Sequence ladder is required to determine the precise positionAATAAG62DNase footprinting （1）The protein protects DNA from attack by DNase. （2）Treat the DNA-protein complex with DNase I under mild conditions, so that an average of only one cut occur per DNA molecule. Bind proteinDNase(mild),then removeprotein and denature DNAElectrophoresis,autoradiograph
展开阅读全文