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bf67caa127387ad6fc7f512e27769ea1.ppt
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Скачать презентацию 基础医学中心实验室 占地面积 400平方米 分子生物学技术平台 细胞生物学平台
Скачать презентацию 基础医学中心实验室 占地面积 400平方米 分子生物学技术平台 细胞生物学平台
基础医学中心实验室 占地面积 400平方米： • 分子生物学技术平台 • 细胞生物学平台 • 免疫学平台 • 形态学技术平台
Principle & Practice of PCR Dept. Histolgy & Embryology 谢小薰 2
Basic knowledge about PCR Part I 3
What is PCR (聚合酶链反应)? Polymerase Chain Reaction A method to amplify selected sections of DNA in vitro (选择性体外扩增DNA片段的方法). A process which “Amplifies” or “Copies” a piece of DNA repeatedly until there is an amount which is great enough to observe visually. 4
How did the PCR develop? * 1930 年正式提出核酸的概念 * 1953 年 Watson & Crick 提出DNA双螺旋 结构及半保留复制模型 * 1985 年 Mullis 发明了PCR 5
History of PCR • an idea by Kary Mullis； • Bachelors degree in Chemistry, 1966, Georgia Institute of Technology； • Ph. D in Biochemistry, in 1972, in California University； • post-doctoral research, 7 years, in University of Kansas Medical School; • a scientist for the Cetus Corporation of Emeryville, California. 6
• The idea was not the product of a painstaking laboratory discipline, but was conceived while cruising on highway. • For this brilliant idea, a $10, 000 bonus from Cetus. • received Nobel Prize for chemistry in 1993. • refused to team up with biotechnology industry. • Currently, he consults and lectures around the world about biotechnology or the development of the scientific method. http: //www. karymullis. com 7
"Dancing Naked in the Mind Field" In the book he writes with passion and humor about a wide range of subjects: - from the scientific method to parapsychology, - from poisonous spiders to the HIV virus and AIDS, - from global warming to astrology, - from the O. J. Simpson trial to how you can turn a light bulb on with your mind.
What do we need for doing the PCR? Basic Components: * Template (模板)：DNA, c. DNA; * Primer (引物): oligonucleotides ( 寡核苷酸); * d. NTP (三磷酸脱氧核苷酸) * Taq Polymerase (聚合酶) * Buffer (缓冲液 ) 9
• Deoxyribonucleic Acid (DNA) • Complementary DNA (c. DNA): single-stranded DNA made from a messenger RNA template with reverse transcriptase (逆转录酶）. 10
• Short preexisting oligonucleotides ( 寡核苷酸 ) to which new deoxyribonucleotides can be added by DNA polymerase (聚合酶). • Two primers have to flag the beginning and end of the gene to be copied and are complementary to it. • 待扩增 DNA片段两翼互补的寡核苷酸 5′ Primer 1 3′ 3′ 5′ 6 Primer 2 11
Deoxynucleotide triphosohates （d. NTP）: Adenine （腺嘌呤） Thymidine （胸腺嘧啶） Cytosine （胞嘧啶） Guanine（鸟嘌呤） 12
Taq Polymerase (Taq 聚合酶) • An enzyme from Thermus aquaticus that survives in hot springs in Yellowstone National Park ； • Original report was published in 1976. • Roughly 10 years later, PCR was developed 13
• a thermostable enzyme; • catalyzing the polymerization of nucleotides into duplex DNA in the 5´->3´ direction; • 5'– 3' DNA polymerase; • lacking 3'– 5' exonuclease activity; • having error rate; • leaves extra single adenine nucleotide overhangs at the 3’end of PCR products.
How did the PCR work? d-a-e cycle. 变性 退火: time? /℃ ? 延伸 15
Denaturation / 变性（First step） • opening double-strand DNA at 94 °C; • Making single-strand DNA accessible to primers. • temperature： 94 °~ 95° C • time： 2~5 mins ( initial step ), 30 -60 s (each cycle）. 16
• Use a longer time or higher denaturing temperature for GC-rich template DNA. • unnecessarily long denaturation times will decrease the activity of Taq DNA Polymerase.
Annealing / 退火 (Second step) a process annealing primers to the template; • annealing temperature（Ta）: 55°~ 65°C *based on the melting temperature （Tm） • annealing time: 30~60 s 18
Extension / 延伸 (Third step) • Polymerase binds and extends a complementary DNA strand from each primer. • temperature: 72 o C ( Taq working best) • time: 2 min (each cycle) / 6 min (final extension) 19
Initial denaturing Denaturing Annealing 30 -35 cycles Extension Final Extension 20
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What is the PCR used for? • analysis of entire genomes (基因组); • gene expression (RT-PCR) • analysis mutagenesis in vitro (体外）; • to detect and quantify minute amounts of a pathogen(病原体)… 24
Disadvantages of PCR • Need to have DNA sequence information • Size limitation of product • Infidelity (失真) of DNA replication *Reducing error rate to use Pfu polymerase 25
The Type of PCR and application Part II 26
The PCR Type • Standard PCR • RT- PCR • Quantitative PCR （定量PCR） • Mathylation PCR • Multiplex PCR (多重PCR） 27
Standard PCR 变性 退火: time? /℃ ? 延伸 28
Reverse-Transcription Polymerase Chain Reaction (RT-PCR) 29
What is RT-PCR? • It is stands for reverse transcription (逆转 录) and polymerase chain reaction (RTPCR). • It is the PCR amplification of a reverse transcription product.
Reverse Transcriptase ( RT, 逆转录酶) • a common name for an enzyme that functions as a RNA-dependent DNA polymerase. • cloned and expressed in E. Coli. in 1985 • modified from: - Moloney Murine Leukemia Virus (M-MLV); - Avian Myeloblastosis Virus (AMV). • Function: - catalyze (催化) the conversion of RNA to DNA;
Activities of Reverse transcriptase • DNA polymerase activity: transcribing both single-stranded RNA / DNA templates with requirement of primer; • RNase H activity: degrading the RNA from RNA-DNA hybrids,
Procedures for RT-PCR： 1. Extraction of total RNA（提取总RNA） 2. Synthesis of c. DNA 3. Quality test of c. DNA synthesized （amplification of house keeping gene) 4. Amplification of target gene with c. DNA 33
-80 ℃保存的组织 / 细胞 提取RNA 合成 c. DNA gene specific pr. d. T 18 pr. RT-PCR random hexamer pr. 34
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d. T 18引物 37
TWO STEP vs ONE STEP RT-PCR • two step is generally more reliable as you have the option of adjusting your PCR parameters. • one step is quicker, less work but also less reliable because your PCR reaction conditions are restricted to the same conditions 38
Real- time Quantitative PCR （实时定量PCR） 39
Real-Time quantitative PCR (实时定量PCR) • 1992年 由一位日本人第一次提出; • 目的：实时地看到PCR反应的整个过程。 40
Working principle 2. Laser excites fluorescent （荧光） 1. Taq Polymerase 2. “finds”the primer …. . . T F Q …. . . DNA 3. Fluorescence （荧光素） absorbed by quencher molecule （淬灭基团） 4. Detector registers “no reaction”. 41
Working principle Fluorescence （荧光素） . …. …. . T . F …. Quencher molecule （淬灭基团） Q 5. Primer is extended 6. by Taq Polymerase 42
Working principle 6. Extension continues until the Polymerase Meets fluorescent molecule. . . …. . . T F Q …. . . … 43
7. Taq polymerase breaks down the probe. The fluorescece and quencher molecule become separated. F Q . . …. . . T . . . … … 44
Bisulfite Conversion Based PCR 45
What is methylation (甲基化）? • an enzyme-mediated chemical modification (修饰). • adds methyl (CH 3) groups at selected sites on proteins, DNA and RNA. 46
DNA methylation only affects the Cytosine (胞嘧啶) when it is followed by a Guanine (鸟嘌呤)，that is Cp. G island . 胞嘧啶 DNA methyltransferase （ DNA甲基转移酶） 47
Cp. G island • Region of genomic DNA rich in the dinucleotide C-G. Methylation of the C is maintained through cell divisions, and profoundly affects the degree of transcription of the nearby genes.
The function of DNA methylation * remains incompletely understood. * has been proposed in multiple systems, including control : - gene expression - chromosomal integrity （染色体完整性） 49
methods used for DNA methylation at methods used in DNA methylation? two major types : * methylation-sensitive restriction enzymes （甲基化敏感性限制性酶） * Methylation Sensitive PCR (MSP): sodium bisulfite unmethylated C U (uracil, 尿嘧啶） （un. MC） sodium bisulfite methylated C methylated C 50
Methylation Sensitive PCR (MSP): * Primers are designed to amplify: - methylated DNA - unmethylated DNA * a simple gel electrophoresis (凝胶电泳） will yield the answer. 51
Design Primers for Methylation PCR http: //www. urogene. org/methprimer • free online program for designing primers for methylation specific PCR (MSP) and bisulfite sequencing PCR. If you have questions, following website may help you. http: //micro. nwfsc. noaa. gov/forums/ 52
PCR-SSCP (Single-Strand Conformation Polymorphism， SSCP单链构象多态性) 原理 单链DNA片段呈复杂的空间折叠构象，这种立体结 构主要是由其内部碱基配对等分子内相互作用力来 维持的，当有一个碱基发生改变时，会或多或少地 影响其空间构象，使构象发生改变，空间构象有差 异的单链DNA分子在聚丙烯酰胺凝胶中受排阻大小 不同. 因此，通过非变性聚丙烯酰胺凝胶电泳 (PAGE)，可以非常 敏锐地将构象上有差异的分子 分离开. 作者称该方法为单链构象多态性 53
基本过程: ①PCR扩增靶DNA; ②将特异的PCR扩增产物变性，而后快速复性，使之 成为具有一定空间结构的单链DNA分子; ③将适量的单链DNA进行非变性聚丙烯酰胺凝胶电泳 ; ④最后通过放射性自显影、银染或溴化乙锭显色分 析结果. 若发现单链DNA带迁移率与正常对照的相比 发生改变，就可以判定该链构 象发生改变，进而推 断该DNA片段中有碱基突变. 54
Principle for Primer Design Part III 55
• Short oligonucleotides (寡核苷酸)to which the new DNA can be added by Taq polymerase ( 聚合酶). • Two primers have to flag the beginning and end of the gene to be copied and are complementary to it. • 待扩增 DNA片段两翼互补的寡核苷酸 5′ Primer 1 3′ 3′ 5′ Primer 2 56
Points for designing primer: • primer length: 18 -24 Nt • Tm and Ta • Specificity 57
Melting temperature (Tm): 在一定盐浓度条件下，50% oligonucleotides (寡核 苷酸)双链解链的温度。 Tm = 2 ( A+T ) + 4 ( G+C ) Annealing temperature (Ta, 退火温度): Primer与Template(模板)结合的温度 Ta = Tm – 5 *** Tm of both primers should be similar. 58
引物长度与 Tm 值的关系 59
Primer Specificity (引物特异性) * Primer length: length↑, Specificity ↑, but Ta↑； * Annealing temperature: Ta↑, Specificity ↑； * Self sequence (自身序列)： • Complementary Primer Sequences • G/C content : 50 -60% • 3’-end Sequence - C/G, CG/GC 结尾，primer结合稳定性↑； -避免任何修饰； 60
Complementary Primer Sequences （互补引物序列） • absolutely no intra-primer homology (同源） beyond 3 base pairs. • no inter-primer homology, to avoid formation of primer dimer(引物二聚). 5'-ACGATTCATCGGACAATGC-3' 3'-CGAAAGAGGCTACTTAGCA-5' 61
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Forward primer 正向引物 5′ 3′ 基因序列 3′ 5′ Reverse primer 反向引物 65
• primer length • Tm and Ta • Specificity - Primer length -Ta - Self sequence
Important points for using primer： • Sequences are always written from 5' to 3‘; • checked for similarities with gene sequences in Genbank: www. ncbi. nlm. gov/blast • Ta 值的实际确定. 67
1 2 3 4 Lane 1: 58 ℃ Lane 2: 60 ℃ Lane 3: 62 ℃ Lane 4: 64 ℃ 引物Ta的实际测定 68
Are there programs available for designing PCR primers? a bunch of good PCR primer design programs on the web 69
Primer Design on the Web – Primer 3 at the MIT Whitehead Institute http: //www. genome. wi. mit. edu/cgibin/primer 3 http: //www. hto. usc. edu/software/procrustes/cassandra/cass_frm. http: //bibiserv. Tech. Fak. Uni-Bielefeld. DE/genefisher/ – Xprimer at the Virtual Genome Center, Univ. Minnesota Medical School http: //alces. med. umn. edu/rawprimer. html
• Primer. Bank is a public resource for PCR primers. These primers are designed for gene expression detection or quantification ( real-time PCR ). • Several ways to search for primers in the Primer. Bank : by Gen. Bank Accession, NCBI protein accession, Locus. Link ID, Primer. Bank ID or Keyword (gene description). • Primer. Bank contains about 180, 000 primers covering most known human and mouse genes. 71
- PCR Primers For Gene Expression Detection or Quantification Search for sp 32 72
The following matches are found for Keyword (All Species): "SP 32" Gene Descriptions: Gen. Bank Accession NCBI Protein Accession NM_032489 NP_115878 Species Human c DNA Length 1632 Gene Description proacrosin binding protein sp 32 precursor [Homo sapiens]. 73
Primer Pair Descriptions: Primer. Bank ID 17999524 a 1 Amplicon Size 112 Sequence (5' -> 3') Length Tm Location Forward Primer TCCCTCACTCCTGAAGGTG 19 59. 9 27 -45 Reverse Primer GAAGCGTTCGTATTCGGTAGG 21 60. 6 138 -118 Location in Coding Sequence (primers and amplicon highlighted) 74
1 atgaggaagc cagccgctgg cttccc tcactcctga aggtgctgct cctgcctctg 61 gcacctgccg cagcccagga ttcgactcag gcctccactc caggcagccctctcct 121 accgaatacg aacgcttctt cgcactgctg actccaacct ggaaggcaga gactacctgc 81 cgtctccgtg caacccacgg ctgccggaat cccacactcg tccagctgga ccaatatgaa 241 aaccacggct tagtgcccga tggtgctgtc tgctccaacc tcccttatgc ctcctggttt 301 gagtctttct gccagttcactaccgt tgctccaacc acgtctacta tgccaagaga 361 gtcctgtgtt cccagt ctctattctc tcacctaaca ctctcaagga gatagaagct 421 tcagctgaag tctcacccac cacgatgacc tcccccatct caccccactt cacagtgaca 481 gaacgccaga ccttccagcc ctggcctgag aggctcagca acaacgtgga agagctccta 541 caatcctcct tgtccctggg aggccaggag caagcgccag agcacaagca ggagcaagga … 75
free online software for MSP (methylationspecific PCR) primer design. http: //www. urogene. org/methprimer/ http: //www. mdanderson. org/departments/methylation 76
Meth. Primer - Design Primers for Methylation PCRs Home Protocols Resources FAQ Help Welcome to Meth. Primer • Meth. Primer is a program for designing bisulfite-conversionbased Methylation PCR Primer. Currently, it can design primers for two types of methylation mapping PCRs: 1) Methylation-Specific PCR (MSP) 2) Bisulfite-Sequencing PCR (BSP) or Bisulfite- Restriction PCR. • Input Sequence: DNA sequences in any format. No editing is required before input. • Output: Meth. Primer returns results in both text and graphic view. Go to Meth. Primer 77
some features of Meth. Primer : Cp. G island prediction Design primer for bisulfite sequence PCR (BSP). Design primer for methylation-specific PCR (MSP) 78
free online software for q. RT-PCR primer design http: //medgen. ugent. be/rtprimerdb/ This is a public database holding real time PCR primers and probes for popular chemistries (SYBR Green I, Taqman, Hybridisation Probes, Molecular Beacon) to prevent time-consuming primer design and experimental optimisation. 79
Key points for designed Primer of RT-PCR 1. Location of the primers； 2. Confirmation of amplifying target: pseudogene(假基因) ? DNA? c. DNA? 3. Primer choice for unknown gene. 80
Exon（外显子）: sequences that will be expressed as amino acids in protein. Intron（内含子）: intervening sequences between exons, not expressed in protein. 81
1. primers in exons on both sides of an intron 2. primers span exon/exon boundaries on the m. RNA 82
P 1 P 3 P 2 P 1 P 3 Exon 1 Exon 2 c. DNA synthesis Exon 3 P 2 83
M 1 M 2 1 32 43 4 84
Pseudogene (假基因）: Non-functional DNA sequences that are very similar to the sequences of known genes. 85
c. DNA 86
“Design and testing of -Actin primers for RT-PCR that do not co-amplify processed pseudogenes” Bio. Techniques 23: 456 -460, 1997 作者报道了检测 Strategene 和 CLONTECH 公 司及一些发表的文章中所用的 -Actin引物的 RT-PCR 结果 87
检测未知基因表达时primer的选用 * 设计多对组合引物。 * 引物最好位于编码区 5`端 300 -400 bp范围。 * 在同样条件下，用DNA和 c. DNA检测引物。 88
c. DNA Neg. DNA c. DNA 89
Lab Practice Part IV 90
• Lab security fire chemical Contamination • Lab facility 91
Avoiding Contamination in PCR Laboratory facilities * Separated working places for: -Template preparation before PCR - Setting up PCR reactions - Post-PCR analysis * Use DNase and RNase free PCR tubes * Special pipette tips and pipettes used only for PCR 92
Avoiding Contamination in PCR Sample handling * Always wear fresh gloves; * Always use new and / or sterilized glassware, and plastic ware; * Always have your own set of PCR reagents and solutions; * Always include control reactions - negative control - positive control 93
Pipette, Pipette Stand Pipette tip
Balances centrifuge 95
Eppendorf Racks 96
Biological Hoods 97
Tissue homogenizer（ 组织匀浆仪） 98
Equipments for Agarose Gel Electrophoresis Sample comb Gel casting tray power supply electrophoresis chamber 99
Lab Practice: • Total RNA exaction • Gel electrophoretic analysis of RNA • Spectrophotometric analysis of RNA 100
80%r. RNA: very stable, combines with proteins to form ribosomes (核糖体） Total RNA 15%t. RNA: stable, carries amino acids to ribosomes <5% m. RNA: very unstable, codes for protein 101
RNA WORK • is not fun !!! • RNases are EVERYWHERE ! • RNases are unharmed by autoclave (高压灭菌器） 102
Total RNA Isolation (Guanidine-based isolation) Equipment and reagents • RNase free glassware and plastic ware • microcentrifuge (微型离心机） • Lysis buffer (裂解液） • 2 M Na. OAC （p. H 4） • saturated Phenol (饱和酚, p. H 4 4. 5） • Chloroform(氯仿 ) • Isoproponal (异丙醇 ) • 75%~80 % ethanol ( Et. OH, 乙醇) • DEPC-treated H 2 O 103
DEPC ( Diethylpyrocarbonate ) • a very useful reagent for making buffers and equipment RNase free • a suspected carcinogen （致癌物质） • reacts with histidine（组氨酸）residues of proteins and will inactivate RNases. • also reacts with RNA • DEPC breaks down to CO 2 and ethanol (乙醇) • autoclave for 15 min to remove residual DEPC. 104
Gel box • Cleaning with 0. 5% SDS. • Rinsing with water, dried with ethanol. • Filling with 3% hydrogen peroxide (10 min). • Rinsing thoroughly with Rnase free d. H 2 O. 105
Tissue homogenizing（匀浆） Exaction with phenol（酚） / chloroform（氯仿） Centrifuge at 4°C 取上清液 (supernatant) RNA precipitation in Isopropanol ( 异丙醇 ) Centrifuge at 4°C Wash RNA pillet in Et. OH Save and dry RNA pillet Resuspend RNA pillet in H 2 O 106
Phenol （酚） • is used for deproteinization of nucleic acids. • Most proteins are more soluble in phenol than in the aqueous phase (水相）. Phenol Homogenized tissue 107
Phenol （酚） -upper aqueous phase contains nucleic acids. -lower phase is organic phase contained protein； Phase partitioning of nucleic acids is p. H dependent p. H 4 -6, DNA in the organic phase and interface, RNA in the aqueous phase. 108
Chloroform （氯仿） • increase efficiency of nucleic acid extractions. • denature proteins aiding separation of nucleic acid from protein. organic phase containing DNA aqueous phase (水相） containing RNA protein 109
Chloroform （氯仿） • increase efficiency of nucleic acid extractions. • denature proteins aiding separation of nucleic acid from protein. organic phase containing DNA aqueous phase (水相） containing RNA protein 110
Method to Check RNA Integrity Agarose Gel stained with Et. Br • Denaturing gel（变性胶） Intact total RNAs have sharp, clear 28 S and 18 S r. RNA bands 28 S: 18 S =2: 1 • Native (non-denaturing) gel - secondary structure of RNA alters its migration pattern - bands are not as sharp 111
Spectrophotometry （分光光度测定法） • Water used for spectrophotometric measurement of RNA should have a p. H of > 7. 5 • Acidic p. H affects UV absorption spectrum of RNA • significantly decreases the A 260/A 280 • Adjust water to a slightly alkaline p. H by adding concentrated Na 2 HPO 4 solution to a final concentration of 1 m. M 112
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What does RNA use? • to synthesis c. DNA * c. DNA library construction * RT-PCR • Northern blot 114
Principles of Gel Electrophoresis （凝胶电泳原理） 115
* a most widely-used technique * a procedure for separating a mixture of molecules through a gel in an electrical field. * charged molecules in an electric field migrate toward according to their charge. 116
Two types of gel (胶）”. - agarose (琼脂糖) - polyacrylamide (聚丙烯酰胺) 117
Agarose Gels (琼脂糖凝胶) • a polysaccharide (多聚糖) extracted from seaweed. • a large range of separation, but relatively low resolving power (分辨能力). • By varying the concentration of agarose, fragments of DNA from about 200 to 50, 000 bp can be separated using standard electrophoretic techniques. 118
DNA solutions are loaded in a well in the gel. 119
The gel acts as a sieve（筛） for DNA molecules. Large molecules have difficulty getting through the holes. Small molecules move easily through the holes。 120
Molecular weight markers are usually a mixture of DNAs with known molecular weights Molecular weight markers are used to estimate the sizes of DNA fragments in your DNA sample 121
Preparation of argose gel First is to prepare a tray to hold the gel. The ends of the tray are taped. 122
The gel comb is placed in the tray. 123
*Agarose powder is mixed with a buffer solution； * Solution is heated until the agarose is dissolved. ＊to pour agarose solution into the tray and allowed to cool. 124
electrophoresis box is filled with buffer, covering the gel. DNA are mixed with a "loading dye". The loading dye allows you to see the DNA as you load it and contains glycerol or sucrose to make the DNA sample heavy so that it will sink to the bottom of the well. 125
* Electrodes（电极） are attached to a power supply. Electrical current is applied. * dye marker indicates that DNA fragments moved through the gel, 126
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DNA is playdough in our hand! 128
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bf67caa127387ad6fc7f512e27769ea1.ppt