1、Molecular Tools基因技术基因技术Molecular Techniques- PCRDNA抽提聚合酶链式反应 Polymerase Chain Reaction (PCR)目标基因的检测“PCR is one of those inventions like the internet, once you have used it, you cannot quite understand how people managed before it existed”Kary Mullis, Inventor, 1983What is PCR?体外扩增特定DNA片段在一个小管子内即可将目标
2、DNA片段在很短的时间内扩增成百万个拷贝数可以有目的性的选择“目标DNA”PCR “Recipe”DNA模板（Template DNA ）引物（Primers）dNTPs扩增缓冲液 Buffer (with Mg2+)TaqDNA聚合酶（Taq DNA polymerase）Isolated from Thermus aquaticusPolymerase Chain Reaction (PCR)TAQ加热，94C，模板DNA的变性变性退火（复性）, 55 C ：引物与模板DNA单链的互补序列配对结合引物的延伸, 72CDenaturationAnnealingExtensionPCR的第一次
3、循环的第一次循环Y染色体线粒体DNAPCRHow Many Cycles?y = N*2x; where y = 最终拷贝数, N = 起始模板的数量, x = 循环数（cycle number）y = N*(1+F)x; Where F = efficiency factor of 0-1，扩增效率typically 0.7+/- End-point PCR: 琼脂糖凝胶电泳琼脂糖凝胶电泳Primer Annealing （引物退火）（引物退火）引物 人工合成的两段寡核苷酸序列一个引物与感兴趣区域一端的一条DNA模板链互补，另一个引物与感兴趣区域另一端的另一条DNA模板链互补e.g., wh
4、at sequence is complementary to AATGCTT ?TTACGAA引物可以和目的DNA的单链结合，然后在DNA聚合酶作用下进行延伸，合成新DNAAnnealing efficiency depends upon specificity and stringency引物的特异性引物的特异性=100%G, guanineA, adenineC, cytosineT, thyminePrimerTARGET H-bondsG ACIIITCIII TIIAIIAIIGIIIGIIIAGCGTaq PolymeraseAT TCGGC引物特异性引物特异性 more str
5、ingentLower temperature - less stringentMg2+Cofactor for Taq polymerase甲酰胺甲酰胺 (Formamide)Lowers the annealing temperatureIf conditions are too stringent, primer will not bind even to a 100% matchPCR ShortcomingsNot quantitative Sample concentration and DNA extractionPCR inhibitorsDNA from non-viable
6、 organisms also amplifiedPCR Variations巢式PCR（Nested PCR）For low target concentration多重PCR（Multiplex PCR）For high throughput反转录PCR（Reverse-Transcription (RT)-PCR)PCR for RNACell culture PCRCulture step first to increase specificity and verify viability/infectivity实时定量PCR (Q-PCR)Provides quantitative
7、information限制性内切酶限制性内切酶可以识别DNA的特异序列，并在识别位点或其周围切割双链DNA的一类内切酶，简称限制酶命名命名重组基因重组基因克隆技术（克隆技术（Cloning Basics）重组克隆载体克隆载体粘性末端重组克隆载体克隆载体粘性末端Typical cloning “vector” or “plasmid”“载体 ”，“质粒”“载体 ”，“质粒”Blue White Screening（蓝白斑筛选）（蓝白斑筛选）StructureFunctionFunctional genesmRNAtranscriptiontranslationproteinsExpression
8、Target: 16S rRNAWho? Cloning, SSCP, DGGEHow many?FISH, qPCR Disadvantages?Difficult to know who is doing what.Target: functional genesPotential to do what?Cloning, SSCP, DGGEHow much potential?FISH, qPCRDisadvantages?Gene may be present but inactive. Target: mRNAWhat are they doing?RT-PCR, Microarra
9、ys, differential displayDisadvantages?Microbial mRNA difficult to work with, techniques still developing.Target: proteinWhat is their final product?2-D gel electrophoresis/ mass spectrometryDisadvantages?Frontier of knowledge, techniques not developed.Oligonucleotides （寡核苷酸）（寡核苷酸）All biomolecular te
10、chniques use oligonucleotides (eg., primer, probes). An oligonucleotide is single-stranded DNA comprised of 15 to 25 bases whose sequence is complementary to a region in the target-cells RNA or DNA. Oligonucleotides can be designed so they are specific to one strain, to a group a similar strains (eg
11、, genus), to an entire domain, or to all life.Oligonucleotide Probing （探针杂交）（探针杂交）Target RNAOligonucleotide probes labeled with fluorescence or radioactive molecule.Wash to remove excess probeDetect fluorescence or radioactivityBiomolecular methods that use oligonucleotide probing:Slot blotting (条带印
12、迹 )Fluorescent in situ hybridization (FISH)Fluorescence In Situ Hybridization（荧光原位杂交）（荧光原位杂交）Use probes to hybridize DNA or RNA present in whole cells, without destroying cell structureUse epifluorescence microscopy to visualizeNo PCR employedQuantitative, can count cellsSubject to probe specificity
13、 and stringency limitationsHybridization: Specificity 100%G, guanineA, adenineC, cytosineU, uracilProbeTARGET hydrogenbondsGAC|GUC|GU|AA|UA|UG|CG|CAGCGHybridization: Specificity illuminaIlluminasequencinguseschain-terminatingnucleotides.Thefluorescent label on the terminating base can be removed to
14、leave anunblocked 3 terminus, making chain termination a reversible process.Because the reversible dye terminator nucleotides are not incorporatedefficiently, the read length of illumina method is less than 454. Also morebase-substitution errors are observed due to the use of modifiedpolymerase and
15、dye terminator nucleotides.Number of reads: illumina 454IlluminasequencingclustersofDNAmoleculesamplifiedfromindividual fragments attached randomly on the surface of a flow cell.Because of the very high densities of clusters that can be analyzed, themachine can reportedly produce more than one billi
16、on bases (1 Gb) of 75base (paired) reads in a single run.Overall advantages of next generation sequencingOvercome the restriction of cloningEnable to generate hundreds of millions of sequencing readsCan sequence target genes from multiple samples in one sequencing run-Use “bar code” tags so know fro
17、m which sample the sequence originated.Collectively, reduce the costs for DNA sequence production. Disadvantages of next generation sequencingRead length: Only obtain 300-500 bp of information per read (pyrosequencing), traditional sanger sequencing provides 500-1000 bp of informationRaw accuracy: l
18、ess accurateMetagenomicsEnvironmental samplesEnvironmental samplesDNA extractionDNA extractionPyrosequencing/illuminasequencingPyrosequencing/illuminasequencingMetagenomeanalysisMetagenomeanalysisvirulence factors, antibiotic functional and metabolic diversity of microbial communities (Proteins, vir
19、ulence factors, antibiotic Metatranscriptomics ( RNA )T-RFLP PyrosequencingExamples:T-RFLPPyrosequencingNumber of T-RFsInverse of the Simpson indexNumber of OTUsInverse of the Simpson index2.3d5.7d2.3d5.7d2.3d5.7d2.3d5.7dIron2032265.10.35.92.698611167.02.56.73.9PVC2001744.20.55.1+4.8811492276.32.68.
20、41.1Cement1431513.30.44.50.4901282205.22.04.31.2Examples:ReferencesReferencesShendure J. and Ji H. Next-generation DNA sequencing. 2008.Nature Biotechnology. 26: 1135-1145Mardis E. R. Next-Generation DNA sequencing methods. 2008. Annu.Rev. Genomics Hum. Genet. 9: 387-402Metzker M. L. Sequencing technologies the next generation. 2010.Nature Reviews: Genetics. 11: 31-46http:/
