老師分子生物學(xué)

1、Gene Manipulation&Genomic AnalysisRecombinant DNA cloning technologynnApplications of recombinant DNA technologynnGenomic analysisnnDNA sequencing1, Maxam-Gilbert sequencing2, Sanger-Coulson sequencing3, Next Generation SequencingMaxam-Gilbert sequencing (chemical cleavage method using double-st

2、randed (ds) DNA)1.Double-stranded DNA to be sequenced is labelled by attaching a radioactive phosphorus (32P) group to the 5' end.2.Using dimethyl sulphoxide and heating to 90oC, the two strands of the DNA are separated and purified3.Single-stranded sample is split into separate samples and each

3、 is treated with one of the cleavage reagents.4.If reactions have been arranged to give only one, or a few, cleavages per DNA molecule, a nested set of end-labelled DNA fragments of different lengths is produced.5.The samples are run together on a sequencing gel which separates the fragments by elec

4、trophoresis depending on their size. DNA bands in the gel are visualized by autoradiography6.The DNA sequence is read directly from the gelThe Maxam-Gilbert method of nucleotide sequence determination is based on preferential, base-specific methylation followed by chemical cleavage to generate a nes

5、ted set of end- labeled derivatives.Maxam-Gilbert sequencingBase specificityChemical used for base alterationChemical used for altered base removalChemical used for strand cleavageGDimethylsulphatePiperidinePiperidineA+GAcidAcidPiperidineC+THydrazinePiperidinePiperidineCHydrazine + alkaliPiperidineP

6、iperidineA>CAlkaliPiperidinePiperidineMaxam-Gilbert sequencingSanger-Coulson sequencing (chain termination method using single-stranded (ss) DNA)1.Sample DNA to be sequenced is clonded into M13 vector DNA to generate ssDNA.2.A short oligonucleotide primer (usually chemically synthesized and somet

7、imes labelled ) is added to the ss recombinant DNA.3.DNA polymerase is then added in the presence of 4 normal nucleotides: d-ATP, d- CTP, d-GTP and d-TTP (one or more of which are labelled with 32P) and a low concentration of 4 analogues of the normal nucleotides in separate incubation mixes.4.Compl

8、ementary strand synthesis occurs away from the primer.5.Each of the 4 mixes is run together on a sequencing gel which separates the fragments by electrophoresis depending on their size. DNA bands in the gel are visualized by autoradiography.6.The DNA sequence is read directly from the gel in a simil

9、ar way to a Maxam-Gilbert sequencing gel.Structures of ribonucleoside triphosphate (NTP), deoxyribonucleoside triphosphate (dNTP), and dideoxyribonucleoside triphosphate (ddNTP)Sanger-Coulson sequencingSanger-Coulson sequencingDideoxy DNA sequencing of a theoretical DNA fragmentDideoxy Sequencing of

10、 DNAAUTOMATED DNA SEQUENCINGIt is based on the Sanger-Coulson chain termination method but the 4 different dideoxy nucleotides (ddA, ddC, ddG and ddT) are fluorescently labelled.4 different fluorophores are used, all 4 reactions can be run in the same tube.Genome SequencingChromosome WalkingGenome s

11、hotgun sequencingStrandOriginalSequenceAGCATGCTGCAGTCATGCTTAGGCTA AGCATGCTGCAGTCATGCT-TAGGCTAAGCATG-CTGCAGTCATGCTTAGGCTAAGCATGCTGCAGTCATGCTTAGGCTAFirst shotgun sequenceSecond shotgun sequenceReconstructionGenome shotgun sequencingHUMAN GENOME PROJECT (HGP)Hierarchical Shotgun (“map-based”, “BAC-base

12、d”, “clone-by-clone”)1990CELERA GENOMICS 1998Whole Genome ShotgunNext Generation Sequencing1. Roche 454 pyrosequencing2. Illumina (Solexa) sequencing3. SOLiD sequencingRoche 454 Sequencing,based on sequencing-by-synthesisNucleotides are flowed sequentially in a fixed order across the PicoTiterPlate

13、device during a sequencing run.During the nucleotide flow, hundreds of thousands of beads each carrying millions of copies of a unique single-stranded DNA molecule are sequenced in parallel.If a nucleotide complementary to the template strand is flowed into a well, the polymerase extends the existin

14、g DNA strand by adding nucelotide(s).Addition of one (or more) nucleotide(s) results in a reaction that generates a light signal that is recorded by the CCD camera in the instrument.The signal strength is proportional to the number of nucleotides incorporated in a single nucelotide flow.ssDNA templa

15、te is hybridized to a sequencing primer and incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase and apyrase, and with the substrates adenosine 5´ phosphosulfate (APS) and luciferin.Addition of one of the four dNTPs, DNA polymerase incorporates the correct, complementary dNTPs

16、 onto the template. This incorporation releases pyrophosphate (PPi) stoichiometrically.ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5´ phosphosulfate.This ATP acts as fuel to the luciferase- mediated conversion of luciferin to oxyluciferin that generates visib

17、le light in amounts that are proportional to the amount of ATP. The light produced in the luciferase- catalyzed reaction is detected by a camera.Unincorporated nucleotides and ATP are degraded by the apyrase, and the reaction can restart with another nucleotide.Roche 454 SequencingIllumina SolexaIll

18、umina SolexaIllumina SolexaIllumina SolexaIllumina SolexaLife/APGs SOLiDThird Generation Sequencing第三代是單技術(shù)是為了解決第二的缺點(diǎn)而開發(fā)的，它的根本特點(diǎn)，不需要任何PCR的過程，這是為了能有效避免因PCR偏向性而導(dǎo)致的系統(tǒng)錯(cuò)誤，同時(shí)提高讀長，并要保持二代技術(shù)的高通量，低成 本的優(yōu)點(diǎn)。1.基于電信號(hào)納米單技術(shù)。當(dāng)DNA堿基通過納米，它們使電荷發(fā)生變化，從而短暫地影響流過納米孔的電流強(qiáng)度（每種堿基所影響的電流變化幅度是不同的），靈敏的電子過的堿基。2.PacBio SMRT技術(shù)。在SMRTCell

19、(單檢測到這些變化從而鑒定所通實(shí)時(shí)反應(yīng)孔)中有許多圓形納米小孔, 即 ZMW(零模波導(dǎo)孔),外徑 100多納米,比檢測激光波長小(數(shù)百納米),激光從底部打上去后不能穿透小孔進(jìn)入上方溶液區(qū),能量被限制在一個(gè) 小范圍(體積20X 10-21 L)里,正好足夠覆蓋需要檢測的部分,使得信號(hào)僅來自這個(gè)小反應(yīng)區(qū)域, 景降到最低。中,從而實(shí)現(xiàn)將背過多游離核苷酸單體依然留在3.Ion Torrent。該技術(shù)使用了一種布滿小孔的高密度半導(dǎo)體， 一個(gè)小反應(yīng)池。當(dāng)DNA聚合酶把核苷酸聚合到延伸中的DNA鏈上孔就是一個(gè)出一個(gè)氫離子，反應(yīng)池中的PH發(fā)生改變，位于池下的離子感受時(shí)，會(huì)器感受到H+離子信號(hào)，H+離子信號(hào)再直

20、接轉(zhuǎn)化為數(shù)字信號(hào)，從而讀出DNA序列。Polymerase Chain Reaction (PCR)polymerase chain reaction (PCR)polymerase chain reaction (PCR)polymerase chain reaction (PCR)Diagnostic Applications of PCR detecting pathogens using genome-specific primer pairs screening specific genes for unknown mutations genotyping using known S

21、TS markersSubcloning DNA targets using PCR T/A Cloning Restriction Site Addition Blunt-end LigationPCR-mediated in vitro mutagenesisGeneration of DNA probesAmplification of differentially-expressed gene sequences Differential display reverse transcriptase PCR (DDRT-PCR) Suppression subtraction hybri

22、dization (SSH) Amplification of cell-specific transcripts using RT-PCRThe use of PCR in forensic science.Subcloning DNA targets using PCRNNN-EcoRIEcoRI-NNNEcoRI digestionClone to EcoRI sitePCR-mediated mutagenesis AATTCCP1 AAP3 AATTTTGGP2P4P1CCGGP4 CCGGPCR-mediated deletionP1P3P2P4P1P4Single strand

23、PCR to generate a DNA probePrimer 1 : Primer 2 = 10 : 1Primer 1Primer 2PCR to generate a template for in vitro transcriptionT7 promoterT7 polymeraseTo apply PCR to the study of RNA, the RNA sample mustfirst be reverse transcribed to cDNA to provide the necessary DNA template for the thermostable pol

24、ymerase. This process is called reverse transcription (RT), hence the name RT-PCR.Schematic diagram of two 5-RACE methods.Schematic diagram of a typical 3-RACE protocol.Quantitative Real-Time PCRQuantitative Real-Time PCRb TaqMan probeTaqman-based SNP genotypingQuantitative Real-Time PCRa representa

25、tive amplification plotQuantitative Real-Time PCRHigh-throughput droplet PCRssDNA template is hybridized to a sequencing primer and incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase and apyrase, and with the substrates adenosine 5´ phosphosulfate (APS) and luciferin.Analysis of Biological Processe