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1、1Welcome to MB Class2Molecular Biology of the Gene, 5/E - Watson et al. (2004)Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the GenomePart IV: RegulationPart V: Methods3Ch 20: Techniques of Molecular Biology Ch 21: Model OrganismsPart V: METHODS4MolecularB
2、iologyCourse Chapter 20 Techniques of Molecular BiologyPreparation, analysis and manipulation of nucleic acids and proteins5The methods depend upon, and were developed from, an understanding of the properties of biological macromolecules themselves.Hybridization-the base-pairing characteristics of D
3、NA and RNADNA cloning- DNA polymerase, restriction endonucleases and DNA ligasePCR-Thermophilic DNA polymerase6Topic 1: Nucleic acidsCHAPTER20:TechniquesofMolecularBiology1.Separation by Electrophoresis (电泳分离电泳分离) 2.Cut by Restriction endonuclease (限制性限制性内切酶切割内切酶切割)3.Identification by Hybridization
4、(杂交鉴杂交鉴定定)4.Amplification by PCR (PCR扩增扩增)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 (基因组序列基因组序列和分析和分析) 71. Gel electrophoresis separates DNA and RNA molecules according to size, shape and topological properties.Topic 1 Nucleic acids-separation81.DNA and RNA mo
5、lecules are negatively charged, thus move in the gel matrix (胶支持物胶支持物) toward the positive pole (正电极正电极).2.Linear DNA molecules2.Linear DNA molecules are separated according to sizessizes. The large DNA molecules move slower than the small molecules.3.The mobility of circular DNA moleculescircular D
6、NA molecules is affected by their topological structures. The mobility of the same molecular weight DNA molecule with different shapes is: supercoiled (超螺旋超螺旋) linear (线性线性) nicked or relaxed (缺刻或松散缺刻或松散) DNA gel mobility (DNA在胶上的迁移性在胶上的迁移性) )9Fig 21-1: DNA is separated by gel electrophoresislargemo
7、derate small10Gel matrix (胶支持物胶支持物) is an inserted, jello-like porous material that supports and allows macromolecules to move through. Gel matrix (胶支持物胶支持物) )11Agarose (琼脂糖琼脂糖): (1)a much less resolving power than polyacrylamide, (2)but can separate DNA molecules of up to tens of kb1 kb0.5 kb2 kb3
8、kb4 kbDNA can be visualized by staining the gel with fluorescent dyes, such as ethidium bromide (EB 溴化乙锭溴化乙锭)12Polyacrylamide (聚丙稀聚丙稀酰胺酰胺): (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
9、 narrow size range (1 to a few hundred bp).13(1)The electric field is applied in pulses that are oriented orthogonally (直角地直角地) to each other. (2)Separate DNA molecules according to their molecule weight, as well as to their shape and topological properties.(3)Can effectively separate DNA molecules
10、over 30-50 kb and up to several Mb in length.Pulsed-field gel electrophoresis (脉冲电泳脉冲电泳) )14Fig. 21-2 pulsed-field gel electrophoresisSwitching between two orientations: the larger the DNA is, the longer it takes to reorient15(1)RNA have a uniform negative charge as DNA does. (2)RNA is single-strand
11、ed and have extensive secondary and tertiary structure, which significantly influences their electrophoretic mobility.(3)RNA can be treated with reagent such as glyoxal (乙二醛乙二醛) to prevent RNA base pairing, so that its mobility correlates with the molecular weight Electrophoresis is also used to sep
12、arate RNAs162. Restriction endonucleases (限限制性内切酶制性内切酶) )cleave DNA molecules at particular sitesnWhy use endonucleases?-To break large DNA molecules into manageable fragments.Nucleic acids-Restriction digestion17n nRestriction endonucleases (RE) are the nucleases that cleave DNA at particular sites
13、 by the recognition of specific sequences.nRE used in molecular biology typically recognize (识别识别) short (4-8bp) target sequences that are usually palindromic (回文结构回文结构), and cut (切割切割) at a defined sequence within those sequences. e.g. EcoRI5.GAATTC.3 .CTTAAG.18the 1st such enzyme foundEscherichia
14、coli Species categoryR13strainHow to name a restriction endonuclease?EcoRI19The random occurrence of the hexameric (六核苷酸的六核苷酸的) sequence: 1/4096 (4-6=1/46)What are the frequencies if the recognition sequences are four (tetrameric) and eight (octameric) nucleotides? homeworkHow to estimate the freque
15、ncy of the RE in a DNA molecule or genome?20(1) Restriction enzymes differ in the recognition specificity: target sites are different.(2) Restriction enzymes differ in the length they recognized, and thus the frequencies differ.(3) Restriction enzymes differ in the nature of the DNA ends they genera
16、te: 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 cleavage activity.21Table 21-1 Table 21-1 Some restriction Endonucleases Some restriction Endonucleases and their recognition sequencesand their recognition sequencesEnzymeEnzymeSequenceSequenceFreq
17、uency Frequency Sau3A1Sau3A1EcoRIEcoRINotI NotI 5-GATC-35-GATC-35-GAATTC-35-GAATTC-35-GCGGCCGC-35-GCGGCCGC-30.25 kb0.25 kb4 kb4 kb65 kb65 kb22sticky ends(粘性末端粘性末端) blunt ends(平末端平末端) Fig 21-4 Recognition sequences and cut sites of various endonucleases23Fig 21-5 Cleavage of an EcoRI site. The 5 prot
18、ruding ends are said to be “sticky” because they readily anneal through base-pairing to DNA molecules cut with the same enzyme243-1. DNA hybridization can be used to identify specific DNA moleculesHybridization: the process of base-pairing between complementary ssDNA or RNA from two different source
19、s.Topic 1 Nucleic acids- DNA hybridization25A labeled, defined sequence used to search mixtures of nucleic acids for molecules containing a complementary sequence. Probe (探针)The mixture being probed has typically either been separated by size on a gel, or is distributed as a library in different col
20、onies26It can be readily located once it has found its target sequence.A probe must be labeled before applied in hybridization (Why?)27End labelingEnd labeling: put the labels at the endsUniform labelingUniform labeling: put the labels internally Radioactive labeling: display and/or magnify the sign
21、als by radioactivity. Non-radioactive labeling: display and/or magnify the signals by antigen labeling: antibody binding enzyme binding - substrate application (signal release)Labeling (标记)of DNA or RNA probes28End labeling5-end labeling using polynucleotide kinase (PNK)3-end labeling using terminal
22、 transferase30How to label one end of a DNA: Labeling at both ends by kinase, then remove one end by restriction digestion. -G-CTTAAp55pAATTC G31Uniformly labeling of DNA/RNANick translation labeling of DNA: DNase I to introduce random nicks into template DNA DNA pol I to remove dNMPs from 3 to 5 an
23、d add new dNMP including labeled nucleotide at the 3 ends.Hexanucleotide primered labeling of DNA: Denature DNA add random hexanucleotide primers and DNA pol synthesis of new strand incorporating labeled nucleotide.32Strand-specific RNA probes: labeled by in vitro transcription of the desired RNA se
24、quence. 333-2 Hybridization probes can identify electrophoretically-separated DNAs and RNAsHybridization: the process of base-pairing between complementary ssDNA or RNA from two different sources.Topic 1 Nucleic acids- DNA hybridizationNorthern/Southern blot analysisgelmembraneElectrophoresisElectro
25、phoresisblottingblottingHybridizationHybridizationFig 21-6Southern 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. Hybrid
26、ization (temperature) 7. Signal detection (X-ray film or antibody) 36Blot type TargetProbeApplicationsSouthern DNA DNA or RNAmapping genomic clonesestimating gene numbers, etcNorthernRNADNA or RNARNA sizes and abundance (gene expression level)WesternProteinAntibodiesprotein size and abundance (gene
27、expression level)Comparison of Southern, Northern and Western bolt hybridization374. 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
28、.Topic 1 Nucleic acids- amplification38Denaturation (变性变性): The target DNA (template) is separated into two stands by heating to 95Primer annealing (退火退火): The temperature is reduced to around 55 to allow the primers to anneal.Polymerization (elongation, extension) (延伸延伸): The temperature is increas
29、ed to 72 for optimal polymerization step which uses up dNTPs and required Mg+.The PCR cycle:Three different steps proceed in each PCR cycle. 39Fig 21-11DenaturationPrimer annealing Polymerization4041The PCR amplificationMany cycles (25-35 in common) are performed to complete one PCR reaction, which
30、resulted in an exponential amplification of the target DNA if both forward and reverse primers pair.42DNA templateAny source of DNAAny source of DNA that provides that provides one or more target molecules can in one or more target molecules can in principle be used as a template for principle be us
31、ed as a template for PCR.PCR.Whatever the source of Whatever the source of template DNA, PCR can only be template DNA, PCR can only be applied if applied if some sequence information some sequence information is knownis known so that primers can be so that primers can be designed.designed.43PCR Prim
32、ers1.Anneal on opposite strands of the target sequence.2.About 18 to 30 nt long and have similar G+C contents so that they anneal to their complementary sequences at similar temperatures.3.Tm=2(a+t)+4(g+c): determine annealing temperature. If the primer is 18-30 nt, annealing temperature can be Tm 5
33、oC445-ATTCCGATCGCTAATCGATGGC- TCCTGTGCA TTTCGCCACTAGAG-33-TAAGGCTAGCGATTAGCTACCG-AGGACACGTAAAGCGGTGATCTC-55-ATTCCGATCGCTAATCGATG-33-CACGTAAAGCGGTGATCTC-5Tm=11x2+9x4=58oCTm=9x2+10x4=58oCDNA sequence is written from 5 to the 3 end if not stated. And only the sense strand is usually given instead of bo
34、th strands. 45Degenerate primers (简并引物简并引物): an oligo pool derived from a protein sequence.E.g. His-Phe-Pro-Phe-Met-Lys can generate a primer 5-CAY TTY CCN TTY ATG AARY= PyrimidineN= any baseR= purine46Enzymes and PCR OptimizationThe most common is Taq polymerase. It has no 3 to 5 proofreading exonu
35、clease activity. Accuracy is low, not good for cloning. High-accuracy DNA polymerase is available commercially.To optimize PCR, the annealing temperature and the Mg+ concentration are varied, or the nested PCR is carried out.47Nested PCR: to increase specificityFirst roundprimersFirst roundPCRSecond
36、 roundprimersSecond roundPCRGene of interest48Reverse transcriptase (RT)-PCRAAA(A)n5-CapmRNA(dT)1218 primeranneal5-CapAAA(A)n35Reverse transcriptiondNTP, RT5-CapAAA(A)n5cDNA:mRNA hybridRegularPCR49PCR mutagenesis (诱变诱变) )PCR can be used to introduce deletion and point mutations1.Two separate PCR rea
37、ctions 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 primers50SP6 primerT7 primerForward mutagenic primerReverse mutagenic primerFirst PCRRemove primersDenature and anne
38、alExtend to full length by DNA polymerase3351Second PCRSP6 primerT7 primerDNA cloning525. DNA cloning, analysis and gene expressionNucleic acids- sequencingThe ability to construct recombinant DNA molecules and maintain them in cells is called DNA cloning.53Processes (过程过程)of DNA cloning：1.Form the
39、recombinant DNA molecules (重组重组DNA) by inserting your interested DNA fragments into a proper vector (载体载体). (Require restriction enzymes and ligase)2.Transform (转化转化) the recombinant DNA molecules into competent cells (感受态细胞感受态细胞).3.Propagation of the cells containing the recombinant DNA to form a c
40、lone (克隆克隆), a set of identical cells containing the same recombinant DNA.4.Select the desired clones using the selective marker. 541.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. c
41、oli. competent cells.4.Grow the cells on a plate containing tetracycline (四环素四环素).Fig 21-7 construction of a DNA library55Host organisms/cells: where the plasmids get multiplied and propagated faithfully, which is crucial for DNA cloning.-Prokaryotic host: E. coli ( most cases)-Eukaryotic host: Yeas
42、t Saccharomyces cerevisiae (large fragments of human genome)56General features of a Vector1.They contain an origin of replication and can autonomously replicating DNA independent of hosts genome.2.Easily to be isolated from the host cell. Most are circular, some are linear (e.g. YAC vector).3.Contai
43、ns at least one selective marker, which allows host cells containing the vector to be selected amongst those which do not. 4.Contains a multiple cloning site (MCS) to be cut by restriction enzymes for DNA manipulation. 57Cloning vectors (克隆载体克隆载体): allowing the exogenous DNA to be inserted, stored,
44、and manipulated at DNA level. E. coli cloning vector (circular): plasmids (质粒质粒)bacteriophages (l and M13) (噬菌体噬菌体)plasmid-bacteriophage l hybrids (cosmids) (考斯考斯质粒粒质粒和噬菌体粒和噬菌体杂和体和体).Yeast cloning vector: yeast artificial chromosomes (YACs，酵母人工染色体酵母人工染色体) (Linear)5859Plasmids: small, extrachromosoma
45、l circular molecules, from 2 to 200 kb in size, which exist in multiple copies within the host cells.Contain an origin of replication, at least one selective marker and multiple cloning site. Example of selective marker: ampr gene encoding the enzyme b-lactamse which degrades penicillin antibiotics
46、such as ampicillin.The commonly used plasmid are small in size ( 3 kb)60Libraries of DNA molecules can be created by cloning (Genomic library and cDNA library)A DNA libraryA DNA library (DNA文库文库) is a population of identical vectors that each contains a different DNA insert. (Fig. 20-8)Genomic Libra
47、ry Genomic Library (基因组文库基因组文库) : the DNA inserts in a DNA library is derived from restriction digestion or physical shearing of the genomic DNA. cDNA library cDNA library (cDNA文库文库) : the DNA inserts in a DNA library is converted from the mRNAs of a tissue, a cell type or an organism. cDNA stands f
48、or the DNA copied from mRNA. (Fig. 20-19) 61Different Insert fragmentsFig 21-8 construction of a DNA library62cDNA library generationThe mRNAs are firstly reverse transcript into cDNA, and these cDNA, both full length and partial, are cloned to make the cDNA library.63annealReverse transcriptionRegu
49、larPCRFig 20-9 Construction of cDNA library64Colony screening 1.Antibiotic screening (抗生素选择抗生素选择)： only the recombinant plasmids grow on the antibiotic-containing plate.2.Blue-white screening (蓝白斑选择蓝白斑选择): DNA insertion in the vector shuts down the LacZ gene expression, and turns the colony to white
50、. 3.Colony hybridization screening (菌落菌落杂交筛选杂交筛选) from a library.Screeningofpositiveclones65Antibiotic screening (抗生素选择抗生素选择)： only intact plasmids grow on the antibiotic-containing plate. X if the vector is in the phosphorylated stateRecombinant DNA moleculesDephosphorylate the vector using alkalin
51、e phosphate can prevent religation of vector molecules68Blue white screening-Allow the discrimination of recombinant plasmid from the religated onesAmproripUC18(3 kb)MCS (Multiple cloning sites,多克隆位点）多克隆位点）Lac promoterlacZInsertion of a DNA fragment interrupts the ORF of lacZ gene, resulting in non-
52、functional gene product that can not digest its substrate x-gal. lacZ encode enzyme b-galactosidase IPTGX-gal(substrate of the enzyme)lac promoter Blue productDuring the experiment, IPTG is added to the selective growth medium. Insertion of the target DNA fragment into the MCS will prevent the forma
53、tion of the blue colony, and white colony is formed instead. 70Recreated vector: blue transformantsRecombinant plasmid: containing inserted DNA: white transformantsRecreated vector (no insert)Recombinant plasmid (contain insert)Transfer to nitrocelluloseor nylon membraneDenature DNA(NaOH)Bake onto m
54、embraneProbe with 32p-labled DNA complementary to gene of interestExpose to filmSelect positive from master plateKeep master plate Screening by plaque hybridizationColony hybridization-Southern blot 72Analysis of a clone 1.Restriction mapping: digestion of the plasmid prepared from a clone with rest
55、riction 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.AnalysisofDNAclones1 Kb+ ladder2 Positive clones digested with different restriction enzymesEmpty vectorRestriction mapping74
56、Sequencing75Expression of a gene from a transformed/transfected plasmid 1.Transformation (转化转化) : introduction of plasmids into bacteria.2.Transfection (转染转染): introduction of plasmids and other exogenous nucleic acids into eukaryotes such as mammalian cells.Geneexpression76Expression vectors: allow
57、ing the exogenous DNA to be stored and expressed in an organism.-E. coli expression vector-Yeast expression vector-Mammalian expression vectorFeatures:In addition to the origin of replication, selective marker, multiple cloning site, expression vector has to contain a promoter and terminator for tra
58、nscription. The inserted gene has to have a start codon and a stop codon for translation77T7 promoterRBSStart codonMCSTranscription terminatorAmproriT7 expression vectorE. coli expression vector78Insert Figure 1MCSH4EukaryoticVectorsYeast expression vector79Fusion proteinsGene cloning and expression
59、 provides a very powerful way to obtain a large amount of the target protein fused with an enzyme, fluorescence protein or a tag for identification (鉴定鉴定) or purification (纯化纯化). 80ExamplesLac fusions (Enzyme) : fuse your target gene with the LacZ coding sequence. His-tag fusions (Tag) : A sequence
60、encodes His-tag was inserted at the N- or C- termini of the target ORF, allowing the fusion protein to be purified by binding to Ni2+ column. GFP fusions (Fluorescence protein): insert your targeted gene at the N- or C- termini of GFP (green fluorescence protein, 绿色荧光蛋白绿色荧光蛋白), and your fusion prote
61、in will give you green fluorescence signal. 816. SequencingTwo ways for sequencing:n1. DNA molecules (radioactively labeled at 5 termini) are subjected to 4 regiments to be broken preferentially at Gs, Cs, Ts, As, separately. (Maxam and Gilbert chemical method, not widely used)n2. Chain-termination
62、method (Sangers method, widely used)Nucleic acids- sequencingMaxam and GilbertSangers enzymic method83Chain-termination method (链终止链终止法法) )nddNTPs are chain-terminating nucleotides: the synthesis of a DNA strand stops when a ddNTP is added to the 3 end84The absence of 3-hydroxyl lead to the ineffici
63、ency of the nucleophilic attack on the next incoming substrate molecule.85If one ddGTP is added to 100 dGTP, DNA synthesis aborts at a frequency of 1/100 every time the polymerase meets a ddGTP Tell from the gel the position of each G86Four separate reactions dNTP+ ddGTP, dNTP+ ddATP dNTP+ ddCTP, dN
64、TP+ ddTTPEach ddNTP carries a fluorescence group, allowing us to “Read” the sequence directly from the gel.Fig 20-15 DNA sequencing gel87Automatic sequencer1.Fluorescence Labeled ddNTP2. Polymerase catalyzed 88Shotgun sequencing of a bacterial genome (鸟枪法测细菌的基因组鸟枪法测细菌的基因组) )1.The bacterial genome wa
65、s randomly sheared into many random fragments with an average size of 1 kb, and cloned intro a vector. (Prepare what you are going to shot)2.Individual recombinant DNA clones are randomly picked to prepare DNA for sequencing on an automated sequencer. (shot)This is called shotgun sequencing.89In use
66、 of shotgun sequencing strategy, multiple sequence coverage is required to obtain all genome sequence.For example: The genome of bacteria Hemophilus influenzae is 1.8 Mb, each sequence read produces 600 bp of sequence. If 33,000 different clones were picked for sequencing, a total of 600 bp x 33,000
67、= 20 Mb sequence was produced.Critical thinking: how to design a pair of primers to sequence the inserts of all the recombinant plasmids in a genomic library? 90The shotgun strategy permits a partial assembly of large genome sequenceThe core techniques for sequencing the large genomes, e.g. human, a
68、re(1) automated shotgun sequencing (obtain sequence)(2) then the subsequent use of computer to assemble the different sequences (analyze sequence, which is the rate-limiting step).91Fig 20-161. Recombinant 1. Recombinant Plasmid LibraryPlasmid Library2. Shotgun 2. Shotgun sequencingsequencing3. Sequ
69、ence 3. Sequence Assembly Assembly Flow chart of Human genome sequencing project92Assembly Step 1: form contigs (A single contig is about 50,000 to 200,000 bp. )Sophisticated computer programs have been developed that assemble the short sequences from random shotgun DNAs into larger contiguous seque
70、nces called contigs.93Assembly Step 2: The paired-end strategy permits the assembly of larger scaffolds (1-2 Mb)94Fig 20-17. Contigs are linked by sequencing the ends of large DNA fragments (plasmid library containing larger DNA fragments). 951.Assemble the contigs from 1kb plasmid shotgun sequence.
71、 (50 kb-200 kb)2.Assemble the contigs to large scaffold by sequencing both ends of 5 kb plasmids. (1 Mb) by sequencing the end of the BAC library.Assembly flowchart96Genome-wide analysisThe purpose of this analysis is to predict the protein coding genes (蛋白质编码基因蛋白质编码基因) and other functional sequence
72、s (其他功能序其他功能序列列) in the genome.97For the genomes of bacteria and simple eukaryotes：Finding protein coding genes = Identification of ORF (open-reading frames). (1)straightforward; (2)fairly effective; (3)but not all ORF=real protein coding genes; (4)key challenge is in identifying the functions of th
73、ese genes.98For animal genomes with complex exon-intron structures, the challenge is far greater：1.A variety of bioinformatics tools are required to identify genes and genetic composition of complex genomes.2.The computer programs identifying potential protein coding genes are based on many sequence
74、 criteria including the occurrence of extended ORFs that are flanked by appropriate 5 and 3 splice sites.99Limitations of the computer methods: 1) one-fourths of genes cannot be identified by this way.2)The failure to identify promoters because the core promoter elements are highly degenerate (退变的退变
75、的). Although the transcription complex is smart enough to identify these elements in cell, we are not yet smart enough to write programs to identify them in silico (硅片，人工硅片，人工).The most important method for validating predicted protein coding genes and identifying those missed by current gene finder
76、 program is the use of cDNA sequence data.100cDNA library sequencing and application:1.Sequence the cDNAs prepared from a cDNA library using shotgun method to generate EST (expressed sequence tag) database. 2.These ESTs are aligned onto genomic scaffolds to help us identify genes and to assemble lar
77、ger scaffolds.101Fig 20-18 Gene finder method: analysis of protein-coding regions in Ciona intestinalis (海鞘海鞘 )A 20-kb genome sequence (scaffold)Predicted by a gene finder program 102The mostly commonly used genome tool BLAST ：Finding regions of similarity between different protein coding genes. 1.I
78、nput a query sequence (询问序列询问序列): a stretch of amino acids or the DNA sequence encoding your interested protein function. 2.Ask the computer to search for the homologous sequences in a protein or DNA database, and you will get all the available genes that may have the similar protein function.103Fig
79、ure 20-21 Example of the BLAST search result104Topic 2: ProteinsCHAPTER20:TechniquesofMolecularBiology1.Protein purification (蛋白质纯化蛋白质纯化) 2.Affinity chromatography can facilitate more rapid protein purification (亲和层析亲和层析纯化纯化)3.Protein separation by PAGE gel electrophoresis (蛋白质分离蛋白质分离) and identific
80、ation by Western analysis4.Protein sequencing (蛋白质测序蛋白质测序)5.Proteomics (蛋白质组学蛋白质组学)105nThe purification of individual proteins is critical to understanding their function.nAlthough there are thousands of proteins in a single cell, each protein has unique properties, such as size, charge (电荷电荷), shap
81、e, and in many instance, function, that make its purification somewhat different from others.1. Protein purification (蛋白质纯化蛋白质纯化) )106nPurification of a protein requires a specific assay to allow you to monitor your purification status, which include a measure of the function of the protein, use of
82、the antibody of the protein.107Column chromatography is an efficient way to purify proteinsIn this approach, protein fractions are passed though glass columns filled with appropriated modified small acrylamide or agarose beads.108Ion exchange chromatographynThe proteins are separated according to th
83、eir surface charge.nThe beads are modified with either negative-charged or positive-charged chemical groups.nProteins bind more strongly requires more salt to be eluted. 109Fig 20-22-a110Gel filtration chromatography nThis technique separate the proteins on the bases of size and shape. nThe beads fo
84、r it have a variety of different sized pores throughout. Small proteins can enter all of the pores, and take longer to elute; but large proteins pass quickly.111Fig 20-22-b1122. Affinity chromatography can facilitate more rapid protein purificationIf the target protein is known to establish a specif
85、ic and high-affinity interaction with a specific protein/nucleic acids/small molecule, we can couple this specific partner of the target protein to the column and thus the target protein will be selectively bound to the column.This method is called affinity chromatography. 113Affinity chromatography
86、Enzyme-substrate bindingReceptor-ligand bindingAntibody-antigen bindingProteinstructureNi2+-His tag-fusion protein binding114Immunoaffinity chromatography(免疫亲和层析免疫亲和层析)nAn antibody that is specific for the target is attached to the bead, and ideally only the target protein can bind to the column.nDi
87、sadvantage: sometimes the binding is too tight to elute our target protein.115nSometimes tags (epitopes, 抗原决定基抗原决定基) can be added to the N- or C- terminal of the target protein, using DNA cloning method, to make the fusion protein. nThis allows the modified fusion proteins to be purified using immun
88、oaffinity purification and a heterologous antibody specific for the tag.nImportantly, the binding affinity can change according to the condition. e.g. the concentration of the Ca2+ in the solution.116Immunoprecipitation (免疫沉淀免疫沉淀)nAttach the antibody to the bead, which is then used to precipitate (沉
89、淀沉淀) a specific protein from a crude cell extract.nIts a useful method to detect what proteins or other molecules are associated with the target protein. 1173. Protein separation by PAGE gel electrophoresis, followed by a western analysisnThe native proteins have neither a uniform charge nor a unifo
90、rm secondary structure.nIf we treat the protein with a strong detergent SDS, the higher structure is usually eliminated. And SDS confers the polypeptide chain a uniform negative charge.118nSometimes, mercaptoethanol (巯基乙醇) is need to break the disulphide bond.nThus, the protein molecules can be reso
91、lved by electrophoresis in the presence of SDS according to the length of individual polypeptide.nAfter electrophoresis, the proteins can be visualized with a stain, such as Coomassie brilliant blue (考马氏亮蓝).nProteins from the PAGE gel can be transferred to a membrane, followed by a western analysis
92、of the target protein by a corresponding antibody.119PTBBeta-actin - P+ P+ P+ P+A protein gel stained by Coomassie Blue Western analysis using two specific antibodies1204. Protein sequencing (蛋白质测序)nTwo sequence method: Edman degradation (Edman切割法切割法)Tandem mass spectrometry (MS/MS) (串连质谱串连质谱).nDue
93、to the vast resource of complete or nearly complete genome, the determination of even a small stretch of protein sequence is sufficient to identify the gene.121Edman degradationA chemical reaction in which the amino acids residues are sequentially release for the N-terminus of a polypeptide chain.12
94、2nStep 1: modify the N-terminal amino with PITC, which can only react with the free -amino group.nStep 2: cleave off the N-terminal by acid treatment, but the rest of the polypeptide remains intact.nStep 3: identify the released amino acids by High Performance Liquid Chromatography (HPLC).The whole
95、process can be carried out in an automatic protein sequencer. 特贵特贵123Fig 20-23124Tandem mass spectrometrynMS is a method in which the mass of very small samples of a material can be determined.125nStep 1: digest your target protein into short peptides.nStep 2: subject the mixture of the peptide to M
96、S, and each individual peptide will be separate.nStep 3: capture the individual peptide and fragmented into all the component peptide.nStep 4: determine the mass of each component peptide.nStep 5:Deconvolution (解析) of these data and the sequence will be revealed. 1265. Proteomics (蛋白质组学)nProteomics
97、is concerned with the identification of the full set of proteins produced by a cell or a tissue under a particular by a particular set of conditions. 127Three principle methods1. 2-D gel electrophoresis for protein protein separationseparation (蛋白质分离蛋白质分离).2. MS spectrometry for the precise determin
98、ationdetermination of the molecular weight and identify of a protein (蛋白蛋白质鉴定质鉴定).3. Bioinformatics for assigning assigning proteins and peptides to the predicted products of protein coding sequence in the genome (蛋白质确定蛋白质确定).128Fig 20-24129Topic 3: Study the interaction between protein and nucleic
99、acidCHAPTER20:TechniquesofMolecularBiology1.Gel retardation assay2.Nuclease protection assay130Gel 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. Then, samples of labeled nucleic acid, with and without being incubat
100、ed with the extract, are run on a gel. The DNA-protein complexes are shown by the presence of slowly migrating bands. DNA bound totwo proteinsDNA-proteincomplexBare DNAA DNA bound with more than one protein to form a larger complex.132DNase I footprinting (DNase I 足迹法足迹法) Identify the actual region
101、of sequence with which the protein interacts.5*Sequence ladder is required to determine the precise positionAATAAG133Bind proteinDNase(mild),then removeprotein and denature DNADNase footprinting （1）The protein protects DNA from attack by DNase. （2）Treat the DNA-protein complex with DNase I under mil
102、d conditions, so that an average of only one cut occur per DNA molecule. Electrophoresis,autoradiograph134015ProteinThe three lanes represent DNA that was bound to 0, 1, and 5 units of protein. The lane with no protein shows a regular ladder of fragments. The lane with one unit shows some protection
103、, and the lane with 5 units shows complete protection in the middle.By including sequencing ladders, we can tell exactly where the protein bound.TCGGAGCAACGCAAACAAACGTGCTTGG135Topic 4: Determining the Structure ofProtein and nucleic acidsCHAPTER20:TechniquesofMolecularBiology1.X-ray crystallography
104、(X-晶晶体衍射体衍射)2.NMR (核磁共振核磁共振)136X-ray crystallographyX-ray crystallography and and NMRNMRDetermining the tertiary structureDetermining the tertiary structureX-ray crystallography:X-ray crystallography: Measuring the pattern of diffraction of a beam of X-rays as it pass through a crystal. The first ha
105、nd data obtained is electron density map, the crystal structure is then deduced. A very powerful tool in understanding protein tertiary structureMany proteins have been crystallized and analyzed137The ribosome structure and its interaction with mRNA and tRNA138Measuring the relaxation of protons aft
106、er they have been excited by radio frequencies in a strong magnetic field. Measure protein structure in liquid but not in crystal. Protein measured are usually smaller than 30 KDa.NMRNMR139CHAPTER20:TechniquesofMolecularBiologyNucleic acids techniques:Electrophoresis; Restriction digestion; Hybridiz
107、ation (southern & northern); PCR amplification; sequencing and genome sequencing; DNA cloning and gene expression.Protein techniques:Protein purification; affinity chromatography; Protein separation and identification by western blot; Protein sequencing; Proteomics.Study the interaction between prot
108、ein and nucleic acidGel retardation & Nuclease protection assaysDetermining the Structure of protein and nucleic acids: X-ray crystallography, NMR140CHAPTER20:TechniquesofMolecularBiology思考题：思考题： 你在本章中学习了哪些核酸和蛋白质技术？它们的作你在本章中学习了哪些核酸和蛋白质技术？它们的作用原理和主要用途各是什么用原理和主要用途各是什么?141Welcome to Welcome to Welcome to Welcome to join in our join in our join in our join in our labslabslabslabs Thanks for Thanks for Thanks for Thanks for your your your your incorporatiincorporatiincorporatiincorporation for the on for the on for the on for the whole class whole class whole class whole class
