1、1,Welcome to MB Class,2,Molecular 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 Genome Part IV: Regulation Part V: Methods,3,Ch 20: Techniques ofMolecular Biology Ch 21: Model Organisms,Part V: METHODS,4,
2、Molecular Biology Course,Chapter 20Techniques of Molecular Biology,Preparation, analysis and manipulation of nucleic acids and proteins,5,The methods depend upon, and were developed from, an understanding of the properties of biological macromolecules themselves. Hybridization-the base-pairing chara
3、cteristics of DNA and RNA DNA cloning- DNA polymerase, restriction endonucleases and DNA ligase PCR-Thermophilic DNA polymerase,6,Topic 1: Nucleic acids,CHAPTER20: Techniques of Molecular Biology,Separation by Electrophoresis (电泳分离) Cut by Restriction endonuclease (限制性内切酶切割) Identification by Hybrid
4、ization (杂交鉴定) Amplification by PCR (PCR扩增) DNA Cloning and gene expression (DNA克隆和基因表达) Genome sequence & analysis (基因组序列和分析),7,1. Gel electrophoresis separates DNA and RNA molecules according to size, shape and topological properties.,Topic 1 Nucleic acids-separation,8,1.DNA and RNA molecules are
5、negatively charged, thus move in the gel matrix (胶支持物) toward the positive pole (正电极). 2.Linear DNA molecules are separated according to sizes. The large DNA molecules move slower than the small molecules. 3.The mobility of circular DNA molecules is affected by their topological structures. The mobi
6、lity 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),DNA gel mobility (DNA在胶上的迁移性),9,Fig 21-1: DNA is separated by gel electrophoresis,large,moderate,small,10,Gel matrix (胶支持物) is an inserted, jello-like porous material that
7、 supports and allows macromolecules to move through.,Gel matrix (胶支持物),11,Agarose (琼脂糖): a much less resolving power than polyacrylamide, but can separate DNA molecules of up to tens of kb,DNA can be visualized by staining the gel with fluorescent dyes, such as ethidium bromide (EB 溴化乙锭),12,Polyacry
8、lamide (聚丙稀酰胺): has high resolving capability, and can resolve DNA that differ from each other as little as a single base pair/nucleotide. but can only separate DNA over a narrow size range (1 to a few hundred bp).,13,The electric field is applied in pulses that are oriented orthogonally (直角地) to ea
9、ch other. Separate DNA molecules according to their molecule weight, as well as to their shape and topological properties. Can effectively separate DNA molecules over 30-50 kb and up to several Mb in length.,Pulsed-field gel electrophoresis (脉冲电泳),14,Fig. 21-2 pulsed-field gel electrophoresis,Switch
10、ing between two orientations: the larger the DNA is, the longer it takes to reorient,15,RNA have a uniform negative charge as DNA does. RNA is single-stranded and have extensive secondary and tertiary structure, which significantly influences their electrophoretic mobility. RNA can be treated with r
11、eagent 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 RNAs,16,Why being used? -To break large DNA molecules into manageable fragments.,Nucleic acids-Restriction digestion,2. Restriction endonucleas
12、es (限制性内切酶) cleave DNA molecules at particular sites by the recognition of specific sequences,17,RE used in molecular biology typically recognize (识别) short (4-8bp) target sequences that are usually palindromic (回文结构), and cut (切割) at a defined sequence within those sequences. e.g. EcoRI,18,the 1st
13、such enzyme found,Escherichia coliSpecies category,R13 strain,How to name a restriction endonuclease?,EcoRI,19,The random occurrence of the hexameric (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?,How
14、 to estimate the frequency of the RE in a DNA molecule or genome?,20,21,(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 natu
15、re of the DNA ends they generate: blunt/flush ends (平末端), sticky/staggered ends (粘性末端). (4) Restriction enzymes differ in the cleavage activity.,22,sticky ends (粘性末端),blunt ends (平末端),Fig 21-4 Recognition sequences and cut sites of various endonucleases,23,3. DNA hybridization can be used to identif
16、y specific DNA molecules,Hybridization: the process of base-pairing between complementary ssDNA or RNA from two different sources.,Topic 1 Nucleic acids- DNA hybridization,24,A labeled, defined sequence used to search mixtures of nucleic acids for molecules containing a complementary sequence.,Probe
17、 (探针),The mixture being probed has typically either been separated by size on a gel, or is distributed as a library in different colonies,25,It can be readily located once it has found its target sequence.,A probe must be labeled before applied in hybridization (Why?),26,End labeling: put the labels
18、 at the ends Uniform labeling: put the labels internally,Radioactive labeling: display and/or magnify the signals 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)
19、 of DNA or RNA probes,27,End labeling,5-end labeling using polynucleotide kinase (PNK)3-end labeling using terminal transferase,29,How to label one end of a DNA: Labeling at both ends by kinase, then remove one end by restriction digestion.,-G -CTTAAp5,5pAATTCG,30,Uniformly labeling,Nick translation
20、 labeling of DNA: DNase I to introduce random nicks into template DNA DNA pol I to remove dNMPs from 3 to 5 and 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 incorpo
21、rating labeled nucleotide.,31,Strand-specific RNA probes: labeled by in vitro transcription of the desired RNA sequence.,Northern/Southern blot analysis,gel,membrane,Electrophoresis,blotting,Hybridization,Fig 21-6,Southern and Northern blotting,DNA on blot,RNA on blot,Genomic DNA preparation RNA pre
22、paration Restriction digestion - Denature with alkali - Agarose gel electrophoresis DNA blotting/transfer and fixation RNA 6. Probe labeling 6. Hybridization (temperature) 7. Signal detection (X-ray film or antibody) ,34,Comparison of Southern, Northern and Western bolt hybridization,35,4. Polymeras
23、e 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.,Topic 1 Nucleic acids- amplification,36,Denaturation (变性): The target DNA (template) is sepa
24、rated into two stands by heating to 95 Primer 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+.,The PCR cycle: T
25、hree different steps proceed in each PCR cycle.,37,Fig 21-11,Denaturation,Primer annealing,Polymerization,38,39,The PCR amplification,Many cycles (25-35 in common) are performed to complete one PCR reaction, which resulted in an exponential amplification of the target DNA if both forward and reverse
26、 primers pair.,40,DNA template,Any source of DNA that provides one or more target molecules can in principle be used as a template for PCR.Whatever the source of template DNA, PCR can only be applied if some sequence information is known so that primers can be designed.,41,PCR Primers,Anneal on oppo
27、site strands of the target sequence. About 18 to 30 nt long and have similar G+C contents so that they anneal to their complementary sequences at similar temperatures. Tm=2(a+t)+4(g+c): determine annealing temperature. If the primer is 18-30 nt, annealing temperature can be Tm 5oC,42,5-ATTCCGATCGCTA
28、ATCGATGGC- TCCTGTGCA TTTCGCCACTAGAG-3 3-TAAGGCTAGCGATTAGCTACCG-AGGACACGTAAAGCGGTGATCTC-5,DNA sequence is written from 5 to the 3 end if not stated. And only the sense strand is usually given instead of both strands.,43,Degenerate primers (简并引物): an oligo pool derived from a protein sequence. E.g. Hi
29、s-Phe-Pro-Phe-Met-Lys can generate a primer 5-CAY TTY CCN TTY ATG AAR Y= Pyrimidine N= any base R= purine,44,Enzymes and PCR Optimization,The most common is Taq polymerase. It has no 3 to 5 proofreading exonuclease activity. Accuracy is low, not good for cloning. High-accuracy DNA polymerase is avai
30、lable commercially. To optimize PCR, the annealing temperature and the Mg+ concentration are varied, or the nested PCR is carried out.,45,Nested PCR: to increase specificity,First round primers,First round PCR,Second round primers,Second round PCR,Gene of interest,46,Reverse transcriptase (RT)-PCR,A
31、AA(A)n,5-Cap,mRNA,(dT)1218 primer,anneal,5-Cap,AAA(A)n,3,5,Reverse transcription,dNTP, RT,5-Cap,AAA(A)n,5,cDNA:mRNA hybrid,Regular PCR,47,PCR mutagenesis (诱变),PCR can be used to introduce point mutations to the target gene in a plasmid,48,49,5. DNA cloning, analysis and gene expression,The ability t
32、o construct recombinant DNA molecules and maintain them in cells is called DNA cloning.,50,Processes (过程) of DNA cloning：Form the recombinant DNA molecules (重组DNA) by inserting your interested DNA fragments into a proper vector (载体). (Require restriction enzymes and ligase) Transform (转化) the recomb
33、inant DNA molecules into competent cells (感受态细胞). Propagation of the cells containing the recombinant DNA to form a clone (克隆), a set of identical cells containing the same recombinant DNA. Select the desired clones using the selective marker.,51,Restriction digestion of your insert and vector using
34、 the same enzyme. Use ligase to join your insert and vector together. Transform the ligation products into E. coli. competent cells. Grow the cells on a plate containing tetracycline (四环素).,Fig 21-7 construction of a DNA library,52,Host organisms/cells: where the plasmids get multiplied and propagat
35、ed faithfully, which is crucial for DNA cloning.-Prokaryotic host: E. coli ( most cases)-Eukaryotic host: Yeast Saccharomyces cerevisiae (large fragments of human genome),53,General features of a VectorThey contain an origin of replication and can autonomously replicating DNA independent of hosts ge
36、nome. Easily to be isolated from the host cell. Most are circular, some are linear (e.g. YAC vector). Contains at least one selective marker, which allows host cells containing the vector to be selected amongst those which do not. Contains a multiple cloning site (MCS) to be cut by restriction enzym
37、es for DNA manipulation.,54,Cloning vectors (克隆载体): allowing the exogenous DNA to be inserted, stored, and manipulated at DNA level. E. coli cloning vector (circular): plasmids (质粒)bacteriophages (l and M13) (噬菌体)plasmid-bacteriophage l hybrids (cosmids) (考斯质粒质粒和噬菌体杂和体). Yeast cloning vector: yeast
38、artificial chromosomes (YACs，酵母人工染色体) (Linear),55,56,Plasmids: small, extrachromosomal 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 m
39、arker: ampr gene encoding the enzyme b-lactamse which degrades penicillin antibiotics such as ampicillin. The commonly used plasmid are small in size ( 3 kb),57,Libraries of DNA molecules can be created by cloning (Genomic library and cDNA library) A DNA library (DNA文库) is a population of identical
40、vectors that each contains a different DNA insert. (Fig. 20-8) Genomic Library (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文库) : the DNA inserts in a DNA library is converted from the mRNAs of a tissue, a
41、 cell type or an organism. cDNA stands for the DNA copied from mRNA. (Fig. 20-19),58,Different Insert fragments,Fig 21-8 construction of a DNA library,59,cDNA library generation The mRNAs are firstly reverse transcript into cDNA, and these cDNA, both full length and partial, are cloned to make the c
42、DNA library.,60,anneal,Reverse transcription,Regular PCR,Fig 20-9 Construction of cDNA library,61,Colony screening,Antibiotic screening (抗生素选择)： only the recombinant plasmids grow on the antibiotic-containing plate. Blue-white screening (蓝白斑选择): DNA insertion in the vector shuts down the LacZ gene e
43、xpression, and turns the colony to white. Colony hybridization screening (菌落杂交筛选) from a library.,Screening of positive clones,62,Antibiotic screening (抗生素选择)： only intact plasmids grow on the antibiotic-containing plate.,Xif the vector is in the phosphorylated state,Recombinant DNA molecules,64,Blu
44、e white screening -Allow the discrimination of recombinant plasmid from the religated ones,Ampr,ori,pUC18 (3 kb),MCS (Multiple cloning sites,多克隆位点）,Lac promoter,lacZ,Insertion of a DNA fragment interrupts the ORF of lacZ gene, resulting in non-functional gene product that can not digest its substrat
45、e x-gal.,lacZ encode enzyme b-galactosidase,IPTG,X-gal,(substrate of the enzyme),lac promoter,Blue product,During the experiment, IPTG is added to the selective growth medium. Insertion of the target DNA fragment into the MCS will prevent the formation of the blue colony, and white colony is formed
46、instead.,66,Recreated vector: blue transformants Recombinant plasmid: containing inserted DNA: white transformants,Recreated vector (no insert),Recombinant plasmid (contain insert),Transfer to nitrocellulose or nylon membrane,Denature DNA(NaOH) Bake onto membrane,Probe with 32p-labled DNA complement
47、ary to gene of interest,Expose to film,Select positive from master plate,Keep master plate,Screening by plaque hybridization,Colony hybridization-Southern blot,68,Analysis of a clone,Restriction mapping: digestion of the plasmid prepared from a clone with restriction enzymes to investigate if the in
48、terested DNA is inserted the recombinant plasmid. Sequencing the cloned DNA to see if the inserted DNA maintains the correct sequence.,Analysis of DNA clones,1 Kb+ ladder,2 Positive clones digested with different restriction enzymes,Empty vector,Restriction mapping,70,Sequencing,71,Expression of a g
49、ene from a transformed/transfected plasmid,Transformation (转化) : introduction of plasmids into bacteria. Transfection (转染): introduction of plasmids and other exogenous nucleic acids into eukaryotes such as mammalian cells.,Gene expression,72,Expression vectors: allowing the exogenous DNA to be stor
50、ed and expressed in an organism. -E. coli expression vector -Yeast expression vector -Mammalian expression vector Features:In addition to the origin of replication, selective marker, multiple cloning site, expression vector has to contain a promoter and terminator for transcription. The inserted gene has to have a start codon and a stop codon for translation,
