分子生物学杨洋第九章分子生物学技术techniquesofmolecularbiology

1、outlinedna-basic principlebasic procedure application1.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技术内切酶切割技术)3.dna cloning and gene expression (基因基因克隆和表达技术）克隆和表达技术）4.分子杂交技术（分子杂交技术（ 5.pcr技术（聚合酶链式反应）技术（聚合酶链式反应）6.基因功能研究方法基因功能研究方法-充分结合自己的研究工作，实战中学习充分结合自己的研究工作，实

2、战中学习1. gel electrophoresis 1. gel electrophoresis （凝胶电泳）（凝胶电泳）1.dna and rna molecules are negatively charged, thus move in the gel matrix (胶胶支持物支持物) toward the positive pole (正电极正电极) are separated according to . the large dna molecules move slower than the small molecules.dna gel mobility (electroph

3、oretic mobility ，dna在胶上的迁移率在胶上的迁移率)largemoderate smallrun gel(跑胶跑胶)3.the mobility of 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 can be visualized b

4、y staining the gel with fluorescent dyes, such as ethidium bromide (eb,溴化溴化乙锭乙锭)dna is separated by gel electrophoresis1 kb0.5 kb2 kb3 kb4 kbdna markergel matrix (胶支持物胶支持物) is a jello( (果冻状果冻状）-like porous material that supports and allows macromolecules to move through. gel matrix (胶支持物胶支持物)polyacr

5、ylamide (聚丙烯酰胺聚丙烯酰胺)agarose (琼脂糖琼脂糖)polyacrylamide (聚丙烯酰聚丙烯酰胺胺): (1)has high resolving capability(分辨力高分辨力高), and can resolve dna/rna that differ from each other as little as a single base pair.(2)but can separate dna over a narrow size range (up to a few hundred bp, 几百几百bp).agarose (琼脂糖琼脂糖): (1)a mu

6、ch less resolving power than polyacrylamide(2)but can separate dna molecules of up to tens of kb1 kb0.5 kb2 kb3 kb4 kb(1)rna have a uniform negative charge as dna does. (2)rna is single-stranded and have extensive secondary and tertiary structure, which significantly influences their electrophoretic

7、 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 only correlates with the molecular weight electrophoresis is also used to separate rnas12pulsed-field gel electrophoresis (脉冲电泳脉冲电泳)pulsed-field gel electrophoresis(pfge)switching

8、between two orientations: the larger the dna is, the longer it takes to reorientnwhy use endonucleases?nto make large dna molecules break into manageable fragmentsnwhy use restriction endonucleases?ncleave dna molecules at particular sitesare the nucleases that dna at particular sites by the of spec

9、ific sequences.(剪刀剪刀)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. 5.gaattc.3 .cttaag.ecori35反向重复序列（回文结构）反向重复序列（回文结构）na linear dna molecule with 6 copies of

10、 gaattcnit will be cut into 7 fragments which could be separated by the gel electrophoresis.(the largest fragment)(the smallest fragment)digestionof a dna fragment with endonuclease ecori(1)restriction enzymes differ in the : target sites are different.(2) restriction enzymes differ in , and thus th

11、e frequencies differ: sau3ai, 5-gatc-3; noti, 5-gcggccgc-3(3) restriction enzymes differ in : blunt ends (平末端平末端), sticky ends (粘性末端粘性末端).sticky ends(粘性末端粘性末端) blunt ends(平末端平末端) recognition sequences and cut sites of various endonucleasescleavage of an ecori site: the 5 protruding ends are said to

12、be “sticky” because they readily anneal (退火退火) through base-pairing to dna molecules cut with the same enzymegene bgene adna连接酶连接酶how does the bacteria prevent its own dna from being cut by the restriction endonucleases?nmodification none of the nucleotides in bacteria dna is modified by an methylas

13、e enzyme (甲基化酶，甲基化酶，modification enzyme) that attaches a methyl group to one of the basesnthe restriction enzyme doesnt bind to sites where one of the bases is modified by methylation restrictionmodificationthe ability to construct recombinant dna molecules and maintain them in cells is called dna c

14、loning.体外构建重组分子体外构建重组分子体内繁殖体内繁殖1. constructing the 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 dna 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. propagatio

15、n of the cells containing the recombinant dna to form a clone (克隆克隆), a set of identical cells containing the same recombinant dna.4. select the desired clones using the selective marker. 1. restriction digestion of your insert and vector using the same enzyme.2. use ligase (连接酶）连接酶） to join your in

16、sert and vector together.3. transform the ligation products into e. coli competent cells.4. select the desired clones: grow the cells on a plate containing tetracycline (四环素四环素).ecoriimportant components of dna cloningnvector (载体）载体）nhost organisms/cells ( (宿主细胞）宿主细胞）nrestriction enzymes and dna lig

17、ase(剪刀剪刀+浆糊浆糊)nscreening method for selecting the desired clones （筛选阳性克隆）（筛选阳性克隆） general features of a vector1. they contain an origin of replication and can autonomously replicate dna independent of hosts genome.2. easily to be isolated from the host cell. 3. contains at least one selective marker

18、, 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. plasmid(质粒质粒): most used vector in dna cloning small, extrachromosomal circular molecules, from 2 to 200 kb in s

19、ize, 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 such as ampicillin-氨苄青霉素抗性基因氨苄青霉素抗性基因multip

20、le cloning site (mcs, 多克隆位点多克隆位点 )mcsselective markerorigin of replicationimportant components of dna cloningnvector (载体）载体）nhost organisms/cells ( (宿主细胞）宿主细胞）nrestriction enzymes and dna ligase(剪刀剪刀+浆糊浆糊)nscreening method for selecting the desired clones （筛选阳性克隆）（筛选阳性克隆） host organisms/cells: where

21、 the plasmids get multiplied and propagated faithfully, which is crucial for dna cloning.-prokaryotic host: e. coli ( most cases)-eukaryotic host: yeast, saccharomyces cerevisiae -vectors can be introduced into competent cells (感受态细胞感受态细胞) by transformation (转化转化)important components of dna cloningn

22、vector (载体）载体）nhost organisms/cells ( (宿主细胞）宿主细胞）nrestriction enzymes and dna ligase(剪刀剪刀+浆糊浆糊)nscreening method for selecting the desired clones （筛选阳性克隆）（筛选阳性克隆） screening of positive clones1.antibiotic screening (抗生素选择抗生素选择)： only the recombinant plasmids grow on the antibiotic-containing plate.2.

23、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. screening methods:(1).antibiotic screening (抗生素选择抗生素选择)： only the recombinant plasmids grow on the antibiotic-containing plate.multiple cloning site(mcs, 多克隆位点多克隆位点 )mcs

24、selective markerorigin of replicationantibiotic-containing platehost cell must be sensitive to this antibiotic!x geneampramprampramprantibiotic screening can not discriminate the clone containing recombinant pasmid and the clonecontaining the religated vector (2). blue-white screening (蓝白蓝白斑选择斑选择)la

25、cz encode enzyme b-galactosidase（b-半乳糖苷酶）半乳糖苷酶） x-gal(substrate of the enzyme)blue producthost e.coli cell : laczm15encoding only the c-terminal of a-peptide of b-galactosidaseplasmid: lacz, a shortened derivative of lacz, encoding n-terminal of a-peptide of b-galactosidase a- complementation (a-互补互

26、补) host e.coli cellplasmidlacz+x-gal(substrate of the enzyme)blue product b-半乳糖苷酶半乳糖苷酶blue white screeningamproripuc18(3 kb)mcs (multiple cloning sites,多克隆位点）多克隆位点）lac promoterlaczamproripuc18(3 kb)mcs (multiple cloning sites,多克隆位点）多克隆位点）lac promoterlaczself-ligated vectoramproripuc18(3 kb)lac promo

27、terlaczrecombinant plasmidinserted geneself-ligated vector: blue transformantsrecombinant plasmid: containing inserted dna, white transformantsself-ligated vector(no insert)recombinant plasmid (contain insert)herbert w. boyer professor in university of california, san francisco stanley cohen profess

28、or in standford university invent recombinant dna technology (genetic engineering)recombined genes that originated from different organisms first experiment about recombinant dna technology amprtetr对四环素和氨苄青霉素都有抗性的新细菌菌株对四环素和氨苄青霉素都有抗性的新细菌菌株overexpression of humaninsulin (胰岛素）胰岛素）gene in e.colibacteria

29、l factory producinglarge amounts of human proteins (insulin)第一个基因工程药物第一个基因工程药物hybridization: the process of base-pairing between complementary ssdna or rna from two different sources.a labeled, defined sequenceused to search mixtures of nucleic acids for specific molecules containing a specific sequ

30、ence which iscomplementary to probe probe (探针探针)radioactive labeling: display and/or magnify the signals by . eg. 32p non-radioactive labeling: display and/or magnify the signals by antibody binding enzyme binding - substrate application (signal release)eg. dig(地高辛标记地高辛标记)probe (探针探针) must be labell

31、ed (why labeling?)end labeling5-end labeling using polynucleotide kinase (pnk)3-end labeling using terminal transferaseuniformly labeling of dna/rna(掺入标记法掺入标记法)denature dna add random hexanucleotide primers and dna pol synthesis of new strand incorporating labeled nucleotide.dntp mixture: 32p-datp,

32、dctp, dgtp, dttp61substrates required for dna synthesissouthern nbasic principle: the base-pairing characteristics of dna and dnanapplication: estimating gene numbersdna 样品 dna 探针变性x-ray 片限制性内切酶消化琼脂糖凝胶电泳转移印迹胶 膜标记杂交杂交暴光硝酸纤维素滤膜，尼龙膜硝酸纤维素滤膜，尼龙膜nprobe: dna sequence specific to the known genelabeledsouthe

33、rn analysissouthern hybridizationnorthern nbasic principle: the base-pairing characteristics of dna and rnanapplication: detecting the expression level of specific genemrna contents (mrna 丰度丰度) expression level of specific genenprobe: dna sequence specific to the known genenorthern analysisthe stren

34、gth of hybridization signalcan reflect the content of specificmrnamrnasouthern 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 label

35、ing 6. hybridization (temperature) 7. signal detection (x-ray film or antibody) western hybridizationnproteins from the page gel can be transferred to a membrane, followed by a western hybridization of the target protein by a corresponding .ndetect the presence of specific protein by antibody-antige

36、n reactionblot type targetprobeapplicationssouthern dna dna or rnamapping genomic clonesestimating gene numbers, etcnorthernrnadna or rnarna sizes and abundance (gene expression level)westernproteinantibodiesprotein size and abundance (gene expression level)comparison of southern, northern and weste

37、rn hybridization (blot)transfer to nitrocelluloseor nylon membranedenature dna (naoh)bake onto membraneprobe with 32p-labled dna complementary to gene of interestexpose to filmselect positive from master platekeep master plate screening by plaque hybridizationcolony hybridization（菌落杂交）（菌落杂交）-souther

38、n blot 5. pcr（聚合酶链式反应）（聚合酶链式反应） the chemistry of dna dna synthesis requires deoxynucleoside triphosphates and a primer:template junction dna is synthesized by extending the 3 end of the primer hydrolysis of pyrophosphate (ppi) is the driving force for dna synthesis substrates required for dna synthe

39、sissemiconservative replication(半保留复制)three hypotheses for dna replicationthe mechanism of dna polymerase (pol)dna polymerase the polymerase chain reaction(pcr) is to used to amplify a sequence of dna in vitro, using a pair of primers each complementary to one end of the the dna target sequence.理论理论

40、技术技术denaturation (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 increased to 72 for optimal polymerization

41、 step which uses dntps and requires mg+.the principle of pcr:three different steps proceed in each pcr cycle. 53535353553353535353535353535353535355335535353353535353denaturationannealingextension (dna polymerase)cycle 1cycle 2cycle 3initialdna8421number of dna molecules (2n): 指数扩增指数扩增many cycles (2

42、5-35 in common) are performed to complete one pcr reaction, which resulted in an of the target dna in which both forward and reverse primers pair.dna template (模模板板) 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

43、 only be applied if so that primers can be designed.figure 8-3 substrates required for dna synthesispcr primers1.anneal on opposite strands of the target sequence: forward and reverse primers （正向引物和反向引物）（正向引物和反向引物）2.have similar g+c contents (tm) so that they anneal to their complementary sequences

44、at similar temperatures (anneal temperature: tm-5c)5-attccgatcgctaatcgatggc- tcctgtgca tttcgccactagag-33-taaggctagcgattagctaccg-aggacacgtaaagcggtgatctc-55-attccgatcgctaatcgatg-33-cacgtaaagcggtgatctc-5forward primerreverse primer5-ctctagtggcgaaatgcac-35353535355335353535353535353535353535533553535335

45、3535353denaturationannealingextension (dna polymerase)cycle 1cycle 2cycle 3pcr enzymesndenaturation (变性变性): the target dna (template) is separated into two stands by heating to 95nprimer annealing (退火退火): the temperature is reduced to around 55 to allow the primers to anneal.npolymerization (elongat

46、ion, extension) (延伸延伸): the temperature is increased to 72 for optimal polymerization step which uses dntps and requires mg2+.ntaq polymerase ：isolated from the thermophilic bacterium thermus aquaticus (嗜热菌）嗜热菌）, 耐热耐热dna聚合酶，耐高温聚合酶，耐高温nit has no 3 to 5 proofreading exonuclease activity nhigh-accuracy

47、 dna polymerase is available commerciallyfigure 8-3 substrates required for dna synthesisdntppcr仪仪pcr扩增的平台期扩增的平台期nmy present research workndegenerate pcr can be used to clone gene coding for enzyme 简并引物简并引物pcrpcr application-example-结合自己的研究工作结合自己的研究工作codon: degenerateanticodon: wobblemany amino acid

48、s are specified by more than one codon-degeneracy (简并性简并性).codons specifying the same amino acid are called synonyms (同义密码子同义密码子).gdegenerate primers (简并引物简并引物): an oligo pool derived from a protein sequence.his-phe-pro-phe-met-lys can generate a primer 5-cay tty ccn tty atg aar-3y= pyrimidine (c or

49、 t)n= any baser= purine (a or g)degenerate pcr( 简并简并pcr)nmy research work: molecular biology research of laccase(漆酶漆酶) produced in fungi -白腐真菌漆酶的分子生物学研究白腐真菌漆酶的分子生物学研究白腐真菌白腐真菌（白腐真菌（white rot fungi)n白腐真菌是一白腐真菌是一类使木材呈白类使木材呈白色腐朽的丝状色腐朽的丝状真菌的总称真菌的总称（主要是担子（主要是担子菌）菌）n已知的唯一能已知的唯一能在纯系培养中在纯系培养中有效地将木质有效地将木质素彻底降

50、解为素彻底降解为co2 和和h2o 的一类微生物的一类微生物木质素：以芳香族为基本结构的高度复杂聚合物木质素：以芳香族为基本结构的高度复杂聚合物 science, 2007, 315(5813): 804-807 n对与木质素结构相似的异生物质环境污染对与木质素结构相似的异生物质环境污染物也具有强大的降解能力物也具有强大的降解能力10710710/21/2021n木质素过氧化物酶（lip） n锰过氧化物酶（mnp） n漆酶（漆酶（laclac）白腐真菌木质素降解酶系白腐真菌木质素降解酶系非立体选择性和非特异性非立体选择性和非特异性 漆酶（漆酶（laccase）n含铜的多酚氧化含铜的多酚氧化酶酶

51、,白腐真菌降解白腐真菌降解木质素的重要酶木质素的重要酶 n具有广泛的底物具有广泛的底物作用范围和独特作用范围和独特的生物降解功能的生物降解功能n环境生物技术环境生物技术biotechnology advances 24 (2006) 500513nuse degenerate pcr( 简并简并pcr) to clone laccase genecopper-binding region is highly conservedcopper-binding region i: his-trp-his-gly-phe-phe-glncopper-binding region iv: his-cys

52、-his-ile-asp-phe-his简并引物简并引物pcr获得获得1.6kb漆酶基因特异性序列漆酶基因特异性序列基因组步移技术获得基因组步移技术获得2118bp漆酶全长结构基因漆酶全长结构基因race和和rt-pcr克隆得到了克隆得到了1566bp 漆酶基因全长漆酶基因全长cdna序列序列1 22kb1kb-我们自己的研究工作我们自己的研究工作question:by degenerate pcr, only the partial sequence of one gene can be obtained, how can we get the entire gene?chromosome

53、walking (染色体步移技术染色体步移技术) long distance inverse pcr technique for efficient cloning of flanking sequences adjacent to known dna fragmentsinverse pcr(反向反向pcr)nreverse transcriptase (rt)-pcr 逆转录逆转录pcr检测基因的转录量检测基因的转录量pcr application-example 2reverse transcriptase (rt)-pcr逆转录逆转录pcraaa(a)n5-capmrna(dt)121

54、8 primeranneal5-capaaa(a)n35reverse transcriptiondntp, rt5-capaaa(a)n5cdna:mrna hybridregularpcrrt-pcr applicationnclone cdna of specific genendetecting the expression level of geneclone cdna of specific gene by rt-pcrnstudy this technique from experiment(实战中学习实战中学习)detecting the expression level of

55、 gene by rt-pcrmrnacdnart-pcr productsemiquantitative rt-pcr （半定量（半定量rt-pcr）right panel: pcr amplification of the cloned lac1 cdna using the cycle number obtainedfrom the left panelvalidation of semiquantitative rt-pcr assays for expression level of lac1(laccase gene)left panel: kinetics ofpcr ampli

56、fication with the electrophoretic image shown at the top. the cycle number (28) that generates half maximal reaction wasused to analyse the expression of the gene.the primers for semiquantitative rt-pcr : plac1f (5-agctttcattcccagtgattg-3) and plac1r (5-aacgagctcaagtacaaatgact-3) were designed accor

57、dingto our cloned cdna for laccase geneeur. j. biochem. (2004) 271, 318328eur. j. biochem. (2004) 271, 318328rt-pcr analysis of transcription of lac1 induced by different concentrations of copperlac1: laccase genegpd: house-keeping genethe expression levels were normalized by using the relative mrna

58、 ratio (lac1/gpd)-semiquantitative rt-pcr rt-pcr(反转录反转录pcr）:effects of various copperconcentrations on lcc mrnameasure the laccase activity:effects of various copper concentrations on laccase activityveratric acidferulic acid(阿魏酸阿魏酸)2,5-xylidinevanillin芳香化合物芳香化合物 allowing the exogenous dna to be sto

59、red 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 transcription. the inserted gene h

60、as to have a start codon and a stop codon for translationt7 promoterrbsstart codonmcs (多克隆多克隆位点）位点）transcription terminatoramprorit7 expression vector1.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 pag