分子生物学课件

1、Molecular BiologyFourth Edition,Chapter 5 Molecular Tools for Studying Genes and Gene Activity,Lecture PowerPoint to accompany,Robert F. Weaver,Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.,5-2,5.1 Molecular Separations 分子的分离,5.2 Labeled Tracers 标记性示踪物,5.

2、4 Mapping and Quantifying Transcripts 转录物的图谱定位与定量,5.3 Using Nucleic Acid Hybridization 核酸杂交,5.5 Measuring Transcription Rates in Vivo 体内转录效率的检测,5.7 Knockouts 基因敲除,5.6 Assaying DNA-Protein Interactions DNA和蛋白互作分析,5-3,5.1 Molecular Separations 分子的分离,5.2 Labeled Tracers 标记性示踪物,5.4 Mapping and Quantifyi

3、ng Transcripts 转录物的图谱定位与定量,5.3 Using Nucleic Acid Hybridization 核酸杂交,5.5 Measuring Transcription Rates in Vivo 体内转录效率的检测,5.7 Knockouts 基因敲除,5.6 Assaying DNA-Protein Interactions DNA和蛋白互作分析,5-4,5.1 Molecular Separations分子的分离,Often mixtures of proteins or nucleic acids are generated during the course

4、of molecular biological procedures A protein may need to be purified from a crude cellular extract(细胞粗提物) A particular nucleic acid molecule made in a reaction needs to be purified,5-5,Gel Electrophoresis,Gel electrophoresis is used to separate different species of: Nucleic acid Protein,凝胶电泳,5-6,DNA

5、 Gel Electrophoresis,Melted agarose is poured into a form equipped with removable comb Comb “teeth” form slots in the solidified agarose DNA samples are placed in the slots An electric current is run through the gel at a neutral pH,5-7,DNA Separation by Agarose Gel Electrophoresis,DNA is negatively

6、charged due to phosphates in its backbone and moves to anode, the positive pole Small DNA pieces have little frictional drag摩擦力 so move rapidly Large DNAs have more frictional drag so their mobility is slower Result distributes DNA according to size Largest near the top Smallest near the bottom DNA

7、is stained with fluorescent dye荧光染料,5-8,DNA Size Estimation,Comparison with standards permits size estimation 与标准分子量相比 Electrophoresis of unknown DNA in parallel with standard fragments permits size estimation 碱基对数目的对数与迁移距离成反比 Same principles apply to RNA separation,5-9,Electrophoresis of Large DNA,

8、Special techniques are required for DNA fragments larger than about 1 kilobasesDNA过大时，对数与迁移距离严重偏离线性关系 Instead of constant current, alternate long pulses of current in forward direction with shorter pulses in either opposite or sideways direction不采用恒定电流，而用脉冲电流，正向长时间脉冲，反方向上短暂的脉冲 Technique is called pu

9、lsed-field gel electrophoresis (PFGE)脉冲场凝胶电泳,5-10,酵母染色体脉冲电泳，0.2M2.2Mbp,5-11,Protein Gel Electrophoresis,Separation of proteins is done using a gel made of polyacrylamide (polyacrylamide gel electrophoresis = PAGE)聚丙烯酰胺 Treat proteins to denature subunits with detergent去垢剂 such as SDS SDS coats polyp

10、eptides with negative charges so all move to anode阳极 Masks natural charges of protein subunits so all move relative to mass not charge遮蔽原有电荷 As with DNA smaller proteins move faster toward the anode,5-12,5-13,5-14,Two-Dimensional Gel Electrophoresis,While SDS-PAGE gives good resolution of polypeptid

11、es, some mixtures are so complex that additional resolution is needed SDS-PAGE可以分离多肽，但过于复杂的多肽混合物则需要更好的方法 Two-dimensional gel electrophoresis can be done如双向电泳,5-15,A Simple 2-D Method简单的双向电泳,Run samples in 2 gels First dimension separates using one concentration of polyacrylamide at one pH Second dim

12、ension uses different concentration of polyacrylamide and pH Proteins move differently at different pH values without SDS and at different acrylamide丙烯酰胺 concentrations,5-16,Two-Dimensional Gel Electrophoresis Details,A two process method: Isoelectric focusing gel: mixture of proteins electrophorese

13、d through gel in a narrow tube containing a pH gradient pH梯度凝胶中进行等电点聚焦 Negatively charged protein moves to its isoelectric point at which it is no longer charged蛋白到达等电点时不在移动 Tube gel is removed and used as the sample in the second process切下，进行下一步,5-17,5-18,Standard SDS-PAGE: Tube gel is removed and

14、used as the sample at the top of a standard polyacrylamide gel进行常规聚丙烯酰胺电泳 Proteins partially resolved by isoelectric focusing are further resolved according to size进一步按分子量分离 When used to a compare complex mixtures of proteins prepared under two different conditions, even subtle differences are visib

15、le通过两种分离方式，细微的差别也可以被找出,5-19,charge,size,When used to a compare complex mixtures of proteins prepared under two different conditions, even subtle differences are visible 通过两种分离方式，细微的差别也可以被找出,5-20,5-21,5-22,Ion-Exchange Chromatography离子交换层析,Chromatography originally referred to the pattern seen after

16、separating colored substances on paper最早是指在纸上将有色物质分离后的图案 Ion-exchange chromatography uses a resin to separate substances by charge利用树脂根据电荷进行分离 This is especially useful for proteins Resin is placed in a column and sample loaded onto the column material树脂被填充成柱状物,5-23,Once the sample is loaded， buffer

17、 is passed over the resin + sample As ionic strength of elution buffer increases, samples of solution flowing through the column are collected离子浓度提高，阴离子与蛋白竞争树脂上的离子结合位点,离子交换层析是用离子交换剂（具有离子交换性能的物质）作固定相，利用它与流动相中的离子能进行可逆的交换性质来分离离子型化合物的层析方法。即溶液中的离子同离子交换剂上功能基团交换的反应过程。带电量小，亲和力小的先被洗脱下来，带电量多，亲和力大的后被洗脱下来。,5-24

18、,Gel Filtration Chromatography凝胶过滤层析,Protein size is a valuable property that can be used as a basis of physical separation Gel filtration uses columns filled with porous resins that let in smaller substances, exclude larger ones多孔树脂类 Larger substances travel faster through the column,5-25,5-26,Affi

19、nity Chromatography亲和层析,In affinity chromatography, the resin contains a substance to which the molecule of interest has a strong and specific affinity树脂含有与目的分子有强专一性的亲和试剂 The molecule binds to a column resin coupled to the affinity reagent Molecule of interest is retained目的分子被吸附了 Most other molecule

20、s flow through without binding杂分子被洗脱了 Last, the molecule of interest is eluted from the column using a specific solution that disrupts the specific binding特异性洗脱液将目标分子洗脱,5-27,5-28,5.1 Molecular Separations 分子的分离,5.2 Labeled Tracers 标记性示踪物,5.4 Mapping and Quantifying Transcripts 转录物的图谱定位与定量,5.3 Using

21、Nucleic Acid Hybridization 核酸杂交,5.5 Measuring Transcription Rates in Vivo 体内转录效率的检测,5.7 Knockouts 基因敲除,5.6 Assaying DNA-Protein Interactions DNA和蛋白互作分析,5-29,5.2 Labeled Tracers标记性示踪物,For many years “labeled” has been synonymous with “radioactive”等同于放射性 Radioactive tracers allow vanishingly small qua

22、ntities of substances to be detected放射性极其灵敏 Molecular biology experiments typically require detection of extremely small amounts of a particular substance极微量,5-30,Autoradiography放射性自显影,Autoradiography is a means of detecting radioactive compounds with a photographic emulsion照相感光乳剂 Preferred emulsion

23、 is x-ray film底片 DNA is separated on a gel and radiolabeled Gel is placed in contact with x-ray film for hours or days Radioactive emissions from the labeled DNA expose the film Developed film shows dark bands,5-31,Autoradiography Analysis,Relative quantity of radioactivity can be assessed looking a

24、t the developed film按黑度分析相对量 More precise measurements are made using densitometer光密度计 Area under peaks on a tracing by a scanner Proportional to darkness of the bands on autoradiogram放射自显影图,5-32,Phosphorimaging磷屏成像,This technique is more accurate in quantifying amount of radioactivity in a substanc

25、e更好的定量 Response to radioactivity is much more linear更加线性化 Place gel with radioactive bands in contact with a phosphorimager plate将放射性膜放于磷屏成像板上 Plate absorbs b electrons that excite molecules on the plate which remain excited until plate is scanned b电子激发板上的分子，直到磷屏成像仪用光束扫描成像板 Molecular excitation is m

26、onitored by a detector这种分子激发被检测仪记录并转换成图像,5-33,5-34,Liquid Scintillation Counting液体闪烁计数器,Radioactive emissions from a sample create photons of visible light are detected by a photomultiplier tube in the process of liquid scintillation counting利用样品放射性发出的射线产生的一种能被光电倍增管检测到的可见光光子 Remove the radioactive m

27、aterial (band from gel) to a vial containing scintillation fluid将放射性样品放到含有闪烁液的小瓶中 Fluid contains a fluor that fluoresces when hit with radioactive emissions放射线轰击后产生荧光 Acts to convert invisible radioactivity into visible light从而将放射线转化成可见光 单位是每分钟闪烁的次数,5-35,Nonradioactive Tracers非放射性示踪剂,Newer nonradioa

28、ctive tracers now rival older radioactive tracers in sensitivity非放射性示踪剂已达到高灵敏度 These tracers do not have hazards: Health exposure健康 Handling方便使用 Disposal废物处理 Increased sensitivity is from use of a multiplier effect of an enzyme that is coupled to probe for molecule of interest利用酶的倍增效应,5-36,对DNA标记来说，

29、DIG通过一个不耐碱的酯键与dUTP相联,杂交后的探针与偶联有碱性磷酸酶的抗体（anti-DIG-AP）结合，然后用显色底物NBT/BCIP或化学发光底物CSPD检测与DIG结合的抗体。,37,38,39,40,41,42,43,44,45,46,47,48,49,5-50,5.1 Molecular Separations 分子的分离,5.2 Labeled Tracers 标记性示踪物,5.4 Mapping and Quantifying Transcripts 转录物的图谱定位与定量,5.3 Using Nucleic Acid Hybridization 核酸杂交,5.5 Measu

30、ring Transcription Rates in Vivo 体内转录效率的检测,5.7 Knockouts 基因敲除,5.6 Assaying DNA-Protein Interactions DNA和蛋白互作分析,5-51,5.3 Using Nucleic Acid Hybridization核酸杂交,Hybridization is the ability of one single-stranded nucleic acid to form a double helix with another single strand of complementary base sequen

31、ce Previous discussion focused on colony and plaque hybridization噬菌班和菌落杂交 This section looks at techniques for isolated nucleic acids离体核酸分子的杂交,5-52,Southern Blots: Identifying Specific DNA Fragments鉴定特异性DNA片段,Digests of genomic DNA are separated on agarose gel酶切分离 The separated pieces are transferre

32、d to filter by diffusion, or more recently by electrophoresing the bands onto the filter转到另一膜上 Filter is treated with alkali to denature the DNA, resulting ssDNA binds to the filter碱变性 Probe the filter using labeled cDNA,5-53,5-54,DNA Fingerprinting and DNA TypingDNA指纹技术和DNA分型,Southern blots are use

33、d in forensic法医的labs to identify individuals from DNA-containing materials Minisatellite DNA is a sequence of bases repeated several times, also called DNA fingerprint几个碱基重复几次出现 Individuals differ in the pattern of repeats of the basic sequence个体不同，重复方式不同 Difference is large enough that 2 people hav

34、e only a remote chance of having exactly the same pattern两个体相同的重复方式几乎不可能,5-55,DNA Fingerprinting,Process really just a Southern blot DNA指纹实质就是Southern杂交 Cut the DNA under study with restriction enzyme Ideally cut on either side of minisatellite but not inside Run digest on a gel and blot Probe with

35、labeled minisatellite DNA and imaged Real samples result in very complex patterns,5-56,Forensicfrensk法医的 Uses of DNA Fingerprinting and DNA Typing,While people have different DNA fingerprints, parts of the pattern are inherited in a Mendelian fashion虽然几乎所有个体都有不同的DNA指纹，但是部分图谱是按照孟德尔定律的 Can be used to

36、establish parentage建立亲子关系 Potential to identify criminals鉴定刑事犯罪 Remove innocent people from suspicion避免冤案 Actual pattern has so many bands they can smear together indistinguishably实际图谱太复杂，难于辩认 Forensics uses probes for just a single locus开发新探针，只与单一的在个体中有很大变异的DNA位点杂交 Set of probes gives a set of simp

37、le patterns杂交后只有一个或者几个条带,5-57,左边两个是妈妈爸爸。 四个孩子里面D1和S1是他们两个亲生的。子女不应该有父母没有的条带（除非突变啦，罕见）。 D2是妈妈和前夫生的，所以有两条红色的条带匹配不到这对夫妇，应该是来自前夫。 S2是领养的，所以一条都没有匹配到。,5-58,嫌疑人A的DNA,嫌疑人B的DNA,受害人衣服上的精液DNA,受害人阴道样本DNA,受害人DNA,5-59,In Situ Hybridization: Locating Genes in Chromosomes原位杂交，基因在染色体上的定位,Labeled probes can be used to

38、 hybridize to chromosomes and reveal which chromosome contains the gene of interest Spread chromosomes from a cell排列染色体 Partially denature DNA creating single-stranded regions to hybridize to labeled probe染色体变性 Stain chromosomes and detect presence of label on particular chromosome杂交定位 Probe can be

39、detected with a fluorescent antibody in a technique called fluorescence in situ hybridization (FISH)荧光标记的核酸探针在变性后与已变性的靶核酸在退火温度下复性；通过荧光显微镜观察荧光信号可在不改变被分析对象（即维持其原位）的前提下对靶核酸进行分析。DNA荧光标记探针是其中最常用的一类核酸探针。,5-60,5-61,5-62,5-63,5-64,5-65,5-66,5-67,5-68,正常,5-69,膀胱癌,5-70,Immunoblots免疫印迹,Immunoblots (also called

40、 Western blots) use a similar process to Southern blots Electrophoresis of proteins蛋白电泳 Blot the proteins from the gel to a membrane转膜 Detect the protein using antibody or antiserum to the target protein一抗 Labeled secondary antibody is used to bind the first antibody and increase the signal二抗,5-71,W

41、estern Blots,5-72,DNA Sequencing,Sanger, Maxam, Gilbert developed 2 methods for determining the exact base sequence of a cloned piece of DNA Modern DNA sequencing is based on the Sanger method,5-73,Sanger Manual Sequencing,Sanger DNA sequencing method uses dideoxy nucleotides to terminate DNA synthe

42、sis双脱氧核苷酸终止DNA合成 The process yields a series of DNA fragments whose size is measured by electrophoresis大小不一的片段 Last base in each fragment is known as that dideoxy nucleotide was used to terminate the reaction双脱氧核苷酸决定各片段最后一个碱基 Ordering the fragments by size tells the base sequence of the DNA按片段顺序可以读出

43、DNA序列,5-74,5-75,5-76,Automated DNA Sequencing,Manual sequencing is powerful but slow Automated sequencing uses dideoxynucleotides tagged with different fluorescent molecules 4种双脱氧核苷酸带上荧光 Products of each dideoxynucleotide will fluoresce a different color Four reactions are completed, then mixed toge

44、ther and run out on one lane of a gel混合物在同一泳道内电泳，电脑记录荧光,5-77,5-78,5-79,Restriction Mapping限制性图谱,Prior to start of large-scale sequencing preliminary work is done to locate landmarks大分子测序先要定位一些标记 A map based on physical characteristics is called a physical map基于物理特征的图谱 If restriction sites are the on

45、ly map features then a restriction map has been prepared基于内切酶为标记的图谱 Consider a 1.6 kb piece of DNA as an example,5-80,Restriction Map Example,Cut separate samples of the original 1.6 kb fragment with different restriction enzymes酶切成片段 Separate the digests on an agarose gel to determine the size of p

46、ieces from each digest电泳分离 Can also use same digest to find the orientation of an insert cloned into a vector通过片段大小确定方向,5-81,5-82,Protein Engineering With Cloned Genes: Site-Directed Mutagenesis基于克隆基因的蛋白质工程：定点突变,Cloned genes permit biochemical microsurgery on proteins基因水平利于对蛋白的显微操作 Specific bases in

47、 a gene may be changed碱基改变 Amino acids at specific sites in the protein product may also be altered相应的氨酸酸改变 Effects of those changes on protein function can be observed蛋白功能改变 Might investigate the role of phenolic group on tyrosine compared to phenylalanine假定要知道酚基的重要性，将酪氨酸改成苯丙氨酸,5-83,Site-Directed M

48、utagenesis With PCR基于PCR的定点诱变,5-84,5.1 Molecular Separations 分子的分离,5.2 Labeled Tracers 标记性示踪物,5.4 Mapping and Quantifying Transcripts 转录物的图谱定位与定量,5.3 Using Nucleic Acid Hybridization 核酸杂交,5.5 Measuring Transcription Rates in Vivo 体内转录效率的检测,5.7 Knockouts 基因敲除,5.6 Assaying DNA-Protein Interactions DNA

49、和蛋白互作分析,5-85,5.4 Mapping and Quantifying Transcripts转录物的图谱定位与定量,Mapping (locating start and end) and quantifying (how much transcript exists at a set time) are common procedures转录的起始和终止位点，转录了多少 Often transcripts do not have a uniform terminator, resulting in a continuum of species smeared on a gel转录

50、终止时长短不一，导致凝胶电泳时弥散 Techniques that specific for the sequence of interest are important所以对目标RNA专一性的检测技术很重要,5-86,Northern Blots,You have cloned a cDNA How actively is the corresponding gene expressed in different tissues?组织差异性 Find out using a Northern Blot Obtain RNA from different tissues提取不同组织的RNA R

51、un RNA on agarose gel and blot to membrane Hybridize to a labeled cDNA probe杂交 Northern plot tells abundance of the transcript转录的丰度 Quantify using densitometer光密度分析,5-87,脾,骨骼肌,睾丸,5-88,S1 Mapping S1图谱定位,Use S1 mapping to locate the ends of RNAs and to determine the amount of a given RNA in cells at a

52、 given time确定启始位点或者特定时间的转录水平 Label a ssDNA probe that can only hybridize to transcript of interest标记探针，可识别目标mRNA Probe must span the sequence start to finish探针需横跨启始点或者终止点 After hybridization, treat with S1 nuclease which degrades ssDNA and RNA S1核酸酶降解单链核酸 Transcript protects part of the probe from d

53、egradation Size of protected area can be measured by gel electrophoresis受保护的大小通过凝胶电泳检测,5-89,S1 Mapping the 5 End,5-90,S1 Mapping the 3 End,5-91,5.1 Molecular Separations 分子的分离,5.2 Labeled Tracers 标记性示踪物,5.4 Mapping and Quantifying Transcripts 转录物的图谱定位与定量,5.3 Using Nucleic Acid Hybridization 核酸杂交,5.5

54、 Measuring Transcription Rates in Vivo 体内转录效率的检测,5.7 Knockouts 基因敲除,5.6 Assaying DNA-Protein Interactions DNA和蛋白互作分析,5-92,5.5 Measuring Transcription Rates in Vivo体内转录效率的检测,Primer extension, S1 mapping and Northern blotting will determine the concentration of specific transcripts at a given time以上方法

55、能检测特定时间里的目标转录浓度 These techniques do not really reveal the rate of transcript synthesis as concentration involves both: Transcript synthesis Transcript degradation 但不能检测转录合成速度和降解速度,5-93,Reporter Gene Transcription报告基因转录分析,Place a surrogatesrt代用品，代替 reporter gene under control of a specific promoter,

56、measure accumulation of product of this reporter gene Reporter genes are carefully chosen to have products very convenient to assay lacZ produces b-galactosidase which has a blue cleavage product b半乳糖苷酶(以无色的ONPG为底物，在-半乳糖苷酶的催化下生成黄色物质，然后通过酶标仪或分光光度计在420nm波长附近测定吸光度，从而实现对-半乳糖苷酶活性的测定) cat produces chloram

57、phenicol acetyl transferase (CAT) which inhibits bacterial growth氯霉素乙酰转移酶(可以将氯霉素乙酞化而使其失活，是细菌产生氯霉素抗性的主要原因) Luciferase produces chemiluminescent compound that emits light 荧光素酶,5-94,5-95,Measuring Protein Accumulation in Vivo体内蛋白质积累水平的检测,Gene activity can be monitored by measuring the accumulation of p

58、rotein (the ultimate gene product)基因的最终产物量 Two primary methods of measuring protein accumulation Immunoblotting / Western blotting免疫印迹 Immunoprecipitation免疫沉淀,5-96,Immunoprecipitation,Label proteins by growing cells with 35S-labeled amino acid蛋白合成时被标记 Bind protein of interest to an antibody结合一抗 Prec

59、ipitate the protein-antibody complex with a secondary antibody complexed to Protein A on resin beads using a low-speed centrifuge 结合二抗或者蛋白A（其上带磁株),低速可沉淀 Determine protein level with liquid scintillation counting 放射自显影（液体闪烁计数器）,5-97,5.1 Molecular Separations 分子的分离,5.2 Labeled Tracers 标记性示踪物,5.4 Mapping and Quantifying Transcripts 转录物的图谱定位与定量,5.3 Using Nucleic Acid Hybridization 核酸杂交,5.5 Measuring Transcription Rates in Vivo 体内转录效率的检测,5.7 Knockouts 基因敲除,5.6 Assaying DNA-Protein Interactions DNA和蛋白互作分析,5-98,