分子生物學課件

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 分子的分離,5.2 Labeled Tracers 標記性示蹤物,5.

2、4 Mapping and Quantifying Transcripts 轉(zhuǎn)錄物的圖譜定位與定量,5.3 Using Nucleic Acid Hybridization 核酸雜交,5.5 Measuring Transcription Rates in Vivo 體內(nèi)轉(zhuǎn)錄效率的檢測,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 分子的分離,5.2 Labeled Tracers 標記性示蹤物,5.4 Mapping and Quantifyi

3、ng Transcripts 轉(zhuǎn)錄物的圖譜定位與定量,5.3 Using Nucleic Acid Hybridization 核酸雜交,5.5 Measuring Transcription Rates in Vivo 體內(nèi)轉(zhuǎn)錄效率的檢測,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分子的分離,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(細胞粗提物) 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,凝膠電泳,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熒光染料,5-8,DNA Size Estimation,Comparison with standards permits size estimation 與標準分子量相比 Electrophoresis of unknown DNA in parallel with standard fragments permits size estimation 堿基對數(shù)目的對數(shù)與遷移距離成反比 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過大時，對數(shù)與遷移距離嚴重偏離線性關(guān)系 Instead of constant current, alternate long pulses of current in forward direction with shorter pulses in either opposite or sideways direction不采用恒定電流，而用脈沖電流，正向長時間脈沖，反方向上短暫的脈沖 Technique is called pu

9、lsed-field gel electrophoresis (PFGE)脈沖場凝膠電泳,5-10,酵母染色體脈沖電泳，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去垢劑 such as SDS SDS coats polyp

10、eptides with negative charges so all move to anode陽極 Masks natural charges of protein subunits so all move relative to mass not charge遮蔽原有電荷 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可以分離多肽，但過于復(fù)雜的多肽混合物則需要更好的方法 Two-dimensional gel electrophoresis can be done如雙向電泳,5-15,A Simple 2-D Method簡單的雙向電泳,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梯度凝膠中進行等電點聚焦 Negatively charged protein moves to its isoelectric point at which it is no longer charged蛋白到達等電點時不在移動 Tube gel is removed and used as the sample in the second process切下，進行下一步,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進行常規(guī)聚丙烯酰胺電泳 Proteins partially resolved by isoelectric focusing are further resolved according to size進一步按分子量分離 When used to a compare complex mixtures of proteins prepared under two different conditions, even subtle differences are visib

15、le通過兩種分離方式，細微的差別也可以被找出,5-19,charge,size,When used to a compare complex mixtures of proteins prepared under two different conditions, even subtle differences are visible 通過兩種分離方式，細微的差別也可以被找出,5-20,5-21,5-22,Ion-Exchange Chromatography離子交換層析,Chromatography originally referred to the pattern seen after

16、separating colored substances on paper最早是指在紙上將有色物質(zhì)分離后的圖案 Ion-exchange chromatography uses a resin to separate substances by charge利用樹脂根據(jù)電荷進行分離 This is especially useful for proteins Resin is placed in a column and sample loaded onto the column material樹脂被填充成柱狀物,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離子濃度提高，陰離子與蛋白競爭樹脂上的離子結(jié)合位點,離子交換層析是用離子交換劑（具有離子交換性能的物質(zhì)）作固定相，利用它與流動相中的離子能進行可逆的交換性質(zhì)來分離離子型化合物的層析方法。即溶液中的離子同離子交換劑上功能基團交換的反應(yīng)過程。帶電量小，親和力小的先被洗脫下來，帶電量多，親和力大的后被洗脫下來。,5-24

18、,Gel Filtration Chromatography凝膠過濾層析,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多孔樹脂類 Larger substances travel faster through the column,5-25,5-26,Affi

19、nity Chromatography親和層析,In affinity chromatography, the resin contains a substance to which the molecule of interest has a strong and specific affinity樹脂含有與目的分子有強專一性的親和試劑 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雜分子被洗脫了 Last, the molecule of interest is eluted from the column using a specific solution that disrupts the specific binding特異性洗脫液將目標分子洗脫,5-27,5-28,5.1 Molecular Separations 分子的分離,5.2 Labeled Tracers 標記性示蹤物,5.4 Mapping and Quantifying Transcripts 轉(zhuǎn)錄物的圖譜定位與定量,5.3 Using

21、Nucleic Acid Hybridization 核酸雜交,5.5 Measuring Transcription Rates in Vivo 體內(nèi)轉(zhuǎn)錄效率的檢測,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標記性示蹤物,For many years “l(fā)abeled” has been synonymous with “radioactive”等同于放射性 Radioactive tracers allow vanishingly small qua

22、ntities of substances to be detected放射性極其靈敏 Molecular biology experiments typically require detection of extremely small amounts of a particular substance極微量,5-30,Autoradiography放射性自顯影,Autoradiography is a means of detecting radioactive compounds with a photographic emulsion照相感光乳劑 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按黑度分析相對量 More precise measurements are made using densitometer光密度計 Area under peaks on a tracing by a scanner Proportional to darkness of the bands on autoradiogram放射自顯影圖,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更加線性化 Place gel with radioactive bands in contact with a phosphorimager plate將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電子激發(fā)板上的分子，直到磷屏成像儀用光束掃描成像板 Molecular excitation is m

26、onitored by a detector這種分子激發(fā)被檢測儀記錄并轉(zhuǎn)換成圖像,5-33,5-34,Liquid Scintillation Counting液體閃爍計數(shù)器,Radioactive emissions from a sample create photons of visible light are detected by a photomultiplier tube in the process of liquid scintillation counting利用樣品放射性發(fā)出的射線產(chǎn)生的一種能被光電倍增管檢測到的可見光光子 Remove the radioactive m

27、aterial (band from gel) to a vial containing scintillation fluid將放射性樣品放到含有閃爍液的小瓶中 Fluid contains a fluor that fluoresces when hit with radioactive emissions放射線轟擊后產(chǎn)生熒光 Acts to convert invisible radioactivity into visible light從而將放射線轉(zhuǎn)化成可見光 單位是每分鐘閃爍的次數(shù),5-35,Nonradioactive Tracers非放射性示蹤劑,Newer nonradioa

28、ctive tracers now rival older radioactive tracers in sensitivity非放射性示蹤劑已達到高靈敏度 These tracers do not have hazards: Health exposure健康 Handling方便使用 Disposal廢物處理 Increased sensitivity is from use of a multiplier effect of an enzyme that is coupled to probe for molecule of interest利用酶的倍增效應(yīng),5-36,對DNA標記來說，

29、DIG通過一個不耐堿的酯鍵與dUTP相聯(lián),雜交后的探針與偶聯(lián)有堿性磷酸酶的抗體（anti-DIG-AP）結(jié)合，然后用顯色底物NBT/BCIP或化學發(fā)光底物CSPD檢測與DIG結(jié)合的抗體。,37,38,39,40,41,42,43,44,45,46,47,48,49,5-50,5.1 Molecular Separations 分子的分離,5.2 Labeled Tracers 標記性示蹤物,5.4 Mapping and Quantifying Transcripts 轉(zhuǎn)錄物的圖譜定位與定量,5.3 Using Nucleic Acid Hybridization 核酸雜交,5.5 Measu

30、ring Transcription Rates in Vivo 體內(nèi)轉(zhuǎn)錄效率的檢測,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核酸雜交,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噬菌班和菌落雜交 This section looks at techniques for isolated nucleic acids離體核酸分子的雜交,5-52,Southern Blots: Identifying Specific DNA Fragments鑒定特異性DNA片段,Digests of genomic DNA are separated on agarose gel酶切分離 The separated pieces are transferre

32、d to filter by diffusion, or more recently by electrophoresing the bands onto the filter轉(zhuǎn)到另一膜上 Filter is treated with alkali to denature the DNA, resulting ssDNA binds to the filter堿變性 Probe the filter using labeled cDNA,5-53,5-54,DNA Fingerprinting and DNA TypingDNA指紋技術(shù)和DNA分型,Southern blots are use

33、d in forensic法醫(yī)的labs to identify individuals from DNA-containing materials Minisatellite DNA is a sequence of bases repeated several times, also called DNA fingerprint幾個堿基重復(fù)幾次出現(xiàn) Individuals differ in the pattern of repeats of the basic sequence個體不同，重復(fù)方式不同 Difference is large enough that 2 people hav

34、e only a remote chance of having exactly the same pattern兩個體相同的重復(fù)方式幾乎不可能,5-55,DNA Fingerprinting,Process really just a Southern blot DNA指紋實質(zhì)就是Southern雜交 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法醫(yī)的 Uses of DNA Fingerprinting and DNA Typing,While people have different DNA fingerprints, parts of the pattern are inherited in a Mendelian fashion雖然幾乎所有個體都有不同的DNA指紋，但是部分圖譜是按照孟德爾定律的 Can be used to

36、establish parentage建立親子關(guān)系 Potential to identify criminals鑒定刑事犯罪 Remove innocent people from suspicion避免冤案 Actual pattern has so many bands they can smear together indistinguishably實際圖譜太復(fù)雜，難于辯認 Forensics uses probes for just a single locus開發(fā)新探針，只與單一的在個體中有很大變異的DNA位點雜交 Set of probes gives a set of simp

37、le patterns雜交后只有一個或者幾個條帶,5-57,左邊兩個是媽媽爸爸。 四個孩子里面D1和S1是他們兩個親生的。子女不應(yīng)該有父母沒有的條帶（除非突變啦，罕見）。 D2是媽媽和前夫生的，所以有兩條紅色的條帶匹配不到這對夫婦，應(yīng)該是來自前夫。 S2是領(lǐng)養(yǎng)的，所以一條都沒有匹配到。,5-58,嫌疑人A的DNA,嫌疑人B的DNA,受害人衣服上的精液DNA,受害人陰道樣本DNA,受害人DNA,5-59,In Situ Hybridization: Locating Genes in Chromosomes原位雜交，基因在染色體上的定位,Labeled probes can be used to

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

39、detected with a fluorescent antibody in a technique called fluorescence in situ hybridization (FISH)熒光標記的核酸探針在變性后與已變性的靶核酸在退火溫度下復(fù)性；通過熒光顯微鏡觀察熒光信號可在不改變被分析對象（即維持其原位）的前提下對靶核酸進行分析。DNA熒光標記探針是其中最常用的一類核酸探針。,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免疫印跡,Immunoblots (also called

40、 Western blots) use a similar process to Southern blots Electrophoresis of proteins蛋白電泳 Blot the proteins from the gel to a membrane轉(zhuǎn)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雙脫氧核苷酸終止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雙脫氧核苷酸決定各片段最后一個堿基 Ordering the fragments by size tells the base sequence of the DNA按片段順序可以讀出

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種雙脫氧核苷酸帶上熒光 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混合物在同一泳道內(nèi)電泳，電腦記錄熒光,5-77,5-78,5-79,Restriction Mapping限制性圖譜,Prior to start of large-scale sequencing preliminary work is done to locate landmarks大分子測序先要定位一些標記 A map based on physical characteristics is called a physical map基于物理特征的圖譜 If restriction sites are the on

45、ly map features then a restriction map has been prepared基于內(nèi)切酶為標記的圖譜 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電泳分離 Can also use same digest to find the orientation of an insert cloned into a vector通過片段大小確定方向,5-81,5-82,Protein Engineering With Cloned Genes: Site-Directed Mutagenesis基于克隆基因的蛋白質(zhì)工程：定點突變,Cloned genes permit biochemical microsurgery on proteins基因水平利于對蛋白的顯微操作 Specific bases in

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

48、utagenesis With PCR基于PCR的定點誘變,5-84,5.1 Molecular Separations 分子的分離,5.2 Labeled Tracers 標記性示蹤物,5.4 Mapping and Quantifying Transcripts 轉(zhuǎn)錄物的圖譜定位與定量,5.3 Using Nucleic Acid Hybridization 核酸雜交,5.5 Measuring Transcription Rates in Vivo 體內(nèi)轉(zhuǎn)錄效率的檢測,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轉(zhuǎn)錄物的圖譜定位與定量,Mapping (locating start and end) and quantifying (how much transcript exists at a set time) are common procedures轉(zhuǎn)錄的起始和終止位點，轉(zhuǎn)錄了多少 Often transcripts do not have a uniform terminator, resulting in a continuum of species smeared on a gel轉(zhuǎn)錄

50、終止時長短不一，導(dǎo)致凝膠電泳時彌散 Techniques that specific for the sequence of interest are important所以對目標RNA專一性的檢測技術(shù)很重要,5-86,Northern Blots,You have cloned a cDNA How actively is the corresponding gene expressed in different tissues?組織差異性 Find out using a Northern Blot Obtain RNA from different tissues提取不同組織的RNA R

51、un RNA on agarose gel and blot to membrane Hybridize to a labeled cDNA probe雜交 Northern plot tells abundance of the transcript轉(zhuǎn)錄的豐度 Quantify using densitometer光密度分析,5-87,脾,骨骼肌,睪丸,5-88,S1 Mapping S1圖譜定位,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確定啟始位點或者特定時間的轉(zhuǎn)錄水平 Label a ssDNA probe that can only hybridize to transcript of interest標記探針，可識別目標mRNA Probe must span the sequence start to finish探針需橫跨啟始點或者終止點 After hybridization, treat with S1 nuclease which degrades ssDNA and RNA S1核酸酶降解單鏈核酸 Transcript protects part of the probe from d

53、egradation Size of protected area can be measured by gel electrophoresis受保護的大小通過凝膠電泳檢測,5-89,S1 Mapping the 5 End,5-90,S1 Mapping the 3 End,5-91,5.1 Molecular Separations 分子的分離,5.2 Labeled Tracers 標記性示蹤物,5.4 Mapping and Quantifying Transcripts 轉(zhuǎn)錄物的圖譜定位與定量,5.3 Using Nucleic Acid Hybridization 核酸雜交,5.5

54、 Measuring Transcription Rates in Vivo 體內(nèi)轉(zhuǎn)錄效率的檢測,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體內(nèi)轉(zhuǎn)錄效率的檢測,Primer extension, S1 mapping and Northern blotting will determine the concentration of specific transcripts at a given time以上方法

55、能檢測特定時間里的目標轉(zhuǎn)錄濃度 These techniques do not really reveal the rate of transcript synthesis as concentration involves both: Transcript synthesis Transcript degradation 但不能檢測轉(zhuǎn)錄合成速度和降解速度,5-93,Reporter Gene Transcription報告基因轉(zhuǎn)錄分析,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(以無色的ONPG為底物，在-半乳糖苷酶的催化下生成黃色物質(zhì)，然后通過酶標儀或分光光度計在420nm波長附近測定吸光度，從而實現(xiàn)對-半乳糖苷酶活性的測定) cat produces chloram

57、phenicol acetyl transferase (CAT) which inhibits bacterial growth氯霉素乙酰轉(zhuǎn)移酶(可以將氯霉素乙酞化而使其失活，是細菌產(chǎn)生氯霉素抗性的主要原因) Luciferase produces chemiluminescent compound that emits light 熒光素酶,5-94,5-95,Measuring Protein Accumulation in Vivo體內(nèi)蛋白質(zhì)積累水平的檢測,Gene activity can be monitored by measuring the accumulation of p

58、rotein (the ultimate gene product)基因的最終產(chǎn)物量 Two primary methods of measuring protein accumulation Immunoblotting / Western blotting免疫印跡 Immunoprecipitation免疫沉淀,5-96,Immunoprecipitation,Label proteins by growing cells with 35S-labeled amino acid蛋白合成時被標記 Bind protein of interest to an antibody結(jié)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 結(jié)合二抗或者蛋白A（其上帶磁株),低速可沉淀 Determine protein level with liquid scintillation counting 放射自顯影（液體閃爍計數(shù)器）,5-97,5.1 Molecular Separations 分子的分離,5.2 Labeled Tracers 標記性示蹤物,5.4 Mapping and Quantifying Transcripts 轉(zhuǎn)錄物的圖譜定位與定量,5.3 Using Nucleic Acid Hybridization 核酸雜交,5.5 Measuring Transcription Rates in Vivo 體內(nèi)轉(zhuǎn)錄效率的檢測,5.7 Knockouts 基因敲除,5.6 Assaying DNA-Protein Interactions DNA和蛋白互作分析,5-98,