1 welcome to mb class 2 molecular biology of the gene, 5/e --- watson et al. (2004) part i:...

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Welcome to

MB Class

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

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Ch 20: Techniques of Molecular Biology

Ch 21: Model Organisms

Part V: METHODSPart V: METHODS

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•Molecular Biology Course

Chapter 20Chapter 20

Techniques of Techniques of

Molecular BiologyMolecular Biology

Preparation, analysis and manipulation of nucleic acids and

proteins

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The methods depend upon, and were developed from, an understanding of the properties of biological macromolecules themselves.

Hybridization---the base-pairing characteristics of DNA and RNA

DNA cloning--- DNA polymerase, restriction endonucleases and DNA ligase

PCR---Thermophilic DNA polymerase

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Topic 1: Topic 1: Nucleic acidsNucleic acids

CHAPTER20: Techniques of Molecular Biology

1.1.SeparationSeparation by Electrophoresis ( 电泳分离 )

2.2.CutCut by Restriction endonuclease ( 限制性内切酶切割 )

3.3.IdentificationIdentification by Hybridization ( 杂交鉴定 )

4.4.AmplificationAmplification by PCR (PCR 扩增 )5.DNA Cloning and gene expression

(DNA 克隆和基因表达 )6.Genome sequence & analysis ( 基因

组序列和分析 )

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1. Gel electrophoresis 1. Gel electrophoresis separates DNA and RNA separates DNA and RNA molecules according to molecules according to sizesize, , shapeshape and and topological topological properties.properties.

Topic 1 Nucleic acids-separation

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1.DNA and RNA molecules are negatively charged, thus move in the gel matrix ( 胶支持物 ) toward the positive pole ( 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 DNA molecules is affected by their topological structures. The mobility 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 )

1.DNA and RNA molecules are negatively charged, thus move in the gel matrix ( 胶支持物 ) toward the positive pole ( 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 DNA molecules is affected by their topological structures. The mobility 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 )

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Fig 21-1: DNA is separated by gel electrophoresis

large moderate small

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Gel matrix ( 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 )

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Agarose ( 琼脂糖 ): (1)a much less

resolving power than polyacrylamide,

(2)but can separate DNA molecules of up to tens of kb

1 kb

0.5 kb

2 kb

3 kb4 kb

DNA can be visualized by staining the gel with fluorescent dyes, such as ethidium bromide (EB 溴化乙锭 )

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Polyacrylamide ( 聚丙稀酰胺 ):

(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 narrow size range (1 to a few hundred bp).

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(1)The electric field is applied in pulses that are oriented orthogonally ( 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 over 30-50 kb and up to several Mb in length.

Pulsed-field gel electrophoresis ( 脉冲电泳 )

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Fig. 21-2 pulsed-field gel electrophoresis

Fig. 21-2 pulsed-field gel electrophoresis

Switching between two orientations: the larger the DNA is, the longer it takes to reorient

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(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 mobility.

(3)RNA can be treated with reagent 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

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Why being used?--To break large DNA molecules

into manageable fragments.

Nucleic acids-Restriction digestion2. Restriction 2. Restriction endonucleases endonucleases (( 限制性内切限制性内切酶酶 )) cleave DNA molecules cleave DNA molecules at particular sites at particular sites by the recognitionrecognition of specific sequences

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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. EcoRI5’….GAATTC.….3’

….CTTAAG….

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the 1st such

enzyme found

Escherichia coli Species category

R13strain

How to name a restriction endonuclease?

EcoRI

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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 to estimate the frequency of the RE in a DNA molecule or genome?

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Table 21-1 Table 21-1 Some restriction Endonucleases Some restriction Endonucleases and their recognition sequencesand their recognition sequences

EnzymeEnzyme SequenceSequence Frequency Frequency Sau3A1Sau3A1EcoRIEcoRINotI NotI

5’-GATC-3’5’-GATC-3’5’-GAATTC-3’5’-GAATTC-3’5’-GCGGCCGC-3’5’-GCGGCCGC-3’

0.25 kb0.25 kb4 kb4 kb65 kb65 kb

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(1) Restriction enzymes differ in the recognition specificityrecognition specificity: target sites are different.(2) Restriction enzymes differ in the length they recognizedthe length they recognized, and thus the frequencies differ.(3) Restriction enzymes differ in the nature of the DNA ends they the nature of the DNA ends they generategenerate: blunt/flush ends ( 平末端 ), sticky/staggered ends ( 粘性末端 ).(4) Restriction enzymes differ in the cleavage activitycleavage activity.

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sticky ends

( 粘性末端 )

blunt ends

( 平末端 )

Fig 21-4 Recognition sequences and cut sites of various endonucleases

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3. DNA hybridization 3. DNA hybridization can be used to identify can be used to identify specific DNA moleculesspecific DNA molecules

Hybridization: the process of base-pairing between complementary ssDNA or RNA from two different sources.

Topic 1 Nucleic acids- DNA hybridization

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A 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 colonies

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It can be readily located once it has found its target sequence.

A probe must be labeled before applied in hybridization (Why?)

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End labelingEnd labeling: put the labels at the endsUniform labelingUniform labeling: put the labels internally

Radioactive labeling: display and/or magnify the signals by radioactivityradioactivity. Non-radioactive labeling: display and/or magnify the signals by antigen labelingantigen labeling: antibody binding – enzyme binding - substrate application (signal release)

Labeling ( 标记 ) of DNA or RNA probes

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5’-end labeling using polynucleotide kinase (PNK)

3’-end labeling using terminal transferase

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How to label one end of a DNA: Labeling at both ends by kinase, then remove one end by restriction digestion.

---------------------G---------------------CTTAAp5’

5’pAATTC G

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Nick translation 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 incorporating labeled nucleotide.

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Strand-specific RNA probes: labeled by in vitro

transcription of the desired RNA sequence.

Northern/Southern blot Northern/Southern blot analysisanalysis

gelmembrane

ElectrophoresisElectrophoresis blottingblotting

HybridizationHybridization

Fig 21-6

Southern and Northern blotting

DNA on blot RNA on blot

1.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. Hybridization (temperature) 7. Signal detection (X-ray film or antibody)

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Blot type

Target

Probe Applications

Southern DNA DNA or RNA mapping genomic clones

estimating gene numbers, etc

Northern RNA DNA or RNA RNA sizes and abundance (gene expression level)

Western Protein Antibodies protein size and abundance (gene expression level)

Comparison of Southern, Northern and Western bolt

hybridization

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4. Polymerase chain reaction 4. Polymerase chain reaction (PCR) amplifies DNAs by (PCR) amplifies DNAs by repeated rounds of DNA repeated rounds of DNA replication in vitro 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

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• Denaturation ( 变性 ): The target DNA (template) is separated 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:Three different steps proceed in each PCR cycle.

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Denaturation

Primer annealing

Polymerization

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The PCR amplification

Many cycles (25-35 in common) are performed to complete one PCR reaction, which resulted in an exponential amplificationexponential amplification of the target DNA if both forward and reverse primers pair.

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DNA templateDNA template

Any source of DNAAny 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 some sequence information is knowninformation is known so that primers can be designed.

Any source of DNAAny 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 some sequence information is knowninformation is known so that primers can be designed.

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PCR PrimersPCR Primers

1.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 5oC

1.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 5oC

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5’-ATTCCGATCGCTAATCGATGGC------- TCCTGTGCA TTTCGCCACTAGAG-3’

3’-TAAGGCTAGCGATTAGCTACCG-------AGGACACGTAAAGCGGTGATCTC-5’

5’-ATTCCGATCGCTAATCGATGGC------- TCCTGTGCA TTTCGCCACTAGAG-3’

3’-TAAGGCTAGCGATTAGCTACCG-------AGGACACGTAAAGCGGTGATCTC-5’

5’-ATTCCGATCGCTAATCGATG-3’

3’-CACGTAAAGCGGTGATCTC-5’

Tm=11x2+9x4=58oC

Tm=9x2+10x4=58oC

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.

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.

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Degenerate primers ( 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= purine

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Enzymes and PCR OptimizationEnzymes 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 available commercially.

• To optimize PCR, the annealing temperature and the Mg++ concentration are varied, or the nested PCR is carried out.

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Nested PCR: to increase specificityNested PCR: to increase specificity

First roundprimers

First roundprimers

First roundPCR Second round

primersSecond round

primers

Second roundPCR

Gene of interest

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Reverse transcriptase (RT)-PCRReverse transcriptase (RT)-PCR

AAA(A)n

5‘-CapmRNA

(dT)12~18 primer annealanneal

5‘-Cap

AAA(A)n

3‘ 5‘

Reverse transcription

Reverse transcriptiondNTP, RT

5‘-Cap

AAA(A)n

5‘

cDNA:mRNA hybrid

RegularPCR

RegularPCR

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PCR mutagenesis ( 诱变 )PCR mutagenesis ( 诱变 )

PCR can be used to introduce point mutations to the target gene in a plasmid

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5. DNA cloning, analysis 5. DNA cloning, analysis and gene expressionand gene expression

The ability to construct recombinant DNA molecules and maintain them in cells is called DNA cloning.

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Processes (Processes ( 过程过程 ) ) of DNA cloningof DNA cloning ：：

1. 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)

2. Transform ( 转化 ) the recombinant DNA molecules into competent cells ( 感受态细胞 ).

3. Propagation of the cells containing the recombinant DNA to form a clone ( 克隆 ), a set of identical cells containing the same recombinant DNA.

4. Select the desired clones using the selective marker.

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1. 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. coli. competent cells.

4. Grow the cells on a plate containing tetracycline ( 四环素 ).

Fig 21-7 construction of a DNA library

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• Host organisms/cells: where the plasmids get multiplied and propagated faithfully, which is crucial for DNA cloning.---Prokaryotic host: E. coli ( most cases)---Eukaryotic host: Yeast Saccharomyces cerevisiae (large fragments of human genome)

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General features of a VectorVector

1. They contain an origin of replication and can autonomously replicating DNA independent of host’s genome.

2. Easily to be isolated from the host cell. Most are circular, some are linear (e.g. YAC vector).

3. Contains 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.

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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 artificial chromosomes (YACs ，酵母人工染色体 ) (Linear)

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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 marker: ampr gene encoding the enzyme b-lactamse which degrades penicillin antibiotics such as ampicillin.

• The commonly used plasmid are small in size (~ 3 kb)

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Libraries of DNA molecules can be created by cloning

(Genomic library and cDNA library)A DNA libraryA DNA library (DNA 文库 ) is a population of

identical vectors that each contains a different DNA insert. (Fig. 20-8)

Genomic Library 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 library (cDNA 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 for the DNA copied from mRNA. (Fig. 20-19)

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Different Insert

fragments

Fig 21-8 construction of a DNA library

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cDNA library generationThe mRNAs are firstly reverse

transcript into cDNA, and these cDNA, both full length and partial, are cloned to make the cDNA library.

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annealanneal



RegularPCR

RegularPCR

Fig 20-9 Construction of cDNA library

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Colony screening 1.Antibiotic screening ( 抗生素选

择 ) ： only the recombinant plasmids grow on the antibiotic-containing plate.

2.Blue-white screening ( 蓝白斑选择 ): DNA insertion in the vector shuts down the LacZ gene expression, and turns the colony to white.

3.Colony hybridization screening ( 菌落杂交筛选 ) from a library.

Screening of positive clones

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Antibiotic screening ( 抗生素选择 ) ： only intact plasmids grow on the antibiotic-containing plate.

X if the vector is in the phosphorylated state

Recombinant DNA molecules

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Blue 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 substrate 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 instead.

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Recreated vector: blue transformantsRecombinant plasmid: containing inserted DNA: white transformants

Recreated vector (no insert)

Recombinant plasmid (contain insert)

Transfer to nitrocelluloseor nylon membrane

Denature DNA(NaOH)Bake onto membrane

Probe with 32p-labled DNA complementary to gene of interest

Expose to film

Select positive from master plate

Keep master plate

Screening by plaque hybridization

Colony hybridization-Southern blot

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Analysis of a clone

1.Restriction mapping: digestion of the plasmid prepared from a clone with restriction 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.

Analysis of DNA clones

1 K

b+ lad

der

2 Positive clones digested with different restriction enzymes

Em

pty

vecto

r

Restriction mapping

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Sequencing

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Expression of a gene from a transformed/transfected

plasmid 1.Transformation ( 转化 ) :

introduction of plasmids into bacteria.

2.Transfection ( 转染 ): introduction of plasmids and other exogenous nucleic acids into eukaryotes such as mammalian cells.

Gene expression

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Expression vectors:Expression vectors: allowing 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 transcription. The inserted gene has to have a start codon and a stop codon for translation

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T7 promoterRBS

Start codon

MCS

Transcription terminator

Ampr

ori

T7 expression

vector

E. coli expression vectorE. coli expression vector

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Insert Figure 1

MCS

H4 Eukaryotic Vectors

Yeast expression vectorYeast expression vector

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Fusion proteins

Gene cloning and expression 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 ) or purification ( 纯化 ).

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ExamplesLac fusions (Enzyme)(Enzyme) : fuse your target gene with the LacZ coding sequence. His-tag fusions (Tag)(Tag) : A sequence 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)(Fluorescence protein): insert your targeted gene at the N- or C- termini of GFP (green fluorescence protein, 绿色荧光蛋白 ), and your fusion protein will give you green fluorescence signal.

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6. 6. SequencingSequencing

Two ways for sequencing: 1. 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)

2. Chain-termination method (Sanger’s method, widely used)

Nucleic acids- sequencing

Maxam and Gilbert

Sanger’s enzymic method

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Chain-termination method ( 链终止法 )

ddNTPs are chain-terminating nucleotides: the synthesis of a DNA strand stops when a ddNTP is added to the 3’ end

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The absence of 3’-hydroxyl lead to the inefficiency of the nucleophilic attack on the next incoming substrate molecule.

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If 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 G

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Four separate reactions

dNTP+ ddGTP,

dNTP+ ddATP

dNTP+ ddCTP,

dNTP+ ddTTP

Each ddNTP carries a fluorescence group, allowing us to “Read” the sequence directly from the gel.

Fig 20-15 DNA sequencing gel

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Automatic sequencer

1.Fluorescence Labeled ddNTP

2. Polymerase catalyzed

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Shotgun sequencing of a bacterial genome ( 鸟枪法测细菌的基因组 )1.The bacterial genome was

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.This is called shotgun sequencing.

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In use 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= 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?

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The shotgun strategy permits a partial assembly of large genome sequence

The core techniques for sequencing the large genomes, e.g. human, are

(1) automated shotgun sequencingautomated shotgun sequencing (obtain sequence)

(2) then the subsequent use of use of computer to assemblecomputer to assemble the different sequences (analyze sequence, which is the rate-limiting step).

87Fig 20-16

1. Recombinant 1. Recombinant Plasmid LibraryPlasmid Library

2. Shotgun 2. Shotgun sequencinsequencingg

3. 3. Sequence Sequence Assembly Assembly

Flow chart of Human genome Flow chart of Human genome sequencing projectsequencing project

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1.Assemble the contigs from 1kb plasmid shotgun sequence. (50 kb-200 kb)

2.Assemble the contigs to large scaffold by sequencing both ends of 5 kb plasmids. (<500 kb)

3.Assemble the larger scaffolds (>1 Mb) by sequencing the end of the BAC library.

Assembly flowchart

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Assembly 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 sequences called contigscontigs.

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Fig 20-17. Contigs are linked by sequencing the ends of large DNA fragments (plasmid library containing larger DNA fragments).

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Assembly Step 2: The paired-end strategy permits the assembly of larger scaffolds (1-2 Mb)

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The purpose of this analysis is to predict the protein coding genes ( 蛋白质编码基因 ) and other functional sequences ( 其他功能序列 ) in the genome.

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For 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 these genes.

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For 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 criteria including the occurrence of extended ORFs that are flanked by appropriate 5’ and 3’ splice sites.

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Limitations 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 ). 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 ).

The most important method for validating predicted protein coding genes and identifying those missed by current gene finder program is the use of cDNA sequence data.

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cDNA 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 larger scaffolds.

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Fig 20-18 Gene finder method: analysis of protein-coding regions in Ciona intestinalis ( 海鞘 )

A 20-kb genome sequence (scaffold)Predicted by a gene finder program

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The mostly commonly used genome tool BLAST ：

Finding regions of similarity between different protein coding genes.

1. Input a query sequence ( 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.

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Figure 20-21 Example of the BLAST search result

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Topic 2: ProteinsProteins


1.Protein purification ( 蛋白质纯化 ) 2.Affinity chromatography can

facilitate more rapid protein purification ( 亲和层析纯化 )

3.Protein separation by PAGE gel electrophoresis ( 蛋白质分离 ) and identification by Western analysis

4.Protein sequencing ( 蛋白质测序 )5.Proteomics ( 蛋白质组学 )

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The purification of individual proteins is critical to understanding their function.

Although there are thousands of proteins in a single cell, each protein has unique properties, such as size, charge ( 电荷 ), shape, and in many instance, function, that make its purification somewhat different from others.

1. Protein purification ( 蛋白质纯化 )

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Purification of a protein requires a a specific assayspecific assay to allow you to monitor your purification status, which include a measure of the function of the protein, use of the antibody of the protein.

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Column chromatography is an efficient way to purify proteins

In this approach, protein fractions are passed though glass columns filled with appropriated modified small acrylamide or agarose beads.

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Ion exchange chromatography

The proteins are separated according to their surface chargesurface charge.

The beads are modified with either negative-charged or positive-charged chemical groups.

Proteins bind more strongly requires more salt to be eluted.

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Fig 20-22-a

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Gel filtration chromatography

This technique separate the proteins on the bases of size and size and shape. shape.

The beads for 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.

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Fig 20-22-b

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2. Affinity chromatography can facilitate more rapid protein purification

If the target protein is known to establish a specific and high-affinity interaction with a specific protein/nucleic acids/small molecule, we can couple this specific partnerspecific 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.

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Affinity chromatography

• Enzyme-substrate binding

• Receptor-ligand binding

• Antibody-antigen binding• Antibody-antigen binding

•Protein structure

• Ni2+-His tag-fusion protein binding

• Ni2+-His tag-fusion protein binding

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Immunoaffinity chromatography

( 免疫亲和层析 )

An antibodyantibody that is specific for the target is attached to the bead, and ideally only the target protein can bind to the column.

Disadvantage: sometimes the binding is too tight to elute our target protein.

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Sometimes tagstags (epitopes, 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 proteinfusion protein.

This allows the modified fusion proteins to be purified using immunoaffinity purification and a heterologous antibody specific for the tag.

Importantly, the binding affinity can change according to the condition. e.g. the concentration of the Ca2+ in the solution.

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Immunoprecipitation ( 免疫沉淀 )

Attach the antibody to the bead, which is then used to precipitate ( 沉淀 ) a specific protein from a crude cell extract.

It’s a useful method to detect what proteins are associated with the target protein. Co-Co-immunoprecipation is extensively immunoprecipation is extensively used to study protein-protein used to study protein-protein interaction***interaction***

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3. Protein separation by PAGE gel electrophoresis, followed by a western analysis

The native proteinsnative proteins have neither a uniform charge nor a uniform secondary structure.

If we treat the protein with a strong detergent SDSSDS, the higher structure is usually eliminated. And SDS confers the polypeptide chain a uniform negative charge.

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Sometimes, mercaptoethanolmercaptoethanol ( 巯基乙醇 ) is need to break the disulphide bond.

Thus, the protein molecules can be resolved by electrophoresiselectrophoresis in the presence of SDS according to the length of individual polypeptide.

After electrophoresis, the proteins can be visualized with a stainstain, such as Coomassie brilliant blue ( 考马氏亮蓝 ).

Proteins from the PAGE gel can be transferred to a membrane, followed by a western analysiswestern analysis of the target protein by a corresponding antibodyantibody.

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PTB

Beta-actin

--- P+ P++ P+++ P++++

A protein gel stained by Coomassie Blue

Western analysis using two specific antibodies

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4. Protein sequencing ( 蛋白质测序 )

Two sequence method: Edman degradation (Edman 切割法 )Tandem mass spectrometry (MS/MS) ( 串连质谱 ).

Due 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.

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Edman degradation

A chemical reaction in which the amino acid’s residues are sequentially release for the N-terminus of a polypeptide chain.

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Step 1: modify the N-terminal amino with PITC, which can only react with the free α-amino group.

Step 2: cleave off the N-terminal by acid treatment, but the rest of the polypeptide remains intact.

Step 3: identify the released amino acids by High Performance Liquid Chromatography (HPLC).

The whole process can be carried out in an automatic protein sequencer. 昂贵

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Fig 20-23

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Tandem mass spectrometry

MS is a method in which the mass of very small samples of a material can be determined.

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Step 1: Digest your target protein into short peptides (<20 aa) by a protease.

Step 2: Dubject the mixture of the peptide to MS, and each individual peptide will be separated by its mass/charge ratio.

Step 3: Capture and fragment individual peptides into all the component peptides.

Step 4: Determine the mass of each component peptide.

Step 5: Collect and deconvolute ( 解析 ) these data through search the protein databases to reveal the sequence of the target protein.

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5. Proteomics ( 蛋白质组学 )

Proteomics is concerned with the identificationidentification of the full setfull set of proteins produced by a cell or a tissue under a particular by a particular set of conditions.

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Three principle methods

1. 2-D gel electrophoresis for protein protein separationseparation ( 蛋白质分离 ).

2. MS spectrometry for the precise determinationdetermination of the molecular weight and identify of a protein ( 蛋白质鉴定 ).

3. Bioinformatics for assigning assigning proteins and peptides to the predicted products of protein coding sequence in the genome ( 蛋白质确定 ).

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Fig 20-24

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Topic 3: Study the Study the interaction between interaction between

protein and nucleic acidprotein and nucleic acid


1.Gel retardation assay2.Nuclease protection

assay

适用于 RNA-protein ，以及DNA-protein 相互作用研究

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Gel retardation ( 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 incubated with the extract, are run on a gel. The DNA-protein complexes are shown by the presence of slowly migrating bands.

DNA bound totwo proteins

DNA-proteincomplex

Bare DNA

A DNA bound with more than one protein to form a larger complex.

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DNase I footprinting (DNase I 足迹法 )

Identify the actual region of sequence with which the protein interacts.

5’ *

Sequence ladder is required to determine the precise position

AATAAG

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Bind protein

DNase(mild),then removeprotein and denature DNA

DNase footprinting （ 1 ） The protein protects DNA from attack by DNase. （ 2 ） Treat the DNA-protein complex with DNase I under mild conditions, so that an average of only one cut occur per DNA molecule.

Electrophoresis,autoradiograph

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0 1 5[Protein]

The 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, and the lane with 5 units shows complete protection in the middle.By including sequencing ladders, we can tell exactly where the protein bound.

TCGGAGCAACGCAAACAAACGTGCTTGG

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Topic 4: Determining the Determining the Structure ofStructure of

Protein and nucleic acids


1.X-ray crystallography (X-晶体衍射 )

2.NMR ( 核磁共振 )

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X-ray crystallography and NMRDetermining the tertiary structure

X-ray crystallography:

•Measuring the pattern of diffraction of a beam of X-rays as it pass through a crystal. The first hand data obtained is electron density map, the crystal structure is then deduced.

•A very powerful tool in understanding protein tertiary structure

•Many proteins have been crystallized and analyzed

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The ribosome structure and its interaction with mRNA and tRNA

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•Measuring the relaxation of protons after 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.

NMR

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Nucleic acids techniques:Electrophoresis; Restriction digestion;Hybridization (southern, northern, clony); PCR amplification (regular, RT-PCR,

mutational PCR); Sequencing (Sanger), genome sequencing

(Shotgun and genome assembly), genomic analysis (protein gene identification in bacteria and animals, BLAST search to predict the function of a new protein)

DNA cloning and gene expression. (How to analyze a clone, and how to make a fusion protein)

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Protein techniques:Protein purification: column

chromatography and affinity chromatography;

Protein separation by PAGE and identification by western blot

Protein sequencing (Edman degradation and tandem mass spectrometry); Proteomics (combine 2-D gel separation and tandem MS ).

Study the interactions: Protein-nucleic acids in vitro: Gel

retardation & Dnase I footprintingProtein-DNA in cells: Chromatin IPProtein-protein: yeast two-hybrid, Co-IPDetermining the Structure of protein and

nucleic acids: X-ray crystallography, NMR

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思考题：

你在本章中学习了哪些核酸和蛋白质技术？它们的作用原理和主要用途各是什么 ?

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God bless you allThanks for your

great corporatio

n throughout the class

God bless you allThanks for your

great corporatio

n throughout the class

1 welcome to mb class 2 molecular biology of the gene, 5/e --- watson et al. (2004) part i:...

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