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Molecular Biology. Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the Genome Part IV: Regulation Part V: Methods. Part V: METHODS. Ch 20: Techniques of Molecular Biology Ch 21: Model Organisms. Molecular Biology Course.

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slide1

Molecular Biology

Part I: Chemistry and Genetics

Part II: Maintenance of the Genome

Part III: Expression of the Genome

Part IV: Regulation

Part V: Methods

slide2

Part V: METHODS

Ch 20: Techniques of

Molecular Biology

Ch 21: Model Organisms

slide3

Molecular Biology Course

Chapter 20

Techniques of

Molecular Biology

Preparation, analysis and manipulation of nucleic acids and proteins

slide4

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
slide5

CHAPTER20: Techniques of Molecular Biology

Topic 1: Nucleic acids

  • Separation by Electrophoresis (电泳分离)
  • Cut by Restriction endonuclease (限制性内切酶切割)
  • Identification by Hybridization (杂交鉴定)
  • PCR
  • Genome sequence & analysis
  • DNA Cloning and gene expression
slide7

DNA gel mobility (DNA在胶上的迁移性)

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 molecules are separated according to sizes. The large DNA molecules move slower than the small molecules.

3.The mobility of circular DNA molecules is affected by their topological structures. The 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)

slide9

Gel matrix (胶支持物)

Gel matrix (胶支持物) is an inserted, jello-like porous material that supports and allows macromolecules to move through.

slide10

Polyacrylamide(聚丙稀酰胺):

has high resolving capability, and can resolve DNA/RNA that differ from each other as little as a single base pair/nucleotide.

but can separate DNA over a narrow size range (up to a few hundred bp/nt).

slide11

4 kb

3 kb

2 kb

1 kb

0.5 kb

Agarose (琼脂糖):

a much less resolving power than polyacrylamide,

but can separate DNA molecules of up to tens of kb

slide12

Pulsed-field gel electrophoresis (脉冲电泳)

The electric field is applied in pulses that are oriented orthogonally (直角地) to each other.

Separate DNA molecules according to their molecule weight, as well as to their shape and topological properties.

Can effectively separate DNA molecules over 30-50 kb and up to several Mb in length.

fig 20 2 pulsed field gel electrophoresis
Fig. 20-2 pulsed-field gel electrophoresis

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

slide14

Electrophoresis is also used to separate RNAs

RNA have a uniform negative charge as DNA does.

RNA is single-stranded and have extensive secondary and tertiary structure, which significantly influences their electrophoretic mobility.

RNA can be treated with reagent such as glyoxal (乙二醛) to prevent RNA base pairing, so that its mobility correlates with the molecular weight

2 restriction endonucleases cleave dna molecules at particular sites

Nucleic acids-Restriction digestion

2. Restriction endonucleases (限制性内切酶)cleave DNA molecules at particular sites
  • Why use endonucleases?

--To make large DNA molecules break into manageable fragments.

slide16

5’….GAATTC.….3’

….CTTAAG….

  • Restriction endonucleases (RE) arethe nucleases that cleave DNA atparticular sites by the recognitionof specific sequences.
  • RE used in molecular biology typically recognize (识别)short (4-8bp) target sequences that are usually palindromic (回文结构), and cut (切割) at a defined sequence within those sequences.

e.g. EcoRI

slide17

How to name a restriction endonuclease?

EcoRI

the 1st such

enzyme found

Escherichia coli

Species category

R13

strain

slide18

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

The random occurrence of the hexameric (六核苷酸的)sequence:

1/4096 (4-6)

What are the frequencies if the recognition sequences are four (tetrameric) and eight (octameric) nucleotides? [homework]

slide19

(The largest fragment)

(The smallest fragment)

  • Consider a linear DNA molecule with 6 copies of GAATTC:

it will be cut into 7 fragments which could be separated by the gel electrophoresis.

Fig 20-3 digestionof a DNA fragment with endonuclease EcoRI

slide20

Use of multiple REs allows different regions of a DNA molecule to be isolated

  • A given molecule will generate a characteristic series of pattern when digested with a set of different enzymes.
    • e.g. the combination of EcoRI + HindIII
slide21

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

slide22

Fig 20-4 Recognition sequences and cut sites of various endonucleases

blunt ends

(平末端)

sticky ends

(粘性末端)

slide23

Fig 20-5 Cleavage of an EcoRI site. The 5’ protruding ends are said to be “sticky” because they readily anneal through base-pairing to DNA molecules cut with the same enzyme

3 dna hybridization can be used to identify specific dna molecules

Nucleic acids- DNA hybridization

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

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

slide25

Probe (探针)

A labeled, defined sequence used to search mixtures of nucleic acids for molecules containing a complementary sequence.

slide26

Labeling (标记) of DNA or RNA probes (why labeling?)

Radioactive labeling: display and/or magnify the signals by radioactivity.

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

End labeling: put the labels at the ends

Uniform labeling: put the labels internally

slide27

End labeling

5’-end labeling usingpolynucleotide kinase (PNK)

3’-end labeling using terminal transferase

slide29

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

slide30

Uniformly labeling of DNA/RNA

Nick translation labeling of DNA:

DNase I to introduce random nicks DNA polI 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.

slide31

Strand-specific RNA probes:

labeled by in vitro transcription of the desired RNA sequence.

slide32

Southern and Northern blotting

DNA on blot

RNA on blot

  • Genomic DNA preparation RNA preparation
  • Restriction digestion -
  • Denature with alkali -
  • Agarose gel electrophoresis 
  • DNA blotting/transfer and fixation RNA
  • 6. Probe labeling 
  • 6. Hybridization (temperature) 
  • 7. Signal detection (X-ray film or antibody) 
slide35

bI1

bI2

bI3

bI4

bI5

Pre-mRNAs

mRNA

Northern analysis COB RNAs in S. cerevisiae

4 polymerase chain reaction

Nucleic acids- PCR

4. Polymerase chain reaction

The polymerase chain reaction(PCR) is to used to amplify a sequence of DNA using a pair of primers each complementary to one end of the the DNA target sequence.

slide38

The PCR cycle:

Three different steps proceed in each PCR cycle.

  • 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++.
slide40

Fig.

Steps of PCR

Template

Primers

Enzymes

slide41

The PCR amplification

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

slide42

DNA template

Any source of DNA that provides one or more target molecules can in principle be used as a template for PCR.

Whatever the source of template DNA, PCR can only be applied if some sequence information is known so that primers can be designed. .

slide43

PCR Primers

  • Anneal on opposite strands of the target sequence.
  • About 18 to 30 nt long and have similar G+C contents so that they anneal to their complementary sequences at similar temperatures.
  • Tm=2(a+t)+4(g+c): determine annealing temperature. If the primer is 18-30 nt, annealing temperature can be Tm  5oC
slide44

Tm=11x2+9x4=58oC

5’-ATTCCGATCGCTAATCGATG-3’

3’-CACGTAAAGCGGTGATCTC-5’

Tm=9x2+10x4=58oC

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

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

DNA sequence is written from 5’ to the 3’ end if not stated. And only the sense strand is usually given instead of both strands.

slide45

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 AAR

Y= Pyrimidine

N= any base

R= purine

slide46

Enzymes and PCR Optimization

  • The most common is Taq polymerase. It has no 3’ to 5’ proofreading exonuclease activity. Accuracy is low, not good for cloning. High-accuracy DNA polymerase is available commercially.
  • To optimize PCR, the annealing temperature and the Mg++ concentration are varied, or the nested PCR is carried out.
slide47

Nested PCR

First round

primers

Gene of interest

Second round

PCR

First round

PCR

Second round

primers

slide48

Reverse transcriptase (RT)-PCR

5‘-Cap

mRNA

AAA(A)n

(dT)12~18

primer

anneal

5‘-Cap

3‘

5‘

AAA(A)n

Reverse transcription

dNTP, RT

5‘-Cap

5‘

Regular

PCR

AAA(A)n

cDNA:mRNA hybrid

slide49

PCR mutagenesis (诱变)

To introduce deletion or point mutations

  • Two separate PCR reactions are performed.
  • One PCR amplifying the 5’-portion of the insert, and the other amplifying the 3’-portion of the insert.
  • The point mutation/deletion mutations are located in the primers
slide50

Forward mutagenic primer

SP6 primer

T7 primer

Reverse mutagenic primer

First PCR

Remove primers

Denature and anneal

3’

3’

Extend to full length by DNA polymerase

slide51

Second PCR

SP6 primer

T7 primer

DNA cloning

5 sequencing

Nucleic acids- sequencing

5. Sequencing

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)
slide53

Sanger’s enzymic method

Maxam and Gilbert

chain termination method
Chain-termination method
  • ddNTPs are chain-terminating nucleotides: the synthesis of a DNA strand stops when a ddNTP is added to the 3’ end
slide55

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

slide56

Tell from the gel the position of each G

If one ddGTP is added to 100 dGTP, DNA synthesis aborts at a frequency of 1/100 every time the polymerase meets a ddGTP

fig 20 15 dna sequencing gel
Fig 20-15 DNA sequencing gel

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.

slide58

Automatic

sequencer

  • Fluorescence
  • Labeled ddNTP
  • 2. Polymerase catalyzed
shotgun sequencing of a bacterial genome
Shotgun sequencing of a bacterial genome
  • 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)
  • DNA was prepared from individual recombinant DNA clones and separately sequenced on automated sequencer. (shot)
  • This is called shotgun sequencing.
  • 3. To obtain all the DNA sequence in the bacterium Hemophilus influenzae genome, 10x sequencecoverage was used.
slide60

10x Coverage example:

If the H. influenzae genome is 1.8 kb, each read produces 600 bp of sequence, and 600 bp x 33,000 different colonies= 20 Mb.

That is to say 33,000 colonies are picked to prepare plasmid for sequencing.

the shotgun strategy permits a partial assembly of large genome sequence
The shotgun strategypermits a partial assembly of large genome sequence
  • The key technical insights that facilitating the sequencing of the human genome was the reliance on
  • automated shotgun sequencing (obtain sequence)
  • then the subsequent use of computer to assemble the different sequences (analyze sequence, which is the rate-limiting step).
slide62

1. Recombinant

Plasmid Library

2. Shotgun sequencing

3. Sequence Assembly

Fig 20-16

slide63
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 contigs.

assembly step 2 the paired end strategy permits the assembly of larger scaffolds 1 2 mb
Assembly Step 2: The paired-end strategy permits the assembly of larger scaffolds (1-2 Mb)
slide65
Fig 20-17. Contigs are linked by sequencing the ends of large DNA fragments (plasmid library containing larger DNA fragments).
slide66

Assembly flowchart

  • Assemble the contigs from 1kb plasmid shotgun sequence. (50 kb-200 kb)
  • Assemble the contigs to large scaffold by sequencing both ends of 5 kb plasmids. (<500 kb)
  • Assemble the larger scaffolds (>1 Mb) by sequencing the end of the BAC library.
genome wide analysis
Genome-wide analysis

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.

slide68

For the genomes of bacteria and simple eukaryotes:

Finding protein coding genes = Identification of ORF (open-reading frames).

straightforward;

fairly effective;

but not all ORF=real protein coding genes;

key change is in identifying the functions of these genes

slide69

For animal genomes with complex exon-intron structures, the challenge is far greater:

A variety of bioinformatics tools are required to identify genes and genetic composition of complex genomes.

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.

slide70

Limitations of the computer methods:

~ one-fourths of genes cannot be identified by this way.

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.

slide71
cDNA library generation, sequencing and application:
  • The mRNAs are firstly reverse transcript into cDNA, and these cDNA, both full length and partial, are cloned to make the cDNA library
  • Sequence the cDNAs using shotgun method to generate EST (expressed sequence tag) database.
  • These ESTs are aligned onto genomic scaffolds to help us identify genes and to assemble larger scaffolds.
slide72

Fig 20-9 RT-PCR

anneal

Reverse transcription

Regular

PCR

slide73

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

comparative genome analysis
Comparative genome analysis

The comparison of different animal genomes:

  • permits a direct assessment of changes in gene structure and sequence;
  • Allows to refine the identification of protein coding genes within a given genome;
  • Helps identify short exons.
slide75

One of the striking findings of comparative genome analysis is the high degree of synteny (conservation in genetic linkage,遗传连锁的保守性) between distantly related animals.

slide77

Other functional sequences under variation constraints (变异限制):

The logic is that “functional sequence cannot be changed randomly”.

Regulatory sequences-transcription factor binding sites and larger elements of gene regulation, such as enhancers.

The computer program VISTA aligns the sequence contained in different genomes over short windows (10-20 bp), and can be used to predict the conserved regulatory sequence.

It is predicted that human and mice contain more like 50,000-100,000 enhancers.

slide79

The mostly commonly used genome tool BLAST :

Finding regions of similarity between different protein coding genes.

Input a query sequence (询问序列): a stretch of amino acids or the DNA sequence encoding your interested protein function.

Ask the computer to search the homologous sequences in the database, and you will get all the available genes that may have the similar protein function.

6 dna cloning analysis and gene expression

Nucleic acids- sequencing

6. DNA cloning, analysis and gene expression

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

slide82

Processes (过程) of DNA cloning:

  • Forming the recombinant DNA molecules (重组DNA) by inserting your interested DNA fragments into a proper vector (载体). (Require restriction enzymes and ligase)
  • Transform (转化) the recombinant DNA molecules into competent cells (感受态细胞).
  • Propagation of the cells containing the recombinant DNA to form a clone (克隆), a set of identical cells containing the same recombinant DNA.
  • Select the desired clones using the selective marker.
slide83

Restriction digestion of your insert and vector using the same enzyme.

  • Use ligase to join your insert and vector together.
  • Transform the ligation productsinto E. coli. competent cells.
  • Grow the cells on a plate containing tetracycline (四环素).
slide84

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)
slide85

General features of a Vector

  • They contain an origin of replication and can autonomously replicating DNAindependent of host’s genome.
  • Easily to be isolatedfrom the host cell. Most are circular, some are linear (e.g. YAC vector).
  • Contains at least oneselective marker, which allows host cells containing the vector to be selected amongst those which do not.
  • Contains amultiple cloning site (MCS) to be cut by restriction enzymes for DNA manipulation.
slide86

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)

slide88

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 commonly small (~ 3 kb)
slide89

Libraries of DNA molecules can be created by cloning

(Genomic library and cDNA library)

A DNA library (DNA文库) is a population of identical vectors that each contains a different DNA insert. (Fig. 20-8)

Genomic Library (基因组文库) : the DNA inserts in a DNA library is derived from restriction digestion or physical shearing of the genomic DNA.

cDNA library (cDNA文库) : the DNA inserts in a DNA library is converted from the mRNAs of a tissue, a cell type or an organism. cDNA stands for the DNA copied from mRNA. (Fig. 20-19)

slide91

Screening of positive clones

Colony screening

  • Antibiotic screening (抗生素选择): only the recombinant plasmids grow on the antibiotic-containing plate.
  • Blue-white screening (蓝白斑选择): DNA insertion in the vector shuts down the LacZ gene expression, and turns the colony to white.
  • Colony hybridization screening (菌落杂交筛选) from a library.
slide92

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

slide93

Recombinant DNA molecules

X

if the vector is phosphorylated

slide94

Dephosphorylate the vector using alkaline phosphate to prevent religation of vector molecules

slide95

Blue white screening

Lac promoter

MCS (Multiple cloning sites,

多克隆位点)

Ampr

pUC18

(3 kb)

lacZ’

ori

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.

slide96

lacZ encode enzymeb-galactosidase

(substrate of the enzyme)

lac promoter

X-gal

IPTG

Blue product

The expression of active b-galactosidase has to be vector dependent for the selection purpose

lacZ’: a shortened derivative of lacZ,

encoding N-terminal a-peptide of b-galactosidase.

Host strain for vectors containing lacZ’:

contains a mutant gene encoding only the C-terminal portion of b-galactosidase which can then complement the a-peptide to produce the active enzyme

slide97

Recreated vector: blue transformants

Recombinant plasmid: containing inserted DNA: white transformants

Recreated vector (no insert)

Recombinant plasmid (contain insert)

slide98

Colony hybridization-Southern blot

Transfer to nitrocellulose

or nylon membrane

Keep master

plate

Select positive

from master plate

Denature DNA(NaOH)

Bake onto membrane

Probe with 32p-labled DNA

complementary to

gene of interest

Expose to film

Screening by plaque hybridization

slide99

Analysis of DNA clones

Analysis of a clone

  • Restriction mapping:digestion of the plasmid prepared from a clone with restriction enzymes to investigate if the interested DNA is inserted the recombinant plasmid.
  • Sequencingthe cloned DNA to see if the inserted DNA maintains the correct sequence.
slide100

2 Positive clones digested with different restriction enzymes

Empty vector

1 Kb+ ladder

Restriction mapping

slide102

Gene expression

Expression of a gene from a transformed/transfected plasmid

  • Transformation (转化): introduction of plasmids into bacteria.
  • Transfection (转染):introduction of plasmids and other exogenous nucleic acids into eukaryotes such as mammalian cells.
slide103

Expression vectors:

allowing the exogenous DNA to be stored and expressed in an organism.

--E. coli expression vector

--Yeast expression vector

--Mammalian expression vector

Features:

In addition to the origin of replication, selective marker, multiple cloning site, expression vector has to contain a promoter and terminator for transcription. The inserted gene has to have a start codon and a stop codon for translation

slide104

T7 promoter

RBS

Start codon

MCS

Ampr

Transcription terminator

T7 expression vector

ori

slide105

H4 Eukaryotic Vectors

A YEp vector

MCS

Insert Figure 1

slide106

Fusion proteins

Lac fusions: fuse your target gene with the LacZ coding sequence

His-tag fusions: A sequence encodes His-tag was inserted at the N- or C- termini of the target ORF, which allows purification of the fusion protein to be purified by binding to Ni2+ column.

GFP fusions: insert your targeted gene at the N- or C- termini of GFP, and your fusion protein will give you green fluorescence signal.

slide107

CHAPTER20: Techniques of Molecular Biology

  • Protein purification (蛋白质纯化)
  • Affinity chromatography can facilitate more rapid protein purification (亲和层析纯化)
  • Protein separation by PAGE gel electrophoresis (蛋白质分离) and identification by Western analysis
  • Protein sequencing (蛋白质测序)
  • Proteomics(蛋白质组学)

Topic 2: Proteins

slide108

1. Protein purification (蛋白质纯化)

  • 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.
slide109
Purification of a protein requires a specific 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.
column chromatography is an efficient way to purify proteins
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.

ion exchange chromatography
Ion exchange chromatography
  • The proteins are separated according to their surface charge.
  • The beads are modified with either negative-charged or positive-charged chemical groups.
  • Proteins bind more strongly requires more salt to be eluted.
gel filtration chromatography
Gel filtration chromatography
  • This technique separate the proteins on the bases of size and 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.
2 affinity chromatography can facilitate more rapid protein purification
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 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.

slide116

Protein structure

Affinity chromatography

  • Enzyme-substrate binding
  • Receptor-ligand binding
  • Antibody-antigen binding
  • Ni2+-His tag-fusion protein binding
immunoaffinity chromatography
Immunoaffinity chromatography(免疫亲和层析)
  • An antibody 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.
slide118
Sometimes tags (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 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.
immunoprecipitation
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 or other molecules are associated with the target protein.
3 protein separation by page gel electrophoresis followed by a western analysis
3. Protein separation by PAGE gel electrophoresis, followed by a western analysis
  • The native proteins have neither a uniform charge nor a uniform secondary structure.
  • If we treat the protein with a strong detergent SDS, the higher structure is usually eliminated. And SDS confers the polypeptide chain a uniform negative charge.
slide121
Sometimes, mercaptoethanol (巯基乙醇) is need to break the disulphide bond.
  • Thus, the protein molecules can be resolved by electrophoresis in the presence of SDS according to the length of individual polypeptide.
  • After electrophoresis, the proteins can be visualized with a stain, 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 analysis of the target protein by a corresponding antibody.
slide122

A protein gel stained by Coomassie Blue

Western analysis using two specific antibodies

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

PTB

Beta-actin

4 protein sequencing
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.
edman degradation
Edman degradation

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

slide125
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. 特贵

tandem mass spectrometry
Tandem mass spectrometry
  • MS is a method in which the mass of very small samples of a material can be determined.
slide128
Step 1: digest your target protein into short peptides.
  • Step 2: subject the mixture of the peptide to MS, and each individual peptide will be separate.
  • Step 3: capture the individual peptide and fragmented into all the component peptide.
  • Step 4: determine the mass of each component peptide.
  • Step 5:Deconvolution (解析) of these data and the sequence will be revealed.
5 proteomics
5. Proteomics (蛋白质组学)
  • Proteomics is concerned with the identification of the full set of proteins produced by a cell or a tissue under a particular by a particular set of conditions.
three principle methods
Three principle methods

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

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

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

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CHAPTER20: Techniques of Molecular Biology

Topic 3: Study the interaction between protein and nucleic acid

  • Gel retardation assay
  • Nuclease protection assay
gel retardation
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.
slide134

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

DNA bound to

two proteins

DNA-protein

complex

Bare DNA

dnase i footprinting dnase i
DNase I footprinting (DNase I 足迹法)

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

Sequence ladder is required to determine the precise position

AATAAG

*

5’

slide136

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.

Bind protein

DNase(mild),then remove

protein and denature DNA

Electrophoresis,

autoradiograph

slide137

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.

[Protein]

T

C

G

G

A

G

C

A

A

C

G

C

A

A

A

C

A

A

A

C

G

T

G

C

T

T

G

G

1

0

5

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CHAPTER20: Techniques of Molecular Biology

Topic 4: Determining the Structure of

Protein and nucleic acids

  • X-ray crystallography (X-晶体衍射)
  • NMR (核磁共振)
slide139

X-ray crystallography and NMR

Determining 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
slide141

NMR

  • 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.
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CHAPTER20: Techniques of Molecular Biology

Nucleic acids techniques:

Electrophoresis; Restriction digestion; Hybridization (southern & northern); PCR amplification; sequencing and genome sequencing; DNA cloning and gene expression.

Protein techniques:

Protein purification; affinity chromatography; Protein separation and identification by western blot; Protein sequencing; Proteomics.

Study the interaction between protein and nucleic acid

Gel retardation & Nuclease protection assays

Determining the Structure of protein and nucleic acids: X-ray crystallography, NMR