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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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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


Chapter 20

Techniques of

Molecular Biology

Preparation, analysis and manipulation of nucleic acids and proteins


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


CHAPTER20: Techniques of Molecular Biology understanding of the properties of biological macromolecules themselves.

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


Gel Electrophoresis ( understanding of the properties of biological macromolecules themselves.凝胶电泳)

1. Gel electrophoresis separates DNA and RNA molecules according to size, shape and topological properties


DNA gel mobility understanding of the properties of biological macromolecules themselves.(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)


Fig 20 1 dna is separated by gel electrophoresis

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

Fig 20-1: DNA is separatedby gel electrophoresis

moderate

large

small


Gel matrix ( dyes, such as ethidium bromide (EB 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.


Polyacrylamide dyes, such as ethidium bromide (EB (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).


4 dyes, such as ethidium bromide (EB 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


Pulsed-field gel electrophoresis dyes, such as ethidium bromide (EB (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 dyes, such as ethidium bromide (EB

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


Electrophoresis is also used to separate RNAs dyes, such as ethidium bromide (EB

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 dyes, such as ethidium bromide (EB

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.


5 dyes, such as ethidium bromide (EB ’….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


How to name a restriction endonuclease? dyes, such as ethidium bromide (EB

EcoRI

the 1st such

enzyme found

Escherichia coli

Species category

R13

strain


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]


(The largest fragment) genome?

(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


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


(1) Restriction enzymes differ in the molecule to be isolatedrecognition 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.


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

blunt ends

(平末端)

sticky ends

(粘性末端)


Fig 20-5 Cleavage of an endonucleasesEcoRI 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 endonucleases

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.


Probe ( endonucleases探针)

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


Labeling ( endonucleases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


End labeling endonucleases

5’-end labeling usingpolynucleotide kinase (PNK)

3’-end labeling using terminal transferase


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

---------------------G

---------------------CTTAAp5’

5’pAATTC

G


Uniformly labeling of DNA/RNA endonucleases

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.


Strand-specific RNA probes: endonucleases

labeled by in vitro transcription of the desired RNA sequence.


Southern and Northern blotting endonucleases

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) 


Southern analysis endonucleases



bI1 endonucleases

bI2

bI3

bI4

bI5

Pre-mRNAs

mRNA

Northern analysis COB RNAs in S. cerevisiae



4 polymerase chain reaction

Nucleic acids- PCR hybridization

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.


The PCR cycle: hybridization

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++.


Fig. hybridization

Steps of PCR

Template

Primers

Enzymes


The PCR amplification hybridization

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.


DNA template hybridization

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. .


PCR Primers hybridization

  • 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


Tm= hybridization11x2+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.


Degenerate primers ( hybridization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


Enzymes hybridization 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.


Nested PCR hybridization

First round

primers

Gene of interest

Second round

PCR

First round

PCR

Second round

primers


Reverse transcriptase (RT)-PCR hybridization

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


PCR mutagenesis ( hybridization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


Forward mutagenic primer hybridization

SP6 primer

T7 primer

Reverse mutagenic primer

First PCR

Remove primers

Denature and anneal

3’

3’

Extend to full length by DNA polymerase


Second PCR hybridization

SP6 primer

T7 primer

DNA cloning


5 sequencing

Nucleic acids- sequencing hybridization

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)


Sanger’s enzymic method hybridization

Maxam and Gilbert


Chain termination method
Chain-termination method hybridization

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


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


Tell from the gel the position of each G the nucleophilic attack on the next incoming substrate molecule.

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 the nucleophilic attack on the next incoming substrate molecule.

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.


Automatic the nucleophilic attack on the next incoming substrate molecule.

sequencer

  • Fluorescence

  • Labeled ddNTP

  • 2. Polymerase catalyzed


Shotgun sequencing of a bacterial genome
Shotgun sequencing of a bacterial genome the nucleophilic attack on the next incoming substrate molecule.

  • 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.


10x Coverage example: the nucleophilic attack on the next incoming substrate molecule.

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 strategy the nucleophilic attack on the next incoming substrate molecule.permits 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).


1. Recombinant the nucleophilic attack on the next incoming substrate molecule.

Plasmid Library

2. Shotgun sequencing

3. Sequence Assembly

Fig 20-16


Assembly Step 1: form contigs the nucleophilic attack on the next incoming substrate molecule.

(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 the nucleophilic attack on the next incoming substrate molecule.permits the assembly of larger scaffolds (1-2 Mb)


Fig 20-17. Contigs are linked by sequencing the ends of the nucleophilic attack on the next incoming substrate molecule.large DNA fragments (plasmid library containing larger DNA fragments).


Assembly flowchart the nucleophilic attack on the next incoming substrate molecule.

  • 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 nucleophilic attack on the next incoming substrate molecule.

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.


For the genomes of the nucleophilic attack on the next incoming substrate molecule.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


For the nucleophilic attack on the next incoming substrate molecule.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.


Limitations of the computer methods: the nucleophilic attack on the next incoming substrate molecule.

~ 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.


cDNA library generation, sequencing and application: the nucleophilic attack on the next incoming substrate molecule.

  • 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.


Fig 20-9 RT-PCR the nucleophilic attack on the next incoming substrate molecule.

anneal

Reverse transcription

Regular

PCR


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 regions in

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.


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.



Other functional sequences under variation constraints ( is the 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.



The mostly commonly used genome tool conserved regulatory sequences 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.


Figure 20-21 Example of the BLAST search result conserved regulatory sequences


6 dna cloning analysis and gene expression

Nucleic acids- sequencing conserved regulatory sequences

6. DNA cloning, analysis and gene expression

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


  • Processes ( conserved regulatory sequences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.



  • Host organisms/cells same enzyme.: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)


  • General features of a Vector same enzyme.

  • 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.


Cloning vectors ( same enzyme.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)


  • Plasmids: same enzyme. 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)


Libraries of DNA molecules can be created by cloning same enzyme.

(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)



Screening of positive clones same enzyme.

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.


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


Recombinant DNA molecules same enzyme.

X

if the vector is phosphorylated


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


Blue white screening prevent religation of vector molecules

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.


lacZ prevent religation of vector molecules 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


Recreated vector prevent religation of vector molecules: blue transformants

Recombinant plasmid: containing inserted DNA: white transformants

Recreated vector (no insert)

Recombinant plasmid (contain insert)


Colony hybridization-Southern blot prevent religation of vector molecules

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


Analysis of DNA clones prevent religation of vector molecules

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.


2 Positive clones digested with different restriction enzymes

Empty vector

1 Kb+ ladder

Restriction mapping


Sequencing enzymes


Gene expression enzymes

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.


Expression vectors: enzymes

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


T7 promoter enzymes

RBS

Start codon

MCS

Ampr

Transcription terminator

T7 expression vector

ori


H4 Eukaryotic Vectors enzymes

A YEp vector

MCS

Insert Figure 1


Fusion proteins enzymes

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.


CHAPTER20: Techniques of Molecular Biology enzymes

  • 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


1. Protein purification ( enzymes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.


  • Purification of a protein requires enzymesa 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 enzymes

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 enzymes

  • 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.


Fig 20-22-a enzymes


Gel filtration chromatography
Gel filtration chromatography enzymes

  • 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.


Fig 20-22-b enzymes


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.


Affinity chromatography

  • Enzyme-substrate binding

  • Receptor-ligand binding

  • Antibody-antigen binding

  • Ni2+-His tag-fusion protein binding


Immunoaffinity chromatography
Immunoaffinity chromatography purification(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.


  • Sometimes purificationtags (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 ( purification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.


  • Sometimes, by a western analysismercaptoethanol (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.


A protein gel stained by Coomassie Blue by a western analysis

Western analysis using two specific antibodies

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

PTB

Beta-actin


4 protein sequencing
4. Protein sequencing ( by a western analysis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 by a western analysis

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


  • 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


Fig 20-23 only react with the free α-amino group.


Tandem mass spectrometry
Tandem mass spectrometry only react with the free α-amino group.

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


  • Step 1: digest your target protein into short peptides. only react with the free α-amino group.

  • 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 ( only react with the free α-amino group.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 only react with the free α-amino group.

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).


Fig 20-24 only react with the free α-amino group.


CHAPTER20: Techniques of Molecular Biology only react with the free α-amino group.

Topic 3: Study the interaction between protein and nucleic acid

  • Gel retardation assay

  • Nuclease protection assay


Gel retardation
Gel retardation ( only react with the free α-amino group.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.


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 complex.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’


DNase footprinting complex.(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


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


CHAPTER20: Techniques of Molecular Biology units of protein.

Topic 4: Determining the Structure of

Protein and nucleic acids

  • X-ray crystallography (X-晶体衍射)

  • NMR (核磁共振)


X-ray crystallography units of protein. 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



NMR tRNA

  • 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.


CHAPTER20: Techniques of Molecular Biology tRNA

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


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