Chapter 20 Chapter 20

Techniques of Techniques of molecular biologymolecular biology

IntronductionIntronduction

The living cell is an extraordinary The living cell is an extraordinary complicated entity, with thousands of complicated entity, with thousands of macromolecules in it.macromolecules in it.

So is important to separate individual So is important to separate individual macromolecules from the myriad macromolecules from the myriad mixtures, to dissect the genome into mixtures, to dissect the genome into manageable size segment for manageable size segment for manipulation and analysis manipulation and analysis

IntronductionIntronduction

Recently ,it has become possible to apply Recently ,it has become possible to apply molecular approaches to the large-scale molecular approaches to the large-scale analysis of the full complement of RNAs analysis of the full complement of RNAs and proteins in the cell and to determine tand proteins in the cell and to determine the sequence of an entire genome. he sequence of an entire genome.

IntronductionIntronduction

Two parts of this chapterTwo parts of this chapter Part 1 Part 1

techniques for the manipulation and techniques for the manipulation and characterization of nucleic acidcharacterization of nucleic acid

Part 2 Part 2

techniques for the isolation and analysis techniques for the isolation and analysis of protein of protein

Part 1 Nucleic acids Part 1 Nucleic acids

1.1. Separate DNAs and RNAs Separate DNAs and RNAs

2.2. Identify specific DNA moleculesIdentify specific DNA molecules

3.3. Isolation of specific DNAs moleculIsolation of specific DNAs moleculeses

4.4. DNA cloningDNA cloning

5.5. DNA sequencingDNA sequencing

1.1.2 Separate DNAs and RNA1.1.2 Separate DNAs and RNAss

Gel electrophoresisGel electrophoresis

separate DNA according to the lengtseparate DNA according to the length, shape, topological propertiesh, shape, topological properties

1.1. Why Why 2.2. How How 3.3. WhenWhen

WhyWhy DNA is negatively charged, so they will migDNA is negatively charged, so they will mig

rate to the positive pole once put in an elrate to the positive pole once put in an electronic field ectronic field

The rates they travel through the gel are difThe rates they travel through the gel are different for their different length. The longferent for their different length. The longer the DNA is ,the slower its rate will beer the DNA is ,the slower its rate will be

After the electrophoresis the DNA can be viAfter the electrophoresis the DNA can be visualized by staining the gel with fluorescsualized by staining the gel with fluorescent dyes, such as ethidiument dyes, such as ethidium

HowHow

Constant electrophoresis Constant electrophoresis

Polyacrylamide Polyacrylamide

high resolving capabilityhigh resolving capability

narrow size rangenarrow size range

Agarose Agarose

less resolving capabilityless resolving capability

large NDA and their diffrenceslarge NDA and their diffrences

HowHow

Pulsed –field Pulsed –field

Very long DNAs are unable to penetrate evVery long DNAs are unable to penetrate even with agarose.en with agarose.

Pulsed –field is used to solve this problem.Pulsed –field is used to solve this problem.

WhenWhenElectrophoresis is used to separate Electrophoresis is used to separate DNAs of different lengthDNAs of different length DNAs of different shapeDNAs of different shape eg.linear and circulareg.linear and circular DNA of different topological propertiesDNA of different topological properties eg. supercoiled and less supercoiled DNAeg. supercoiled and less supercoiled DNA RNARNA single strand single strand secondary and tertiary structuresecondary and tertiary structure

Restriction endonuclease can find the Restriction endonuclease can find the specific site of DNA and cut it to sepspecific site of DNA and cut it to separate the DNA from the genome to foarate the DNA from the genome to fo

rm manageable fragmentsrm manageable fragments

Eg . EcoR1Eg . EcoR1

1.1.2 Restriction endonu1.1.2 Restriction endonuclease cleavageclease cleavage

restriction enzymes differ inrestriction enzymes differ in recognition sequence recognition sequence cut frequency(=1/4cut frequency(=1/4nn))

cut sitecut site

1.2 Identify specific 1.2 Identify specific DNA moleculesDNA molecules

DNA hybridizationDNA hybridization Base-pair between two single -stranded Base-pair between two single -stranded

polynucleotide from different sources is called polynucleotide from different sources is called hybridizationhybridization

ProbeProbe The defined sequence is called probe, either a The defined sequence is called probe, either a

purified fragment or a chemically synthesized. purified fragment or a chemically synthesized. The probe must be labeled so that it can be The probe must be labeled so that it can be readily located.readily located.

Two basic methods to label a probeTwo basic methods to label a probe

1.synthesis of a new DNA in the presence of the labeled prec1.synthesis of a new DNA in the presence of the labeled precursor.ursor.

Use PCR with a labeled precursor ,or hybridize short random hexUse PCR with a labeled precursor ,or hybridize short random hexameric oligonucleotides to DNA and allow a DNA polymerase tameric oligonucleotides to DNA and allow a DNA polymerase to extend themo extend them

The label precursors are most commonly nucleotide modified with The label precursors are most commonly nucleotide modified with a fluorescent moiety or radioactivity atoms.a fluorescent moiety or radioactivity atoms.

The DNA labeled with fluorescent precursors can be detected by rThe DNA labeled with fluorescent precursors can be detected by radiating the sample with UVadiating the sample with UV

DNA Labeled with radioactive atoms can be detected by exposed DNA Labeled with radioactive atoms can be detected by exposed to X-ray film or by photomultipliersto X-ray film or by photomultipliers

2.adding a label to the end of an intact DNA molec2.adding a label to the end of an intact DNA moleculesules

UsageUsage

Southern blotSouthern blot

1.1. restriction enzyme cut a specific fragmentsrestriction enzyme cut a specific fragments

2.2. electrophoresis of these fragment electrophoresis of these fragment

3.3. transfer to a filter and detect a specific sequence transfer to a filter and detect a specific sequence with a probe homologous to itwith a probe homologous to it

This can detect the amount of the specific sequenceThis can detect the amount of the specific sequence

UsageUsage

Northern blot Northern blot

To monitor the amount of a specific To monitor the amount of a specific mRNA.mRNA.

This a reflection of the expressing level of a This a reflection of the expressing level of a gene.gene.

1.4 Isolation of specific DN1.4 Isolation of specific DNAs moleculesAs molecules

Much molecular analysis requires the Much molecular analysis requires the separation of specific segment of DNA separation of specific segment of DNA from much larger DNA molecules and from much larger DNA molecules and their amplification. This is important totheir amplification. This is important to

DNA analysisDNA analysis

DNA sequencingDNA sequencing

DNA manipulationDNA manipulation

1.4.1 DNA cloing1.4.1 DNA cloing

DNA cloningDNA cloningThe ability to construct recombinant DNA molecules and maintain them The ability to construct recombinant DNA molecules and maintain them

in the cell is called.in the cell is called.

Vector Vector provide the information necessary to propagate the cloned DNAprovide the information necessary to propagate the cloned DNA

Inserted DNAInserted DNA inserted into the vector and include the DNA of interestinserted into the vector and include the DNA of interest

1.4.2 Cloning DNA in 1.4.2 Cloning DNA in plasmid vectorplasmid vector

Vector DNA has three characteristics:Vector DNA has three characteristics:

1.1. origin of replication to allow them to replicate origin of replication to allow them to replicate independentlyindependently

2.2. selectable marker to allow cells contain the selectable marker to allow cells contain the vector be identifiedvector be identified

3.3. single site for one or more restriction enzyme single site for one or more restriction enzyme to allow DNA fragments to be inserted into itto allow DNA fragments to be inserted into it

PlasmidPlasmid

From bacteria ,single-cell eukaryotesFrom bacteria ,single-cell eukaryotesPropagate independentlyPropagate independentlyCarry gene encode resistance to Carry gene encode resistance to

antibioticsantibioticsCarry useful restriction siteCarry useful restriction siteSome drive the expression of Some drive the expression of

gene( express vector)gene( express vector)

Vector is carried into the Vector is carried into the host cellhost cell

Transformation:Transformation: a host organism can take up DNA from the ena host organism can take up DNA from the en

vironmentvironmentGenetic competenceGenetic competence only a bacteria of genetic competence can exeonly a bacteria of genetic competence can exe

cute transformationcute transformation E.coli can be reddened competent to take up DE.coli can be reddened competent to take up D

NA by treatment with calcium ions. An antibiotiNA by treatment with calcium ions. An antibiotic to with the plasmid impacts resistance is the c to with the plasmid impacts resistance is the use to select transformations.use to select transformations.

1.4.3 Libraries of DNA 1.4.3 Libraries of DNA molecules can be created molecules can be created by cloningby cloning

For complex starting DNAFor complex starting DNA

A population of identical vector that each A population of identical vector that each contain a different DNA insertscontain a different DNA inserts

Restriction enzyme :give a desired average Restriction enzyme :give a desired average insert sizeinsert size

Genomic libraries :DNA libraries for a Genomic libraries :DNA libraries for a whole genomewhole genome

cDNA librarycDNA library

A mRNA is converted to DNA strands A mRNA is converted to DNA strands

mRNA DNA mRNA DNA

Hybridization can be used to identify a Hybridization can be used to identify a specific clone in a DNA library, which is specific clone in a DNA library, which is called cloning hybridization.called cloning hybridization.

Positively charged membrane filter is used Positively charged membrane filter is used in this processionin this procession

Reverse transcription

1.4.4 Chemical synthesized oligo1.4.4 Chemical synthesized oligonucleotidenucleotide

Short ,custom-designed segment of DNA knows as oligonuShort ,custom-designed segment of DNA knows as oligonucleotide.cleotide.

Solid supports using –machines automate the processSolid supports using –machines automate the processThe protected resides are called phosphoamidinesThe protected resides are called phosphoamidines30 bp is of enough accuracy, however, longer DNA synthes30 bp is of enough accuracy, however, longer DNA synthes

is final product is less uniform due to the inherent failsis final product is less uniform due to the inherent failsUsage Usage site-directed mutagenesissite-directed mutagenesis probe in hybridizationprobe in hybridization primer in PCRprimer in PCR

1.4.5 The Polymerase 1.4.5 The Polymerase Chain Reaction( PCR)Chain Reaction( PCR)

AcquireAcquire DNA polymeraseDNA polymerase oligonucleotideoligonucleotide single –strand templatesingle –strand templateStepsSteps 1.heat denature single strands1.heat denature single strands 2.add primers primer-template junction2.add primers primer-template junction 3.add DNA polymerase DNA synthesis 3.add DNA polymerase DNA synthesis 4.goto Step 14.goto Step 1

1.5 DNA sequencing1.5 DNA sequencing Nested set of DNA fragments Nested set of DNA fragments

Two methods Two methods

1 DNA molecules are redioactively l1 DNA molecules are redioactively labeled at their 5’termis, and then suabeled at their 5’termis, and then subjected to four different regions of cbjected to four different regions of chemical treatment that cause them themical treatment that cause them to break preferentially.o break preferentially.

2 chain-termination nucleotides2 chain-termination nucleotides

2’-,3’ didexynucleotide (ddNTPs)2’-,3’ didexynucleotide (ddNTPs)

ddNTP:2’-dexoy-NTP=1:100ddNTP:2’-dexoy-NTP=1:100

1.5.1 Shotgun 1.5.1 Shotgun sequencingsequencing

For large genome For large genome DNA was prepared from a bacteria genome individual recombinant DNA was prepared from a bacteria genome individual recombinant

DNA clone and separately sequence on a sequenators.DNA clone and separately sequence on a sequenators. 10Xsequence coverage10Xsequence coverage fast and less expensive than systematically sequencing every definfast and less expensive than systematically sequencing every defin

ed restriction DNA fragment on the physical map of that bacteria ced restriction DNA fragment on the physical map of that bacteria chromosome hromosome

setbackssetbacks once a single site is not correctly identified ,the whole genome maonce a single site is not correctly identified ,the whole genome ma

y be wrongy be wrong contigs are linked by sequencing the end of large DNA fragments.contigs are linked by sequencing the end of large DNA fragments.

1.5.2 Genome-wide Analysis1.5.2 Genome-wide Analysis

Bacteria and single eukaryotes

Straightformed, effective

Key challenge is in identification the function of the genes

Animal genome

Complex exon-intron structure

No 100% accuracy to final exon

Fail to identify promoters

1.5.3 Comparative 1.5.3 Comparative genome analysisgenome analysis

High degree of syntenyHigh degree of synteny

BLATS( basic local alignment search tool ) BLATS( basic local alignment search tool ) is used to find region of similarity betweeis used to find region of similarity between different protein coding gene, search thn different protein coding gene, search the genome to find query sequencee genome to find query sequence

Part 2 ProteinsPart 2 Proteins

2.1.Purification2.1.Purification

require a specific array, based on any of their fearequire a specific array, based on any of their features ,including weight, shape, charges they ctures ,including weight, shape, charges they carrying, other specific features.arrying, other specific features.

incorporation assays:incorporation assays: immunoblotting( specific interaction between Aimmunoblotting( specific interaction between A

b and Ag)b and Ag) a specific DNA for an DNA binding proteina specific DNA for an DNA binding protein

StepsSteps

Preparation of cell extracts contain active pPreparation of cell extracts contain active proteins .roteins .

To protect the activation of the proteinsTo protect the activation of the proteins

1.fitting temperature :4 1.fitting temperature :4 ooCC

2.fitting ionic salt2.fitting ionic salt

2.2 Separate protein using 2.2 Separate protein using column chromatographycolumn chromatography

Many way columns can be used :Many way columns can be used :

1 Icon exchange chromatography1 Icon exchange chromatography

separate proteins by their surface ionic separate proteins by their surface ionic charge charge

2 gel filtration chromatography2 gel filtration chromatography

separate proteins on the basic of size and separate proteins on the basic of size and shapeshape

Affinity chromatographyAffinity chromatography

rapid protein purification rapid protein purification

ATP -----beads ATP binding proteinsATP -----beads ATP binding proteins

immunoaffinity chromatographyimmunoaffinity chromatography

Ab-----beads AgAb-----beads Ag

Ag------Beads AbAg------Beads Ab

modified protein modified protein

Polycacylamide gelPolycacylamide gel

Sodium Dodecyl sulphate ( SDS)Sodium Dodecyl sulphate ( SDS)The mercartoethanol ,secondary, tertiary, qThe mercartoethanol ,secondary, tertiary, q

uarternary structure is usually eliminated.uarternary structure is usually eliminated. The proteins are separated on the basic The proteins are separated on the basic of weight.of weight.

visualize the proteins:visualize the proteins: Coomassie brilliant blue Coomassie brilliant blue immunoblottingimmunoblotting

2.3 Protein molecule can be dire2.3 Protein molecule can be directly equencedctly equenced

Two widely used ways Two widely used ways Edman degradationEdman degradation chemical reaction, in which the amino acid residues are chemical reaction, in which the amino acid residues are

sequentially released from the N-terminus of a polypeptsequentially released from the N-terminus of a polypeptide chainide chain

Tanden mas spectrometry( MS/MS)Tanden mas spectrometry( MS/MS) principle: material travels through the instrument in a maprinciple: material travels through the instrument in a ma

nner that is sensate to its mass/charge rationner that is sensate to its mass/charge ratio protein of interest must be digested into short pettides.protein of interest must be digested into short pettides.

2.4 Proteomics2.4 Proteomics

Proteomics is concerned with the Proteomics is concerned with the identification of the full set of protein identification of the full set of protein produced by a cell or tissue under a produced by a cell or tissue under a particular set of condition ,that relative particular set of condition ,that relative abundance ,and their interacting partner abundance ,and their interacting partner proteins. proteins.

Proteomics base on three principal method:Proteomics base on three principal method:

1 Two-dimensional gel electrophoresis1 Two-dimensional gel electrophoresis

2 mass spectronetry for precise determina2 mass spectronetry for precise determination tion

3 bioinformatics to assigning proteins and 3 bioinformatics to assigning proteins and peptides and to the predicted products of peptides and to the predicted products of protein coding sequencesprotein coding sequences