genecloninggauthaman

8/3/2019 Genecloninggauthaman

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Gene cloningGene cloning


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1. What does it mean toclone a gene?

2. Cutting and pasting DNAmoleculesa. Restriction enzymes

b. Ligases

3. Vectorsa. Plasmids

b. Phage4. Cloning genes

a. Genomic versus cDNA clones

b. Overall strategy for cloninga gene

c. Generating cDNA libraries Generating cDNA

Ligating into vector Vectors for cDNA libraries

Introduce recombinant vectorinto E. coli

Evaluating a cDNA library Screening a cDNA library for a

gene of interest

Analysis of cDNA clones

d. Cloning genomic DNA Cloning capacity of vectors

Generating randomly cleavedgenomic DNA

Cosmid vectors

Screening genomic libraries

e. Generating labeled DNA probes

for screening libraries and otherapplications

OutlineOutline


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Whole organism cloning


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What is a cloned gene?What is a cloned gene? A particular stretch

of DNA isolated froman entire genome

In a bacterial plasmid can produce many

copies of the gene

can produce the protein

plasmid

RECOMBINANTPLASMID

promoter

cloned gene


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Cutting and pasting DNACutting and pasting DNA

Recombinant DNA

Break DNA non randomly

Restriction enzymes


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Restriction enzymesRestriction enzymes

Stewart Linn and Werner Arber1960's

Natural phenomema in bacteria

Host controlled restriction andmodification


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How do we see a phage? After lysis progeny

phage infectneighboring cells

On bacterial lawnexponential

infection giveplaques


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E. colilawn plaque

E. colistrain A transfected withphage

Good infection Poor infection:phage is restricted on

E. colistrain B

E. colistrain B transfected withphage


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Take daughter phagefrom this infection andre-infect strain B

Now we get good infectionWhy?


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E. colistrain A transfected withphage

E. colistrain B transfected withphage

Strain B produces an enzyme which cuts upand destroys foreign DNA

Strain B methylates its own DNA to protectit from degredation by restriction enzyme

Phage that has been propagated in strain B becomes

methylated and thus protected from degredationin strain B in the future


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H.O. Smith, K.W. Wilcox, andH.O. Smith, K.W. Wilcox, and

T.J. KelleyT.J. Kelley Isolated first useful restriction

enzyme 1968

Sequence specific

Hind II5 G T( pyrimidine: T or C) ( purine: A or G) A C 3

3 C A (purine: A or G) (pyrimidine: T or C) T G 5


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Type II enzymes Type III

enzymes

Type I enzymes

Proteinstructure

separate endonucleaseand methylase

bifunctional enzymeof 2 subunits

bifunctional enzyme of 3subunits


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enzymes

Type I enzymes

Proteinstructure




bipartite and

asymmetrical

(e.g TGAN(8)TGCT)

Recognition

site

short sequence (4-6

bp) often palindromic

asymmetrical

sequence of 5-7 bp


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enzymes

Type I enzymes

Proteinstructure




bipartite and

asymmetrical

(e.g TGAN(8)TGCT)

Cleavage site same or very close to

recognition site

24-26 bp

downstream ofrecognition site

non-specific>1000bp

from recognition site

Recognition

site

short sequence (4-6


asymmetrical

sequence of 5-7 bp


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enzymes

Type I enzymes

Proteinstructure




bipartite and

asymmetrical

(e.g TGAN(8)TGCT)


recognition site

24-26 bp


non-specific>1000bp


Restriction

andmethylation

separate reactions same enzyme

catalyzes both

same enzyme catalyzes

both

Recognition

site

short sequence (4-6


asymmetrical

sequence of 5-7 bp


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enzymes

Type I enzymes

Proteinstructure




bipartite and

asymmetrical

(e.g TGAN(8)TGCT)


recognition site

24-26 bp


non-specific>1000bp


Restriction

andmethylation

separate reactions same enzyme

catalyzes both

same enzyme catalyzes

both

ATP needed

forrestriction

no yes yes

Recognition

site

short sequence (4-6


asymmetrical

sequence of 5-7 bp


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Type II enzymes

Proteinstructure



recognition site

Restriction

andmethylation

separate reactions

ATP needed

forrestriction

no

Recognition

site

short sequence (4-6



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Four and six base pairFour and six base pair

cutterscutters

How often will a 4 bpcutter cut a piece

of DNA?

How often will a 6 bpcutter cut a piece

of DNA?

()4 = 1 every 256 ()6

= 1 every 4096


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Sticky endsSticky ends

Example: Eco R1

GAATTCCTTAAG G AATTCCTTAA G3OH

5P

5P

3OH

Sticky end

Eco R1


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Blunt endsBlunt ends

Example: HaeIII

GGCCCCGG

GG CCCC GG

5P3OH

Blunt end

5P 3.OH


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Cla I AT/CGAT Ban III AT/CGAT

Isoschizomers


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1. What does it mean toclone a gene?

2. Cutting and pasting DNAmoleculesa. Restriction enzymes

b. Ligases3. Vectors

a. Plasmids

b. Phage4. Cloning genes

a. Genomic versus cDNA clones

b. Overall strategy for cloninga gene

c. Generating cDNA libraries Generating cDNA

Ligating into vector Vectors for cDNA libraries

Introduce recombinant vectorinto E. coli

Evaluating a cDNA library Screening a cDNA library for a

gene of interest

Analysis of cDNA clones

d. Cloning genomic DNA Cloning capacity of vectors

Generating randomly cleavedgenomic DNA

Cosmid vectors Screening genomic libraries

e. Generating labeled DNA probes

for screening libraries and otherapplications

OutlineOutline


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Pasting DNAPasting DNA


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PlasmidsPlasmids

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/Potter-Dressler.gif


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Individual plasmid molecules visualized by platinum

shadowing and electron microscopy.from Dr. David Summers lab Cambridge UK

relaxed

supercoiled


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Features which make plasmidsFeatures which make plasmids

useful as cloning vectorsuseful as cloning vectors

Antibiotic resistance

High copy number relaxed controlled

stringently controlled

Readily isolated from cells Readily introduced into cells


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TransformationTransformation

Introduction of plasmid DNAinto bacterial cells

Gives colonies


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pBR322

unique sites


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pUC18/19


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-galactosidase

X-galGal +Blue dye

blue colonies

InterruptedLac Z gene

White colonies

-galactosidase

Lac Z gene

EcoR1


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Blue/white selection


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LigationLigation

Ligase

T4 DNA LigaseE. coli DNA Ligase

Potential problems with ligations

Self-ligating vector


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Use CIP to

avoid problems

with self ligation


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Use directional cloningUse directional cloning

Two different restriction enzymes

Give non-complementary sticky ends

Prevents vector from self-ligating


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Bacteriophage vectors

Bacteriophage =eaters of

bacteria

Are always doublestranded DNAviruses

Most derivedfrom

bacteriophage

(lambda)


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Lysogenyo Phage exits as prophage within bacterial cell

o UV light or certain chemical can activateprophage

o inducing lysis and phage release

DifferentDifferent


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DifferentDifferent

LambdaLambdalifestyleslifestyles

http://www.accessexcellence.org/


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Life cycle of temperatephage e.g. Lambda

as a cloning


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as a cloningvector

packagingtransfecting


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Features which makeFeatures which makeuseful as a cloning vectoruseful as a cloning vector

Lytic cycle

Lysogeny dispensible

Packaged in vitro

Size restraints for packaging


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Can accommodatelarger DNAfragments 20-40kb

Difficult tomanipulate in thelab

Phage

Can accommodate

relatively smallDNA fragments toto 10 kb

Easy to manipulate

in the lab

Plasmids


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