Recombinant DNA Technology

Restriction Endonucleases; cloning and Transformation

Restriction Endonucleases

• Restriction endonucleases RESTRICT viruses Viral genome is destroyed upon entry

• Restriction endonuclease = Restriction enzymes Endo (inside), nuclease (cuts nucleic acid)

• Restriction endonuclease recognizes a short and specific DNA sequence and cuts it from inside.

• The specific DNA sequence is called recognition sequence

• 1952-53: Luria and Human discovered the phenomenon of restriction and modification Named as host-induced, or host-controlled, variation

Few Restriction Enzymes

Enzyme Organism from which derived

Target sequence

(cut at *)

5' -->3'

Bam HI Bacillus amyloliquefaciens G* G A T C C

Eco RI Escherichia coli RY 13 G* A A T T C

Hind III Haemophilus inflenzae Rd A* A G C T T

Mbo I Moraxella bovis *G A T C

Pst I Providencia stuartii C T G C A * G

Sma I Serratia marcescens C C C * G G G

Taq I Thermophilus aquaticus T * C G A

Xma I Xanthamonas malvacearum C * C C G G G

Protection of Self-DNA

Bacteria protect their self DNA from restriction digestion by methylation of its recognition site.

Methylation is adding a methyl group (CH3) to DNA.

Restriction enzymes are classified based on recognition sequence and methylation pattern.

Palindrome, Restriction Enzyme, Sticky Ends

GAATTCGAATTC

GAATTC G

AATTC

Sticky Ends(Cohesive Ends)

EcoRI

CIVIC, Madam

Get An Apple To The Class

G

AATTC G

AATTC

Restriction Mapping of DNA

A B 10 kb

8 kb2 kb

A

7 kb3 kb

B

5 kb3 kb2 kb

A+B

CK A B A+B M

Restriction enzymes

Juang RH (2004) BCbasics

The Specific Cutting and Ligation of DNA

GAATTC

CTTAAG

GAATTC

CTTAAG

G

CTTAA

AATTC

G

AATTC

G

G

CTTAA

G

CTTAA

AATTC

G

G

CTTAA

AATTC

G

G

CTTAA

AATTC

G

EcoRI

DNA LigaseEcoRI sticky end EcoRI sticky end

CLONING

CLONING

CLONING VECTORS

PLASMIDS

• Extra-chromosomal DNA found in bacteria.• Loops of double-stranded DNA and some of them present

in multiple copies.• Independently replicate inside bacteria.• Purpose of a vector is to carry DNA into a cell.

• Foreign DNA must be cut with same enzyme as plasmid so

the ‘ends’ are compatible – plasmid and foreign DNA are

then joined by ligase enzyme.• Artificial plasmids have been genetically engineered for the

purpose of cloning.• Used as vectors, i.e., to transfer DNA from one cell

to another.

Features of plasmids

Plasmids also contain a selectable marker.

• Usually an antibiotic resistance gene:– required for maintenance of plasmid in the

cell– advantageous for bacteria to keep the

plasmid (can then grow in presence of antibiotic).

• Commonly used selectable markers are ampicillin, neomycin and chloramphenicol.

Cloning vectors

• In gene cloning, once recombinant DNA (rDNA) has been constructed it is introduced into a host.

• In the host, rDNA has to be:

maintained

replicated

passed from one generation to another.• This is achieved by introducing rDNA into a cell

on a DNA vehicle called a cloning vector – most commonly used are plasmid cloning vectors.

Drug Resistance Gene Transferred by Plasmid

Plasmid gets out and into the host cell

Resistant Strain

New Resistance Strain

Non-resistant Strain

Plasmid

EnzymeHydrolyzingAntibiotics

Drug Resistant Gene

mRNA

Target Genes Carried by Plasmid

1 plasmid1 cellRecombinant

PlasmidTransformation

Target GeneRecombination

Restriction

Enzyme

Restriction

Enzyme

Ch

rom

oso

mal

DN

ATarget Genes

DNA Recombination

TransformationHost Cells

What is Bacterial Transformation?

The transformation of bacteria!

The genetic information of a bacterial cell actually takes in new genetic information and makes it a part of itself! It can then copy that sequence over and over and over and over and over ………………

Steps in transforming bacteria

1. Bacteria incubated with plasmid (with appropriate promoter and antibiotic resistance gene).

2. Bacteria plated out onto agar plates containing antibiotic.

3. Only those bacteria that have taken up plasmid will be able to grow on agar plus antibiotic.

4. Transformed bacteria can then be isolated and grown in bulk with appropriate antibiotic.

5. Bacteria multiply to produce genetically identical offspring – clones.

Amplification and Screening of Target Gene

1

1 cell line, 1 colonyX100

X1,000

PlasmidDuplicationBacteria

Duplication

Plating

Pick the colonycontaining target gene

=100,000

Questions?

THANKS