recombinant dna technology restriction endonucleases; cloning and transformation
TRANSCRIPT
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