Recombinant DNA Technology - Tools and Techniques

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Recombinant DNA Technology Tools and Techniques
R. C. Gupta M.D. (Biochemistry) Jaipur,
India
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Recombinant DNA, having unrelated genes, is also
known as chimeric DNA
Chimera is a mythological creature having the
head of a lion, the trunk of a goat and the tail
of a serpent
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Restriction endonucleases
The most important tools of recombinant DNA
technology
Discovered by Arber, Smith and Nathans in the
early 1970s
Opened the doors of recombinant DNA
research
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A palindrome is a word or a sentence which reads
the same from left to right and from right to
left e.g. DAD, MADAM, RADAR etc
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5 GGCC 3 5 TTTAAA 3 3 CCGG 5
3 AAATTT 5
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Blunt ends
Sticky ends
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Restriction sites of some enzymes
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Amplification
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Cloning of recombinant DNA
Cloning of DNA was pioneered by Herbert Boyer and
Stanley Cohen
Stanley Cohen
Herbert Boyer
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A clone is a population of identical organisms or
cells or molecules derived from the same source
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Plasmids
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Technique
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Multiplication of the bacterial cell
and independent replication of the plasmid
produce multiple copies of the foreign
DNA within a short time
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An ideal vector should replicate rapidly, should
have several restriction sites for different
restriction enzymes and more than one
antibiotic-resistance genes
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Ampicillin-resistance gene
pBR322
Tetracycline-resistance gene
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A foreign gene/DNA fragment can be inserted
in the middle of an antibiotic-resistance
gene
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Agar plate containing ampicillin
Replica plate containing tetracycline
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Bacteriophages
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The virus infects a bacterial cell by
injecting its DNA into the bacterial cell
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Lysogenic pathway
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Lytic pathway
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Cosmids
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Bacterial artificial chromosome (BAC)
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Restriction sites
BAC
Chloramphenicol- resistance gene
Origin of replication
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Yeast artificial chromosome (YAC)
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Plasmid 10 kb Phase 20 kb Cosmid 45
kb BAC 100-300 kb YAC 3,000 kb
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Polymerase chain reaction (PCR)
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Nested PCR
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First primer
Second primer
Nested PCR
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Multiplex PCR
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Multiplex PCR
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Reverse transcriptase-PCR
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Quantitative PCR
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Techniques for identification of
DNA and RNA
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Southern blotting
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Samples
DNA ladder
Gel
Nitrocellulose sheet
Paper towels
Nitrocellulose sheet
Gel
Gel
Sponge
Buffer
The fragments are transferred to a nitrocellulose
sheet
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Northern blotting
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Northern blot transfer
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Western blotting
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Western blot transfer
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Techniques for determining base sequence of
DNA
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Maxam-Gilbert method
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Sangers dideoxy method
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Frederick Sanger
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CH2
2
3
2
3
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• The relative concentrations of the
  dideoxynucleotide and its normal counterpart are
  such that the dideoxy analogue enters randomly at
  different sites in different cycles of
  replication

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