Genetic engineering-1

Dr Malpe Surekha Bhat

 Introduction
Genetic engineering includes techniques
used for
DNA sequencing
DNA cloning
Detection of mutations
Prenatal diagnosis
Tracing chromosomes from parents to
offspring
 Three challenges

1. To cleave huge DNA molecules into

defined fragments – restriction
endonucleases
2. To amplify a fragment into multiple copies
of detectable amount – Cloning, PCR
3. To detect and isolate a fragment from
multiple fragments – Probes and Blotting
techniques
Cleavage of DNA to fragments
- Use of Restriction Endonucleases

Amplifying DNA fragments

- Gene cloning
- Polymerase chain reaction
Detection and isolation
- Probes
- Blotting techniques
- RFLP
 Restriction Endonucleases
Enzymes that
 Are present in bacteria, restricts the
expression of foreign DNA
 Are named based on the bacteria of origin
Eg. EcoR1 – Eco = Escherichia coli
R1 = Strain RY13.
 Recognises specific palindromic sequences on
DNA (restriction sites)
 cuts DNA at restriction sites to generate DNA
fragments (restriction fragments)
 Produces sticky or blunt ends on restriction
fragments
 Sticky ends
 One strand extends beyond the complementary region, to produce an overhang
 Two different DNA
fragments with sticky
ends, produced from
same restriction
endonuclease can join
easily to form a hybrid
DNA
 Blunt ends
 Both complementary strands of DNA are of
equal length
 Outline of DNA cloning
 Isolation of two kinds of DNA
- foreign DNA that contains gene of
interest ,
- Vector (Carrier) DNA

 Treatment of vector and foreign DNA with

the same restriction enzyme

 Mixture of foreign DNA with vector

 Outline of DNA cloning – contd.
 Addition of DNA ligase to form recombinant
DNA
 Introduction of recombinant DNA into
bacterial cells by transformation
 Bacteria are grown on culture plates,
multiple gene copies are produced
 Amplified gene copies stored as libraries and
retrieved when needed OR
genes are allowed to expressed in bacteria
to produce industrial amounts of proteins
DNA libraries
 Carriers used in cloning
(Self study)
 Plasmids – define, properties
 Phage DNA
 Retrovirus DNA
 YAC vectors
 BAC vectors
Polymerase chain reaction