Restriction Enzyme

(Restriction Endonuclease)
 Example of restriction
endonucleases
Enzyme Producing oganism Cleavage Site

PvuI Proteus vulgaris …….CGATCG……..

GCTAGC
HpaI Haemophilus parainfluenzae …….GTTAAC……..
CAATTG
EcoRI Escherichia coli …….GAATTC……..
CTTAAG
Sau3AI Staphylococcus aureus …….GATC…….
CTAG
 Types of restriction sites :
1. Sticky end
2. Blunt end
 Polymerase Chain Reaction
 The standard polymerase chain reaction (PCR)
amplifies a specific DNA segment many fold
 This is necessary to have enough starting template for
sequencing.
 It uses two oligonucleotide primers complementary to
opposite strands of a duplex DNA
 The primer 3' ends when annealed to the target DNA
are closer to one another than the 5' ends are.
 A PCR consists of multiple cycles of denaturation of
the synthesized DNA, annealing of the
oligonucleotides, synthesis of a DNA chain
 PCR is used widely in:
 Molecular cloning
 Pathogen detection
 Genetic engineering
 Mutagenesis
 Genetics, producing molecular markers
 The cycling reactions of PCR:
 Denaturation at 94°C
 Annealing at 54°C

 extension at 72°C

 To amplify RNA, a cDNA copy of the RNA

can be made by reverse transcription prior to
PCR. The combination is called RT-PCR.
 Is there a gene copied during PCR
and is it the right size ?
 Before the PCR product is used in further
applications, it has to be checked if :
 There is a product formed
 not every PCR is successful
 the quality of the DNA is poor

 the quality of the DNA is poor

 there is too much starting template

 The product is of the right size
 expected gene should be 1800 bases long but
showed a band of 500 bases
 one of the primers probably fits on a part of the
gene closer to the other primer or both primers fit
on a totally different gene
 Only one band is formed
 the primers fit on the desired locations, and also on
other locations
 have different bands in one lane on a gel.
 RAPD Analysis
 Random Amplification of Polymorphic DNA
 described by Williams et al. (1990)
 commonly used molecular marker in genetic
diversity studies.
 The principle involved in generating RAPDs is
 a single, short oligonucleotide primer, which binds to
many different loci
 used to amplify random sequences from a complex
DNA template
 RAPDs are dominant markers and therefore cannot be
used to identify heterozygotes
 advantages (Karp and Edwards,1997):
 no DNA probes
 sequence information is not required for the design of
specific primers
 no blotting or hybridisation steps on the procedure
 require small amounts of DNA (about 10 ng per reaction)
 detect higher levels of polymorphism
 RAPD reactions are PCR reactions, but they amplify
segments of DNA which are essentially unknown
 in RAPD analysis, the target sequence(s) (to be
amplified) is unknown
 a primer is an arbitrary sequence
 RAPDs exhibit polymorphism and thus can be used as
genetic markers
 RAPD bands of differing lengths can be assigned to
the same locus, these RAPD bands are codominant
Standard PCR:
 In this example, only 2 RAPD PCR products
are formed:
 Product A is produced by PCR amplification of the
DNA sequence which lies in between the primers
bound at positions 2 and 5.
 Product B is the produced by PCR amplification of
the DNA sequence which lies in between the
primers bound at positions 3 and 6.
 no PCR product is produced by the primers
bound at positions 1 and 4 because these
primers are too far apart to allow completion of
the PCR reaction.
 no PCR products are produced by the primers
bound at positions 4 and 2 or positions 5 and 3
because these primer pairs are not oriented
towards each other.
there was a change in sequence at primer annealing site #2
the primer is no longer able to anneal to site #2, and thus the PCR
product A is not produced