RLFP , RAPD AND AFLP DESCRIPTION

1. REASTRICTION FRAGMENT LENGTH
POLYMORPHISM ANALYSIS

2. • Definition : used to detect variation in length of
DNA (polymorphisms) in order to distinguish
individuals, populations, or species or to pinpoint
the locations of genes within a sequence.
• Restriction endonuclease enzyme recognisation
site : in DND
• Variations in pattern of restriction endonuclease
enzyme recognisation in two different DNA is due
to variable number of tandem repeats(VNTRs)

3. • Hybridization based method
• Cannot run pcr as require more amount of
DNA

4. • If DNA is incubated with specific restriction
endonuclease enzyme produce cut after
recognising specific sequence on DNA
sequence.
• EcoRV restriction endonuclease site
TWO DIFFERENT DNA SEGMENT
INCUBATE WITH SAME
RESTRICTION
ENDONUCLEASE ENZYME
DIFFERENT DNA FRAGMENTS
agarose gel electrophoresis , Southern
blotting respectively

5. Applications
1) genetic fingerprinting : criminal
identification
2) Paternity testing
3) Diversity or phylogeny study

6. Random amplification of polymorphic
DNA (RAPD)
• PCR based method
• Random primer is used to compare
polymorphism in different DNA
• 1990 BY WILLIUM et.al
• Lab technique by which amplify unknown
sequence

7. STEPS RAPD
• DNA ISOLATION
DENATURATION
ADD RANDOM PRIMER
PUT IN PCR MACHINE
GELE ELECTROPHORESIS

8. Amplified fragment length
polymorphism (AFLP)
• AFLP is a technique used to detect
polymorphisms in DNA when no information
about the genome is known
• Method based on pcr
• Involve use of rflp and pcr techniques
• Faster and less labor require
• Use for geneting mapping

9. • Developed in 1990s by Keygene
• Uses two different restriction enzyme (6 base
cutter and 4 base cutter)

10. • 6 base cutter (EcoRI) and 4 base cutter (Mse1)
cut only complementary DNA sequences.

11. Restriction digestion by two different restriction enzyme
(6 base cutter (EcoR1) and 4 base cutter (Mse1)
DNA isolation
5’ GAATTC 3’
3’CTTAAG5’
5’ TTAA 3’
3’AATT 5’
DNA with complementary sites
GAATTC…..//……TTAA…..//
CTTAAG…//….AATT…//
RESTRICTIO
N PRODUCT
AATTC…//…T
G….//…AAT
LIGATIONS WITH ADOPTORS TO
COMPLEMENTORY DNA
Pcr amplification 2 times to get more
numbers of fragments
EcoR1 and Mse1 Adaptor specific primers (consisting of
complementary to restriction ,adaptors . Add adenine at 3
prime end and cytocin at 5 prime end on both primers
respectively
Selective amplifications
EcoR1: consisting of complementary to
restriction ,adaptors and AGC
Mse1: : consisting of complementary to
restriction ,adaptors and CAG
GEL ELECTROPHORESIS

12. • THANK YOU