Restriction fragment length polymorphism (RFLP) analysis detects variations in DNA fragment length to distinguish individuals. It involves incubating DNA with restriction endonucleases that cut DNA at specific recognition sites. Differences in the resulting fragment patterns between individuals arise from variations in restriction enzyme recognition sites. RFLP analysis can be used for genetic fingerprinting, paternity testing, and studying diversity. Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) are PCR-based methods that detect polymorphisms without prior DNA sequence knowledge and are faster than RFLP analysis.

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