PCR-RFLP

1. Restriction Fragment Length
Polymorphism (RFLP)
Dr. Abdul Hameed,PhD
Chief Scientific Officer
Institute of Biomedical and Genetic Engineering (IBGE),
24-Mauve Area, G-9/1, Islamabad, Pakistan
ahameed0786@hotmail.com

2. RFLP – Restriction Fragment Length Polymorphism
 Variation in the DNA sequence of a genome
detected by cutting DNA into pieces with
restriction enzymes.
 Important tool in genome mapping, localization of
genetic disease genes, determination of risk for a
disease, genetic fingerprinting and paternity testing
etc.

3. Restriction Enzymes

4. Restriction Endonucleases
Also called restriction enzymes
1962: “molecular scissors” discovered in bacteria
3,000 enzymes have been identified, around 200 have
unique properties, many are purified and available
commercially

5. Restriction Endonucleases
Named for bacterial genus, species, strain, and type
Example: EcoR1
Genus: Escherichia
Species: coli
Strain: R
Order discovered: 1

6. Restriction Endonucleases
Enzymes recognize specific 4-8 bp sequences
Some enzymes cut in a staggered fashion - “sticky ends”
EcoRI 5’…GAATTC…3’
3’…CTTAAG…5’
Some enzymes cut in a direct fashion – “blunt ends”
PvuII 5’…CAGCTG…3’
3’…GTCGAC…5’

7. Uses for Restriction Enzymes
RFLP analysis (Restriction Fragment Length
Polymorphism)
DNA sequencing
DNA storage – libraries
Transformation
Large scale analysis – gene chips

8. Restriction Fragment Length
Polymorphism Analysis
1. DNA digestion with restriction enzyme(s)
2. Separation of Digested DNA on gel
electrophoresis
• Smear - Many DNA fragments with
slight differences in length
3. Denaturing the double-stranded DNA to make
it single-stranded by treating the gel
chemically
4. Southern blotting (Transfer of single stranded
DNA on to a positively charged nylon
membrane

9. RFLP Analysis
4. Southern blotting:
i. Transfer DNA from gel to
nylon membrane
ii. Expose nylon membrane to
solution with radioactive
complementary nucleotide
probes that hybridize to
specifically chosen DNA
sequences on nylon
membrane
iii. Place nylon membrane
against X-ray film, where
hybridized radioactive
probes cause exposure of
X-ray film, producing an
autoradiogram
http://www.cbs.dtu.dk/staff/dave/roanoke/genetics980211.html

10. Genotyping a biallelic RFLP
marker in a family.
• PCR amplification
• Digestion with restriction enzyme
• Separation of digested DNA fragments by agarose gel
electrophoresis
• Staining the gel with ethidium Bromide (Fluoresce under
UV-illumination only when bound to DNA

11. NPHS2 Mutation Identified By
DNA Sequencing

12. NPHS2 Gene: NORMAL Exon 5 Sequence
 cataggaaaggagcccaagaatcaagcctgtcatccaaacttttttctgcctagATCGTGACCAAAGA
CATGTTTATAATGGAGATAGATGCCATTTGCTACTACCGAATGGAAAATGCC
TCTCTTCTCCTAAGCAGTCTTGCTCATGTATCTAAAGCTGTGCAATTCCTTG
TGCAAACCACTATGAAGCGTCTCCTAGCACATCGATCCCTCACTGAAATTCT
TCTAGAGAGGAAGAGCATCGCCCAAGATGCAAAGgtacttagataaacataatggcca
atatgctgaaa
NPHS2 Gene: MUTANT exon 5 Sequence (9bp Del)
 cataggaaaggagcccaagaatcaagcctgtcatccaaacttttttctgcctagATCGTGACCAAAGA
CATGTTTATAATGGAGATAGATGCCATTTGCTACTACCGAATGGAAAATGCC
TCTCTTCTCCTAAGCAGTCTTGCTCATGTATCTAAAGCTGTGCAATTCCTTG
TGCAAACCACTATGAAGCGTCTCCTAGCACATCGATCCCTCACTGAAATTCT
GAAGAGCATCGCCCAAGATGCAAAGgtacttagataaacataatggccaatatgctgaaa
PCR-RFLP (NPHS2 Exon 5)

13. R.E Mapping (NEBcutter)
http://nc2.neb.com/NEBcutter2/

14. NPHS2, Ex.5 Restriction Maps

15. PCR-RFLP Steps
 PCR Amplification (NPHS2 Gene Ex.5)
 Digestion with R.E (XbaI)
 Agarose Gel Electrophoresis
 Gel Staining with Ethidium Bromide
 Gel Photograph Under UV-illumination

16. Figure 2: Molecular basis of nephrotic syndrome. (A) Pedigree of NPHS family
shows consanguineous family with three male patients (filled boxes). (B) RFLP
analysis of PCR fragment amplified from exon 5 of the NPHS2 gene utilizing Xba I
restriction enzyme. The 293bp PCR product of exon 5 for all family members was
digested with Xba I restriction enzyme. Fragments were resolved on 2.0% agarose
gel. Individuals with a single fragment of 284bp were identified as homozygous
mutant since they did not contain the restriction site for XbaI. Those with two
fragments (225bp and 68 bp) were heterozygous normal/carriers for the mutation. M:
DNA size standard, UC: Undigested control PCR product.

18. Choosing the Restriction Enzyme for your SNP
http://watcut.uwaterloo.ca/template.php

19. Paste FASTA sequence here

20. SNP in the sequence
[g/a]
Click

21. Click

22. Restriction Database (rebase)
 http://rebase.neb.com/rebase/rebase.ht
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