1. IMBB 2017 RAB, Kigali - Rwanda May 02 – 13, 2017
PCR-RFLP
IMBB 2017 RAB, Kigali - Rwanda May 02 – 13, 2017
Martina Kyalo

2. Molecular Markers SSR, RFLP RAPD, AFLP Single locus marker
Multi-locus marker
RAPD, AFLP

3. What are restriction enzymes?
Restriction endonucleases are enzymes that cleave the sugar-phosphate backbone of DNA.
Evolved primitive immune system in bacteria.
Why is the bacterial DNA affected??
Restriction endonuclease & DNA methyltransferase (restriction- modification system).
> 3,000 RE discovered.
Classes:
Endonucleases: cleave within DNA strands.
Exonucleases: cleave from ends of DNA strands.

4. Restriction enzymes Named after their host of origin e.g.
EcoRI was isolated from Escherichia coli (strain RY13).
Hind II and Hind III from Haemophilus influenza.
XhoI from Xanthomonas holcicola.
Specific-palindromic sequences.
Cleave dsDNA though few cleave ssDNA, although usually at low efficiency.
Recognize 4-, 6- or 8- base pairs.

5. Restriction enzymes
The recognition site for one enzyme may contain the restriction site for another e.g. BamHI /Sau3AI.
Isoschizomers recognize identical sequences
SphI (CGTAC/G) and BbuI (CGTAC/G).
Different optimum reaction conditions, stabilities and costs.

6. Restriction enzymes Restriction recognitions sites can be: Unambiguous

7. Restriction enzymes Produce 3 different types of cuts: Blunt cut
3' overhangs
5' overhangs

8. Requirements for restriction digestion
Template DNA.
Restriction enzyme(s).
Restriction Buffer.
Water Bath/PCR machine.
Enhancers e.g. BSA

9. Procedure for PCR-RFLP
Total DNA extraction
PCR
Restriction digestion
Agarose electrophoresis
Data analysis

10. Restriction Digestion

11. PCR-RFLP

12. NEB cutter

13. Applications of PCR-RFLP
DNA Cloning.
Recombinant DNA Technology.
Molecular breeding.
Constructing DNA Libraries.
Southern Blot Hybridization.
Forensics (CSI).

14. IMBB 2017 RAB, Kigali - Rwanda May 02 – 13, 2017
Thank you
IMBB 2017 RAB, Kigali - Rwanda May 02 – 13, 2017
Martina Kyalo