IMBB 2017 RAB, Kigali - Rwanda May 02 – 13, 2017

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Presentation transcript:

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

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

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.

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.

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.

Restriction enzymes Restriction recognitions sites can be: Unambiguous

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

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

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

Restriction Digestion

PCR-RFLP

NEB cutter http://www.labtools.us/nebcutter-v2-0/

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

IMBB 2017 RAB, Kigali - Rwanda May 02 – 13, 2017 Thank you IMBB 2017 RAB, Kigali - Rwanda May 02 – 13, 2017 Martina Kyalo m.kyalo@cgiar.org