1. Restriction Fragment Length Polymorphism

2. Definition The variation in the length of DNA fragments produced by a restriction endonuclease that cuts at a polymorphic locus.  Such variations are generated by mutations that create or abolish recognition sites for these enzymes.

3. Contents RFLP technology  Isolating DNA  Restriction digestion and gel electrophoresis  DNA transfer by Southern blotting  DNA hybridisation Interpreting RFLP bands Advantages and disadvantages of RFLPs Applications

4. RFLP Technology

5. RFLP technology

6. DNA isolation Total DNA of very high molecular weight  Plants – Chloroplast/mitochondria/nucleus of plant cells  Humans- Any human tissue Eg: Buccal cells, saliva, blood, hair follicle, semen etc. Complications  Breakage during isolation  Nucleases degrading DNA

7. Restriction enzyme digestion and gel electrophoresis

8. Southern Blotting

9. Hybridisation

10. DNA probes

11. DNA probes sources Nuclear DNA:  Genomic libraries  cDNA Cytoplasmic DNA The species specificity of many single-locus probes requires that libraries be built when studying new species. However, probes from related genera can often be used

12. DNA probes sources Repetitive sequences or minisatellite-type: Basic ‘motif’ of 10 to 60 bp in tandem Highly variable between human individuals Polymorphisms in the number of repeated units (also called VNTRs

13. Criteria for choosing a RFLP marker Highly polymorphic alleles with equivalent frequencies (Co-dominant markers or dominant-recessive). The RFLP markers most commonly used for DNA profile analysis are found on chromosomes 1, 2, 4, 5, 10 and 17. These RFLP markers are named after their locations on these chromosomes. For example, the marker on chromosome 2 is called D2S44 (section 44 of chromosome 2). These chromosomal locations are also referred to as DNA loci. 393 RFLPs : Donnis Keller (1987)

15. Summary

16. Animation http://bcs.whfreeman.com/thelifewire/content/chp16/ 1602001.html http://bcs.whfreeman.com/thelifewire/content/chp16/ 1602001.html http://www.biostudio.com/d_%20RFLP%20Analysis. html http://www.biostudio.com/d_%20RFLP%20Analysis. html http://www.biostudio.com/d_%20Detecting%20RFLP s.html http://www.biostudio.com/d_%20Detecting%20RFLP s.html

17. RFLP Analysis

18. Restriction enzymes Restriction enzymes are produced by bacteria as a defense against viruses. These enzymes cut DNA at specific base sequences called recognition sites. Results in smaller pieces of DNA called RFLP’s

19. Restriction site example: EcoR1

20. Interpretation: Case1

21. Interpretation: Case2

22. Interpretation: Case3

23. Interpretation: Case4

24. RFLP Applications Advantages and Disadvantages

25. RFLP Applications Agriculture – direct method for selecting desirable genes such as disease resistance Forensics Paternity Genetic mapping  Determine disease status of an individual, ie, Huntington’s chorea  Cystic fibrosis  Sickle cell anemia Genetic counselling – very important when discussing results with patients or parents who use this technology to have children who are free of genetic disease.

26. Genetic Mapping : Sickle-cell Anaemia

27. Genetic mapping : Cystic fibrosis Cystic Fibrosis is an autosomal recessive disorder.

28. Paternity issues

29. Forensics: VNTR Molecular biologists have identified regions of the human genome where restriction fragment lengths are highly variable between individuals VNTR stands for ‘variable number of tandem repeats. A tandem repeat is a short sequence of DNA that is repeated at a specific chromosomal locus. Tandem repeats are interspersed throughout the human genome.

30. Forensics: VNTR The number of repeats at a given place on a certain chromosome is highly variable from one person to another. The number of such repeats is usually different on the paternal and maternal members of the same person’s chromosome pair.

31. VNTR Red boxes represent the repeat unit and the blue lollipops represent cut sites for a restriction endonuclease. (Here 3 different variants, may be 50 in reality).

32. VNTR: 3 alleles A,B,C The possible genotypes are AA, BB, CC, AB, BC, and AC

33. VNTR When profiles from a single VNTR locus from unrelated individuals are compared, the profiles are normally different. However, it is possible for two individuals to have the same profile at one or two loci. But the chance of more than one person having the same DNA profile at 4, 5, or 6 different VNTR loci is extremely small. FBI- 13 STRs

34. VNTR probability The frequency of an allele pattern at a single VNTR locus : 2pq; frequency of allele p and q in population The frequency of an allele pattern at a multiple VNTR locus  In most cases, a "product rule" calculation can be done by multiplying each individual probability together

35. Problem Assume that the frequency of the DNA profiles for the 5 individual loci were 0.01, 0.02, 0.06, 0.10, and 0.03. How common or rare would this 5 locus DNA profile be in the reference population? Answer: Thus the frequency of the profile is 0.01 x 0.02 x 0.06 x 0.10 x 0.03 = 3.6 x 10 8. Another way to express this probability is take the reciprocal of this number, 1/3.6 x 108 = 27.8 million. Therefore the probability is 1 in 27.8 millions For 13 STR identified by FBI: 1 in 7.7 x 10 15

36. Advantages Highly robust methodology with good transferability between laboratories Codominantly inherited and, as such, can estimate heterozygosity No sequence information required Because based on sequence homology, highly recommended for phylogenetic analysis between related species Well suited for constructing genetic linkage maps Discriminatory power—can be at the species and/or population levels (single-locus probes), or individual level (multi-locus probes) Simplicity—given the availability of suitable probes, the technique can readily be applied

37. Disadvantages Large amounts of DNA required Automation not possible Low levels of polymorphism in some species Need a suitable probe library Time consuming, especially with single-copy probes Costly