DNA Polymorphisms: DNA markers a useful tool in biotechnology

1. DNA Polymorphisms: DNA markers a useful tool in biotechnology • Any section of DNA that varies among individuals in a population, “many forms”. • Examples include: SNPs, RFLPs, STRPs, and AFLPs; • RFLPs include VNTRs and STRPs • microsatellites (STRs) = SSLPs = STRPs = SSRs • Useful for finding, mapping genes involved in disease, and • Individual identification, epidemiology, anthropology, population/ecology studies, taxonomy.

2. SNPs Single nucleotide polymorphisms: regions of DNA where one base pair is different. Occur evenly spread over all the DNA. 1/ 1000-3000 bp Detected by sequencing. If SNP occurs in a restriction enzyme site, it generates an RFLP. Could be in coding or non-coding regions. Over 300,000 human SNPs known and are being mapped.

3. SNP

4. RFLPs • Restriction fragment length polymorphism. • Any difference in the DNA that results in a restriction enzyme producing a different sized piece. • Mutation at a restriction site prevents recognition & cutting. • Results in one band of larger DNA instead of 2 smaller ones. • Different numbers of repeats between restriction recognition sites also generates an RFLP

5. RFLPs www.ncbi.nlm.nih.gov/.../doc/TechRFLP.shtml DNA marker may be associated with genetic condition.

6. VNTRs and STRPs as RFLPs: Minisatellites and Microsatellites • These are RFLPs because they are defined by or visible following restriction enzyme cuts. • Variable Number Tandem Repeats • Groups (10-100) of nucleotides repeated 2 – 100 times (depending on individual and locus). • Restriction sites on both sides of repeated DNA • The more repeats, the longer the fragment. • Simple Tandem Repeat Polymorphisms • Shorter, 2-9 nucleotides repeated • Small enough number for PCR amplification • Also called STRs, SSLPs, etc.

7. Use of VNTRs Restriction sites are on either side; fragment length depends on number of repeats in between sites.

8. STRPs Primers for both sides of repeated region allow PCR amplification of DNA; generates PCR products that differ in length depending on number of repeats. Becoming the standard method for DNA testing in forensics labs. Cheaper, easier, more sensitive.

9. Hardy-Weinberg meets Gil Grissom • Simple tandem repeats • 13 have been chosen for use in forensic work • The 13 independently assort, meaning they are on different chromosomes or far apart on the same. • Product law can be used • Each of the 13 have a number of different alleles • Alleles differ by number of repeats • Repeats vary from 3 to 5. vWA is a tetranucleotide. • Allele frequencies: p1, p2, etc. for each allele • Humans are diploid, have 2 alleles for each locus

10. STRs in forensics Locus vWA • 14 0.081 • 15 0.107 • 15.2 0.179 • 16 0.306 • 17 0.192 • 18 0.089 • 19 0.047 Alleles in different ethnic and racial groups examined, used as database. Panel of 13 different STRs are used. Because the odds of a particular combination of the 13 is product of the frequencies, numbers like 1 in 10 billion can be generated. Hardy-Weinberg: 2pq Band frequency

11. THE 13 CODIS STRs and “probabilities of Identity” http://expertpages.com/news/dna.htm

12. Allele frequencies for D1S80 among US population groups Chance of a white person being heterozygous for alleles 19 and 20: 2 x 0.003 x 0.018 (One in 9,259)

13. RAPD: using PCR to find polymorphisms “Random amplified polymorphic DNA” Screen DNA from individuals by doing PCR with random short primers. (about 8-12 nucleotides) By random chance, primers will amplify many different sections of DNA. Look for bands on gel that are not present in each individual tested. avery.rutgers.edu/.../ archives/onions/rapd.html

14. RAPD: using PCR to find polymorphisms-2