1DNA 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.

2SNPs

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

4RFLPs

• 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

RFLPs5

DNA marker may be associated with genetic condition.

www.ncbi.nlm.nih.gov/.../doc/TechRFLP.shtml

6VNTRs 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.

7Use of VNTRs

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

8STRPs

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.

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

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10STRs 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: 2pqBand frequency

11THE 13 CODIS STRs and “probabilities of Identity”

STRAfrican-American U.S. Caucasian

D3S1358 0.102 0.078

vWA 0.058 0.065

FGA 0.035 0.036

TH01 0.102 0.094

TPOX 0.081 0.211

CSF1PO 0.070 0.122

D5S818 0.097 0.140

D13S317 0.131 0.074

D7S820 0.081 0.061

D8S1179 0.075 0.067

D21S11 0.033 0.045

D18S51 0.028 0.030

D16S539 0.066 0.103

http://expertpages.com/news/dna.htm

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Repeat # Caucasian Hispanic African American

Asian

14 0 0 0 0

15 0 0.001 0 0

16 0.001 0.010 0.002 0.034

17 0.002 0.009 0.028 0.025

18 0.237 0.224 0.073 0.152

19 0.003 0.005 0.003 0.022

20 0.018 0.013 0.032 0.007

21 0.021 0.028 0.115 0.034

22 0.038 0.024 0.081 0.017

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)

13RAPD: 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

14RAPD: using PCR to find polymorphisms-2