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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
9
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
12
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