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Commonly Used Short Tandem Repeat Markers

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Presentation on theme: "Commonly Used Short Tandem Repeat Markers"— Presentation transcript:

1 Commonly Used Short Tandem Repeat Markers
Chapter 5 Commonly Used Short Tandem Repeat Markers ©2002 Academic Press

2 Types of STR Repeats Dinucleotide Trinucleotide Tetranucleotide
Microsatellite = simple sequence repeat (SSR) = short tandem repeat (STR) Dinucleotide Trinucleotide Tetranucleotide Pentanucleotide Hexanucleotide (CA)(CA)(CA)(CA) (GCC)(GCC)(GCC) (AATG)(AATG)(AATG) (AGAAA)(AGAAA) (AGTACA)(AGTACA) Require size based DNA separation to resolve different alleles; dinucleotide repeats require higher resolution ©2002 Academic Press

3 STR Repeat Nomenclature
International Society of Forensic Haemogenetics (ISFH) -- Int. J. Legal Med. (1997) 110: For sequences within genes, use the coding strand For other sequences, select the first GenBank database entry or original literature description Define the repeat sequence which will provide the largest number of consecutive repeats If two sequences are repeated, include both motifs in determining the repeat number ©2002 Academic Press

4 Why STRs are Preferred Genetic Markers
Rapid processing is attainable Abundant throughout the genome Highly variable within various populations Small size range allows multiplex development Discrete alleles allow digital record of data Allelic ladders simplify interpretation PCR allows use of small amounts of DNA material Small product size compatible with degraded DNA ©2002 Academic Press

5 Short Tandem Repeats (STRs)
Fluorescent dye label AATG AATG 7 repeats 8 repeats the repeat region is variable between samples while the flanking regions where PCR primers bind are constant Homozygote = both alleles are the same length Heterozygote = alleles differ and can be resolved from one another Primer positions define PCR product size ©2002 Academic Press

6 Allelic Ladder Formation
Separate PCR products from various samples amplified with primers targeted to a particular STR locus Combine Re-amplify Find representative alleles spanning population variation Polyacrylamide Gel ©2002 Academic Press

7 STR genotyping is performed by comparison of sample data to allelic ladders
Microvariant allele ©2002 Academic Press

8 TH01 Sequence from GenBank with Several PCR Primer Positions
195 bp 170 bp 1 2 4 3 5 6 7 8 9 AATG repeat unit 9 8 7 6 5 4 3 2 1 ©2002 Academic Press

9 STR Markers Used in DNA Typing
Tetranucleotide repeats lower stutter Multiplexed with fluorescent dyes better for mixtures increases genotyping throughput 13 STRs required in U.S. for CODIS Commercial kits from Promega Corporation and Applied Biosystems ©2002 Academic Press

10 Position of Forensic STR Markers on Human Chromosomes
13 CODIS Core STR Loci CSF1PO D5S818 D21S11 TH01 TPOX D13S317 D7S820 D16S539 D18S51 D8S1179 D3S1358 FGA VWA AMEL Sex-typing ©2002 AP, Elsevier

11 Information on 13 CODIS STRs
Locus Name Chromosomal Location Repeat Motif ISFH format GenBank Accession Allele in GenBank Allele Range Number of Alleles Seen CSF1PO 5q TAGA X14720 12 6-16 15 FGA 4q28 CTTT M64982 21 69 TH01 11p15.5 TCAT D00269 9 3-14 20 TPOX 2p23-pter GAAT M68651 11 6-13 10 VWA 12p12-pter [TCTG][TCTA] M25858 18 10-24 28 D3S1358 3p [TCTG][TCTA] Not available -- 9-20 20 D5S818 5q21-31 AGAT G08446 11 7-16 10 D7S820 7q GATA G08616 12 6-15 22 D8S1179 8 [TCTA][TCTG] G08710 12 8-19 13 D13S317 13q22-31 TATC G09017 13 5-15 14 D16S539 16q24-qter GATA G07925 11 5-15 10 D18S51 18q21.3 AGAA L18333 13 7-27 43 D21S11 21q21 Complex [TCTA][TCTG] AP000433 29 24-38 70 ©2002 Academic Press

12 Commercially Available STR Kits
Name Source Release Date STR Loci Included *Power of Discrimination TH01, TPOX, CSF1PO monoplexes Promega Feb 1993 TH01, TPOX, CSF1PO 1:410 AmpFlSTR Blue Applied Biosystems Oct 1996 D3S1358, VWA, FGA 1:5000 AmpFlSTR Green I Jan 1997 Amelogenin, TH01, TPOX, CSF1PO CTTv CSF1PO, TPOX, TH01, VWA (vWF) 1:6600 FFFL F13A1, FES/FPS, F13B, LPL 1:1500 GammaSTR D16S539, D13S317, D7S820, D5S818 1:1.8x104 PowerPlex (version 1.1 and 1.2 later) CSF1PO, TPOX, TH01, VWA, D16S539, D13S317, D7S820, D5S818 1:1.2x108 AmpFlSTR Profiler May 1997 D3S1358, VWA, FGA, Amelogenin, TH01, TPOX, CSF1PO, D5S818, D13S317, D7S820 1:3.6x109 AmpFlSTR Profiler Plus Dec 1997 D3S1358, VWA, FGA, Amelogenin, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820 1:9.6x1010 AmpFlSTR COfiler May 1998 D3S1358, D16S539, Amelogenin, TH01, TPOX, CSF1PO, D7S820 1:8.4x105 AmpFlSTR SGM Plus Feb 1999 D3S1358, VWA, D16S539, D2S1338, Amelogenin, D8S1179, D21S11, D18S51, D19S433, TH01, FGA 1:3.3x1012 PowerPlex 2.1 Jun 1999 D3S1358, TH01, D21S11, D18S51, VWA, D8S1179, TPOX, FGA, Penta E 1:8.5x1010 PowerPlex 16 May 2000 CSF1PO, FGA, TPOX, TH01, VWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, Penta D, Penta E, amelogenin 1:1.8x1017 AmpFlSTR Identifiler May 2001 CSF1PO, FGA, TPOX, TH01, VWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, D19S433, D2S1338, amelogenin 1:2.1x1017 *Caucasian population (rounded to 2 significant figures) ©2002 Academic Press

13 Multiplex PCR with 15 STR Loci and Amelogenin Gender ID
PowerPlex® 16 Result Multiplex PCR with 15 STR Loci and Amelogenin Gender ID DNA Size (bp) D3S1358 TH01 D21S11 D18S51 Penta E D5S818 D13S317 D7S820 D16S539 CSF1PO Penta D Sex-typing AMEL vWA D8S1179 TPOX FGA ©2002 Academic Press JMB

14 Currently 2 Suppliers: Applied Biosystems and Promega Corporation
Commercial STR Kits Kit Contents: Allelic Ladders for Genotyping PCR Component Mix Primer Mix Positive Control DNA Sample Cost to User: $15-30 per DNA sample tested Currently 2 Suppliers: Applied Biosystems and Promega Corporation ©2002 Academic Press

15 AmpFlSTR SGM Plus™ Allelic Ladders
D3S1358 VWA D16S539 D2S1338 Amel D8S1179 D21S11 D18S51 D19S433 TH01 FGA low FGA high 100 bp 150 bp 160 bp 200 bp 250 bp 300 bp 340 bp 350 bp 139 bp DNA Standard ©2002 Academic Press

16 PowerPlex® 1.1 with Amelogenin
FMBIO Image 585 nm (TMR) 505 nm (FL) PowerPlex® 1.1 with Amelogenin 100 bp 400 bp 300 bp 200 bp D13 TH01 D5 D7 D16 vWA TPOX CSF ILS-400 A ©2002 Academic Press

17 PowerPlex® 2.1 D3 TH01 D21 D18 Penta E vWA D8 TPOX FGA ILS-600
FMBIO Image 585 nm (TMR) 505 nm (FL) PowerPlex® 2.1 100 bp 400 bp 300 bp 200 bp D3 TH01 D21 D18 Penta E vWA D8 TPOX FGA ILS-600 ©2002 Academic Press

18 PowerPlex® 16 FL JOE TMR CXR D3 TH01 D21 D18 Penta E D5 D13 D7 D16 CSF
100 bp 400 bp 300 bp 200 bp FL Blue JOE Green TMR Yellow CXR Red D3 TH01 D21 D18 Penta E D5 D13 D7 D16 CSF Penta D CSF A vWA D8 TPOX FGA ABI 310 Data AMEL D3 TH01 TPOX Penta D Penta E FGA D21 D18 CSF D16 D7 D13 D5 VWA D8 ILS-600 ©2002 AP, Elsevier

19 Genotype Results with Profiler Plus™ kit
D18S51 D21S11 D3S1358 vWA D5S818 D8S1179 Amel D13S317 D7S820 FGA GS500-ROX (red dye) Internal Size Standard NED-labeled (yellow dye) PCR products JOE-labeled (green dye) PCR products FAM-labeled (blue dye) PCR products PCR Product Size (bp) Relative Fluorescence Units Sex-typing ©2002 AP, Elsevier

20 STRBase: NIST STR Database
©2002 Academic Press

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