05~forensic_dna_chapter_05_slides (1)

8/11/2019 05~Forensic_DNA_Chapter_05_Slides (1)

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Chapter 5

Commonly Used Short Tandem

Repeat Markers

2002 Academic Press


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Types of STR Repeats

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

Microsatellite = simple sequence repeat (SSR) = shorttandem repeat (STR)

2002 Academic Press


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STR Repeat Nomenclature

International Society of Forensic Haemogenetics (ISFH)

-- Int. J. Legal Med.(1997) 110:175-176

For sequences within genes, use the coding strand

For other sequences, select the first GenBankdatabase 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 indetermining the repeat number

2002 Academic Press


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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


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Short Tandem Repeats (STRs)

the repeat region is variable between samples while the

f lanking regions where PCR pr imers bind are constant

AATG

7 repeats

8 repeats

AATG AATG

Homozygote = both alleles are the same length

Heterozygote = alleles differ and can be resolved from one another

Primer positions def ine PCR product size

Fluorescentdye label

2002 Academic Press


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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


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Microvariantallele

STR genotyping is performed bycomparison of sample data to allelic ladders

2002 Academic Press


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124 3

56789

124 3

56789

AATG repeat unit

170 bp195 bp

TH01 Sequence from GenBank with

Several PCR Primer Positions

2002 Academic Press


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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


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CSF1PO

D5S818

D21S11

TH01

TPOX

D13S317

D7S820

D16S539 D18S51

D8S1179

D3S1358

FGA

VWA

13 CODIS Core STR Loci

AMEL

AMEL

Sex-typing

Position of Forensic STR Markerson Human Chromosomes

2002 AP, Elsevier


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Information on 13 CODIS STRs

D18S51 18q21.3 AGAA L18333 13 7-27 43

Locus

Name

Chromosomal

Location

Repeat Motif

ISFH formatGenBank

Accession

Allele in

GenBank

Allele

Range

Number of

Alleles Seen

CSF1PO 5q33.3-34 TAGA X14720 12 6-16 15

FGA 4q28 CTTT M64982 21 15-51.2 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 7q11.21-22 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

D21S11 21q21 Complex

[TCTA][TCTG]

AP000433 29 24-38 70

2002 Academic Press


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Commercially Available STR Kits

*Caucasian population (rounded to 2 significant figures)

Name Source ReleaseDate

STR Loci Included *Power ofDiscrimination

TH01, TPOX, CSF1PO

monoplexes

Promega Feb 1993 TH01, TPOX, CSF1PO 1:410

AmpFlSTRBlue Applied

Biosystems

Oct 1996 D3S1358, VWA, FGA 1:5000

AmpFlSTRGreen I Applied

Biosystems

Jan 1997 Amelogenin, TH01, TPOX, CSF1PO 1:410

CTTv Promega Jan 1997 CSF1PO, TPOX, TH01, VWA (vWF) 1:6600

FFFL Promega Jan 1997 F13A1, FES/FPS, F13B, LPL 1:1500

GammaSTR Promega Jan 1997 D16S539, D13S317, D7S820, D5S818 1:1.8x104PowerPlex

(version 1.1 and 1.2 later)Promega Jan 1997 CSF1PO, TPOX, TH01, VWA, D16S539, D13S317,

D7S820, D5S8181:1.2x108

AmpFlSTRProfiler Applied

Biosystems

May 1997 D3S1358, VWA, FGA, Amelogenin, TH01, TPOX,CSF1PO, D5S818, D13S317, D7S820

1:3.6x109

AmpFlSTRProfiler

PlusApplied

Biosystems

Dec 1997 D3S1358, VWA, FGA, Amelogenin, D8S1179,D21S11, D18S51, D5S818, D13S317, D7S820

1:9.6x1010

AmpFlSTRCOfiler Applied

Biosystems

May 1998 D3S1358, D16S539, Amelogenin, TH01, TPOX,CSF1PO, D7S820

1:8.4x105

AmpFlSTR

SGM Plus AppliedBiosystems

Feb 1999 D3S1358, VWA, D16S539, D2S1338, Amelogenin,D8S1179, D21S11, D18S51, D19S433, TH01, FGA 1:3.3x1012

PowerPlex2.1 Promega Jun 1999 D3S1358, TH01, D21S11, D18S51, VWA, D8S1179,TPOX, FGA, Penta E

1:8.5x1010

PowerPlex16 Promega May 2000 CSF1PO, FGA, TPOX, TH01, VWA, D3S1358,D5S818, D7S820, D8S1179, D13S317, D16S539,

D18S51, D21S11, Penta D, Penta E, amelogenin

1:1.8x1017

AmpFlSTRIdentifiler Applied

Biosystems

May 2001 CSF1PO, FGA, TPOX, TH01, VWA, D3S1358,D5S818, D7S820, D8S1179, D13S317, D16S539,

D18S51, D21S11, D19S433, D2S1338, amelogenin

1:2.1x1017

2002 Academic Press


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D3S1358TH01

D21S11 D18S51 Penta E

D5S818 D13S317D7S820 D16S539 CSF1PO

Penta D

AMEL vWA D8S1179TPOX

FGA

DNA Size (bp)

JMB

Multiplex PCR with 5 STR Loci and Amelogenin Gender ID

Sex-typing

PowerPlex16 Result

2002 Academic Press


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Commercial STR Kits

Kit Contents:

Allelic Ladders for Genotyping

PCR Component Mix

Primer Mix

Positive Control DNA Sample

Currently 2 Suppliers: Applied Biosystems and Promega Corporation

Cost to User: $15-30 perDNA sample tested

2002 Academic Press


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D3S1358 VWA D16S539 D2S1338

D8S1179 D21S11 D18S51Amel

D19S433 FGA lowTH01 FGA high

100 bp 340 bp139 bp 250 bp 300 bp200 bp 350 bp150 bp 160 bp

DNA Standard

AmpFlSTR SGM Plus Allelic Ladders

2002 Academic Press


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FMBIO Image

585 nm(TMR)

505 nm(FL)

100 bp 400 bp300 bp200 bp

D13

TH01

D5 D7 D16

vWA TPOX CSF

ILS-400

A

PowerPlex1.1with Amelogenin

2002 Academic Press


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100 bp 400 bp300 bp200 bp

D3 D21TH01 D18 Penta E

vWA D8 TPOX FGA

ILS-600

FMBIO Image585 nm(TMR)

505 nm(FL)PowerPlex2.1

2002 Academic Press


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100 bp 400 bp300 bp200 bp

D13

D3 D21TH01

D5 D7 D16

D18 Penta E

A vWA D8

CSF Penta D

FGA

ILS-600

TPOX

CSF

ABI 310 Data

AMEL

D3

TH01 TPOX

Penta DPenta E

FGA

D21D18

CSFD16D7

D13D5VWA

D8

FLBlue

JOEGreen

TMRYellow

CXRRed

PowerPlex16

2002 AP,

Elsevier


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D18S51D21S11

D3S1358vWA

D5S818

D8S1179Amel

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)

RelativeFluoresce

nceUnits

Sex-typing

Genotype Results with Profiler Pluskit

2002 AP,

Elsevier


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STRBase: NIST STR Database

2002 Academic Press

05~forensic_dna_chapter_05_slides (1)

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