maximize information from your mixture samples using a ... · rohaizah james, phd; martin...

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www.promega.com 0 10 20 30 40 50 60 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 0 16.3 16,17 17,18 18,19 15.2,18.2 Frequency, % DYS576 0 10 20 30 40 50 60 10 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 0 17.3 18.2 18.3 19.3 20.3 16,17 16,20 17,18 18,19 19,20 19,21 Frequency, % DYS570 PowerPlex® Fusion 6C System High Sensitivity = More Information at Low DNA Input 3 Y-STR’s = Better Determination of Male Contributors (1 Male : 1 Male : 20 Female Mixture; 1ng Total; 29 cycles) 23 Autosomal STR’s + 3 Y-STR’s + Amelogenin = High Discriminatory Power Direct Amp + Fast Cycling + 12.5μl Reaction Volume = Improved Lab Efficiency References Maximize Information from Your Mixture Samples Using a Combined Autosomal STR and Y-STR Multiplex System Rohaizah James, PhD; Martin Ensenberger, PhD; Patricia Fulmer, PhD; Kristy Lenz, MS; Dawn Rabbach, PhD; Cynthia J. Sprecher, PhD; Douglas R. Storts, PhD Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711-5399 Abstract #B7 February 18 th 2015 STR Kit Number of Loci PI 2 (N = 1036) How Much Better than CODIS 20 CODIS 13 13 5.02 x 10 -16 - CODIS 20 20 9.35 x 10 -24 - CODIS 20 + TPOX + D22S1045 + SE33 23 7.73 x 10 -28 10 4 CODIS 20 + TPOX + D22S1045 + Penta D + Penta E 24 6.58 x 10 -29 10 5 PowerPlex® Fusion 6C System 27 2.30 x 10 -32 10 8 0 10 20 30 40 50 60 6 7 8 9 10 11 12 13 14 10,11 Frequency, % DYS391 1. D. Hares (2012) Expanding the CODIS core loci in the United States; Forensic Science International: Genetics; 6: e52 2. J. Butler et al. (2012) Variability of New STR Loci and Kits in US Population Groups; Profiles in DNA 3. J. Purps et al. (2014) A global analysis of Y-chromosomal haplotype diversity for 23 STR loci; Forensic Science International: Genetics; 12: 12 4. K. Ballantyne et al. (2010) Mutability of Y-Chromosomal Microsatellites: Rates, Characteristics, Molecular Bases, and Forensic Implications; The American Journal of Human Genetics; 87: 341 29 cycles for extracted DNA 24 – 26 cycles for direct amp 48 alleles 37 alleles Number of Het. Alleles 252 252 252 239 151 Average PHR 0.92 0.90 0.86 0.74 0.72 Y-STR Number of Alleles 3 Gene Diversity 3 Mutation Rate 4 DYS570 28 0.803 17 / 1429 1.24 x10 -2 DYS576 21 0.789 24 / 1727 1.43 x10 -2 DYS391 10 0.521 5 / 1759 3.23 x10 -3 * 12.5μl PCR reaction volume 6-dye STR amplification system designed to meet expanded CODIS core loci guidelines 1 Contains 23 autosomal STR markers, including SE33 Contains 3 Y-STR loci (DYS391, DYS570, DYS576) 2 Rapidly Mutating (RM) Y-STR markers improve determination of contributors in mixtures One kit for both casework and database use streamlines QC process Capable of direct-amplification of FTA® punch, nonFTA punch, and swab in 25μl or 12.5μl reaction volumes Short PCR cycling time of about 1 hour Compatible with the GeneAmp® 9700 and Veriti® thermal cyclers Compatible with OSIRIS and GeneMarker® HID fragment analysis software PCR setup methods available on the Hamilton MicroLab STAR, Beckman Biomek®, and Tecan Freedom EVO® workstations. Sample Type Pre-treatment 5X AmpSolution™ Reagent Number of Cycles* Amplification Time (min)* Blood FTA® Punch None 25 58 Buccal FTA® Punch Blood nonFTA Punch PunchSolution™ Reagent 24 56 Buccal nonFTA Punch Buccal Swab SwabSolution™ Reagent × 25 58 Buccal FTA® Punch 45pg of each male DNA Error bars in top panel show the standard deviation of the mean from triplicate amplification reactions. All amplification reactions were performed using a GeneAmp® PCR System 9700 thermal cycler One μl of amplified products was separated on an Applied Biosystems® 3500 Genetic Analyzer (1.2kV, 15- second injection).

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Page 1: Maximize Information from Your Mixture Samples Using a ... · Rohaizah James, PhD; Martin Ensenberger, PhD; Patricia Fulmer, PhD; Kristy Lenz, MS; Dawn Rabbach, PhD; Cynthia J. Sprecher,

www.promega.com

0102030405060

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PowerPlex® Fusion 6C System High Sensitivity = More Information at Low DNA Input 3 Y-STR’s = Better Determination of Male Contributors (1 Male : 1 Male : 20 Female Mixture; 1ng Total; 29 cycles)

23 Autosomal STR’s + 3 Y-STR’s + Amelogenin = High Discriminatory Power

Direct Amp + Fast Cycling + 12.5µl Reaction Volume = Improved Lab Efficiency

References

Maximize Information from Your Mixture Samples Using a Combined Autosomal STR and Y-STR Multiplex System Rohaizah James, PhD; Martin Ensenberger, PhD; Patricia Fulmer, PhD; Kristy Lenz, MS; Dawn Rabbach, PhD; Cynthia J. Sprecher, PhD; Douglas R. Storts, PhD

Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711-5399

Abstract #B7

February 18th 2015

STR Kit Number of

Loci PI2

(N = 1036) How Much Better

than CODIS 20

CODIS 13 13 5.02 x 10-16 -

CODIS 20 20 9.35 x 10-24 -

CODIS 20 + TPOX + D22S1045 + SE33

23 7.73 x 10-28 104

CODIS 20 + TPOX + D22S1045 + Penta D + Penta E

24 6.58 x 10-29 105

PowerPlex® Fusion 6C System 27 2.30 x 10-32 108

0102030405060

6 7 8 9

10

11

12

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,11

Fre

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cy, %

DYS391

1. D. Hares (2012) Expanding the CODIS core loci in the United States; Forensic Science International: Genetics; 6: e52

2. J. Butler et al. (2012) Variability of New STR Loci and Kits in US Population Groups; Profiles in DNA

3. J. Purps et al. (2014) A global analysis of Y-chromosomal haplotype diversity for 23 STR loci; Forensic Science International: Genetics; 12: 12

4. K. Ballantyne et al. (2010) Mutability of Y-Chromosomal Microsatellites: Rates, Characteristics, Molecular Bases, and Forensic Implications; The American Journal of Human Genetics; 87: 341

29 cycles for extracted DNA 24 – 26 cycles for direct amp

48 alleles 37 alleles

Number of Het. Alleles 252 252 252 239 151

Average PHR 0.92 0.90 0.86 0.74 0.72

Y-STR Number

of Alleles3

Gene Diversity3 Mutation Rate4

DYS570 28 0.803 17 / 1429 1.24 x10-2

DYS576 21 0.789 24 / 1727 1.43 x10-2

DYS391 10 0.521 5 / 1759 3.23 x10-3

* 12.5µl PCR reaction volume

• 6-dye STR amplification system designed to meet expanded CODIS core loci guidelines1

• Contains 23 autosomal STR markers, including SE33

• Contains 3 Y-STR loci (DYS391, DYS570, DYS576)

• 2 Rapidly Mutating (RM) Y-STR markers improve determination of contributors in mixtures

• One kit for both casework and database use streamlines QC process

• Capable of direct-amplification of FTA® punch, nonFTA punch, and swab in 25µl or 12.5µl reaction volumes

• Short PCR cycling time of about 1 hour

• Compatible with the GeneAmp® 9700 and Veriti® thermal cyclers

• Compatible with OSIRIS and GeneMarker® HID fragment analysis software

• PCR setup methods available on the Hamilton MicroLab STAR, Beckman Biomek®, and Tecan Freedom EVO® workstations.

Sample Type Pre-treatment 5X AmpSolution™

Reagent Number

of Cycles* Amplification Time (min)*

Blood FTA® Punch None √ 25 58

Buccal FTA® Punch

Blood nonFTA Punch PunchSolution™ Reagent

√ 24 56 Buccal nonFTA Punch

Buccal Swab SwabSolution™

Reagent × 25 58

Buccal FTA® Punch

45pg of each male DNA

• Error bars in top panel show the standard deviation of the mean from triplicate amplification reactions.

• All amplification reactions were performed using a GeneAmp® PCR System 9700 thermal cycler

• One μl of amplified products was separated on an Applied Biosystems® 3500 Genetic Analyzer (1.2kV, 15-second injection).