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Integrating the

Quantifiler®

Duo DNA Quantification Kit with the

AmpFℓSTR®

Yfiler®

PCR Amplification Kit in Sexual Assault Investigations

Lisa Calandro, M.P.H.Sr. Manager, Technical Marketing

2 © 2007 Applied Biosystems

Outline

●

Introduction●

Y STR analysis

●

Quantifiler®

Duo assay specificity and efficiency●

Performance with mixture samples

●

Performance with inhibited and degraded samples●

Correlation to AmpFℓSTR®

Yfiler®

kit performance●

Conclusions

Y STR Analysis


When Are Y STRs Used in Forensic Casework?*

●

Autosomal testing yields little or no male DNA profile–

Samples where female DNA is in vast excess of male DNA–

Low level minor male -

interpretation tricky or impossible●

Very few or no sperm are detected●

Differential extraction is not possible –

non sexual assault samples●

Clarify the number of male contributors●

“Last resort”

type of testing:–

Cold cases–

Post-conviction cases

*Source: Cassie Johnson, Orchid


Detecting Male DNA in Forensic Samples

●

Autosomal analysis may reveal presence of small amelogenin

Y peak

●

However, autosomal analysis does not always reveal presence of male DNA in mixed samples

●

Quantifiler Y quantitation has capability to specifically detect male DNA in the presence of >1:9000 female:male mixture ratio


AmpFℓSTR®

Yfiler®

Amplification Kit Overview

●

Amplifies 17 loci in a single reaction using 5-dye chemistry for increased discrimination capacity–

Same dye set used with the AmpFℓSTR®

Identifiler®

Kit

●

Provides specific and sensitive detection of male DNA in the presence of high concentrations of female DNA

●

Developed and validated specifically with the Quantifiler™

Human and Y Human Male Assays

●

User-friendly YfilerTM

haplotype database

●

Validated for use with ABI PRISM®

310, 3100, 3100-Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers


YDYS19

DYS389I/II

DYS390

DYS391

DYS392

DYS393

DYS385a/b

DYS437

DYS438

DYS439

DYS448

DYS456

DYS458

DYS635

Y GATA H4

European

Minimal

Haplotype

SWGDAM

ABI

AmpFSTR®

Yfiler®

Kit

17 Y-STR Loci


Identifiler Analysis of Casework Sampleª

* Since no male DNA detected, a range of input DNA will be amplified in Yfiler

* Lip/breast swabbing from female victim of oral assault

ªSource: Joe Varlaro, Spencer Laboratories, Inc.


Yfiler Analysis of Casework Sampleª


Why Y-STR Analysis? – simulated forensic mixture sample

●

3 male DNA samples mixed at equal ratios

●

Female DNA mixed with 3-male mixture at ratio of 500:1

●

Mixture sample quantified with both Quantifiler™

Human and Male assays

●

1ng input DNA added to an Identifiler®

reaction based on the Quantifiler™

Human results

●

1ng input DNA added to a Yfiler™

reaction based on the Quantifiler™

Male results


AmpFℓSTR®

Identifiler®

Amplification Result –

sample quantified with the human assay only

Only single source female sample detected

Amelogenin –

X only


AmpFℓSTR®

Yfiler®

Amplification Result –

sample quantified with the male assay

Three male profiles detected


Discriminatory capacity†

for three U.S. populations

Y-STR marker combination

African American (N=786)

Caucasians (N=778)

Hispanics (N=381)

European minimal haplotype (9)

75.8% 61.7% 79.8%

“US Haplotype”

(11)

86.8% 74.3% 85.6%

“US Haplotype”

+ DYS437 (12)

87.6% 76.7% 88.2%

AmpFSTR®

Yfiler®

kit (17) 97.6% 95.5% 95.8%

†DC= (# of different haplotypes / pop. size) x 100


Most common Haplotype identified by the European Minimal Haplotype markers (20 individuals)

European Minimal Haplotype

0# of differenthaplotypes

20Yfiler®

Kit

Sample Info DYS19 1DYS385 DYS389 DYS389 DYS390 DYS391 DYS392 DYS393 DYS438 DYS439 DYS437 DYS448 DYS456 DYS458 Y GATA DYS635 C37 14 11,14 13 29 24 11 13 13 13 12 14 19 15 17 12 23C330 14 11,14 13 29 24 11 13 13 13 11 15 19 15 19 11 23ATCC 14 11,14 13 29 24 11 13 13 12 13 15 19 15 17 13 23C304 14 11,14 13 29 24 11 13 13 12 13 15 19 17 18 12 23C327 14 11,14 13 29 24 11 13 13 12 13 15 19 16 16 12 24C63 14 11,14 13 29 24 11 13 13 12 13 15 17 16 17 11 23C177 14 11,14 13 29 24 11 13 13 12 12 15 21 17 19 12 24C198 14 11,14 13 29 24 11 13 13 12 12 15 18 16 17 12 23C276RL 14 11,14 13 29 24 11 13 13 12 12 15 19 15 19 12 23C294 14 11,14 13 29 24 11 13 13 12 12 15 19 15 17 11 24C236 14 11,14 13 29 24 11 13 13 12 12 14 19 15 18 12 23C85RL 14 11,14 13 29 24 11 13 13 12 12 14 19 16 16 11 24C12 14 11,14 13 29 24 11 13 13 12 11 15 19 17 18 12 23C194 14 11,14 13 29 24 11 13 13 12 11 15 19 17 17 12 23C197 14 11,14 13 29 24 11 13 13 12 11 15 19 15 16 >13 24C318 14 11,14 13 29 24 11 13 13 12 11 15 19 16 17 12 23C345 14 11,14 13 29 24 11 13 13 12 11 15 19 15 18 12 23C66RL 14 11,14 13 29 24 11 13 13 12 11 15 19 16 19 12 23C205 14 11,14 13 29 24 11 13 13 12 11 14 19 15 17 12 23C158 14 11,14 13 29 24 11 13 13 12 10 15 19 16 20 13 23

6SWGDAM




Caucasian35%

Hispanic17%

Filipino3%

Asian9%

African American28%

Vietnamese3%

Black African2%

Native American3%

Yfiler Population Data by Ethnic Group3,561 Samples

Samples from:

Austria

CA DOJ

Italy

Minn. BCA

NIST

OCME

Orchid

Poland

Santa Clara County

SeraCare

SLED

South Africa


Key Features of Yfiler Haplotype Database

●

Ability to search all 17 Yfiler loci ●

Ability to search microvariant

Y STR types

●

Ability to perform batch searches using outputs from either Genotyper

or GeneMapper ID

v3.2


Concordant with NIST 20plex and 11plex data

Microvariant Example


AmpFSTR®

Yfiler®

Kit

Input Haplotypes from Genotyper

Search results for individual samples

Quantifiler®

Duo DNA Quantification Kit: Quantitative and Qualitative Assessment of

Total Human and Human Male DNA


Objectives

●

Develop a multiplexed real-time PCR assay to:–

Quantify total human DNA and human male DNA in a single PCR reaction

–

Determine the ratio of human male and female DNA–

Detect PCR inhibitors

–

Allow selection of appropriate STR amplification kit–

Predict success with downstream STR amplification


Quantifiler®

Duo Kit: A Guide to Sample Assessment

IPC Ct Value

DNA Extract

Mixture Ratio

Autosomal STR Y-Filer®

kit

Heavy Inhibition

Re-Purification STR Analysis

Partial Inhibition

MiniFiler™ kit Identifiler®

kit

Quantification


Comparison to Quantifiler®

Kits

●

DNA standard is pooled human male genomic DNA●

PCR Reaction Mix optimized for expanded multiplex performance

●

Ability to obtain ratio of human and male DNA in a mixture sample in a single reaction–

Dual quantification of human and male DNA minimizes sample consumption

●

Assay designed to guide selection of appropriate AmpFℓSTR®

Kit chemistry–

Maximizes recovery of interpretable STR profiles


Configuration of Assay

Target Marker Size Dye

Human DNA

RPPH1(Ribonuclease

P RNA component H1)140 bp VIC®

Human Male DNA

SRY(Sex determining region Y)

130 bp FAM™

IPC Artificial Template 130 bp NED™

The multiplex assay is run on the 7500 Real Time PCR System using SDS v1.2.3 software.


Std. Curve Amplification Plots and ΔRn (SRY/RNaseP/IPC)

SRY

RPPH1

IPC

Sample ng/µl Ave Ct Ave Ct Ave Ct Ave ∆Rn Ave ∆Rn Ave ∆RnRPPH1 SRY IPC RPPH1 SRY IPC

std 1 50 22.9 23.7 29.7 5.4 6.8 1.4std 2 16.7 24.6 25.5 29.5 5.3 6.7 1.4std 3 5.56 26.2 27.0 29.4 5.2 6.7 1.4std 4 1.85 27.8 28.7 29.3 5.0 6.7 1.5std 5 0.62 29.4 30.3 29.4 4.7 6.4 1.4std 6 0.21 30.9 31.8 29.4 4.3 5.8 1.4std 7 0.068 32.5 33.1 29.4 3.4 4.6 1.4std 8 0.023 33.7 34.8 29.3 2.7 2.7 1.4


Standard Curves and IPC Ct Values

Multiplex Standard Curve Human Genomic DNA

20.022.024.026.028.030.032.034.036.038.0

-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0Log of [ ]

Ave

Ct

SRY-FAMRPPH1-VICIPC-NED


RPPH1 Assay Specificity

Male & Female

Gorilla

RPPH1

RNAse P.6.4s (140bp)Species Ave.Ct

Orangutan 40.0Chimp 40.0Gorilla 37.9Macaque 40.0Dog 40.0Cow 40.0Pig 40.0Cat 40.0Chicken 40.0Fish (Salmon) 40.0Rabbit 40.0Mouse 40.0Rat 40.0Hamster 40.0Male 26.5Female 26.2E.coli 40.0Pseudomonas 40.0Neisseria 40.0Staphylococcus 40.0Saccharomyces 40.0

RPPH1

10 ng of DNA from the indicated species was tested.


SRY Assay Specificity

Male

Chimp

SRY

SRY.2s 130 bpSpecies Ave. Ct

Orangutan 40.0Chimp 30.7Gorilla 40.0Macaque 40.0Dog 40.0Cow 40.0Pig 40.0Cat 40.0Chicken 40.0Fish 40.0Rabbit 40.0Mouse 40.0Rat 40.0Hamster 40.0Male (Raji) 28.8Female (NA14672a) 40.0E.coli 40.0pseudomonas 40.0Neisseria 40.0Staphylococcus 40.0Saccharomyces 40.0

.

10 ng of DNA from the indicated species was tested.


Analysis of Male & Female MixturesQuantitation of Mixtures

-1.000

0.000

1.000

2.000

3.000

4.000

5.000

6.000

1:0 1:2 1:5 1:10 1:20

Male/female DNA ratio

Qua

ntity

(ng/

ul)

SRYRPPH1

Male DNA added at a constant concentration of 0.2 ng/µl


1:0

1:2

1:20

1:10

1:5

Mixture study: Identifiler kit profile (1 ng DNA/rxn)

3000 rfu

Male component


Analysis of Male & Female Mixtures 50 pg of male DNA

Male/female ratio

SRY measured

conc (ng/µl)

RPPH1 measured

conc (ng/µl)

SRY/RPPH1 expected

ratio

SRY/RPPH1 measured

ratio1:0 0.028 0.031 1:1 1:11:1 0.032 0.078 1:2 1:2.41:5 0.037 0.198 1:6 1:5.4

1:10 0.034 0.356 1:11 1:10.61:100 0.035 3.140 1:101 1:901:500 0.037 15.220 1:501 1:408 1:1000 0.033 33.540 1:1001 1:1007

Male DNA added at a constant concentration of 0.025 ng/µl


Mixture study: YFiler

kit profile (1 ng DNA/rxn)

1:20

1:100

1:500

1:1000

5000 rfuscale

M:F Ratio


Post-coital samples Quantitation results (ng/μl) after differential extraction

Sample type Sample name RPPH1 SRY Quantifiler H Quantifiler YE-cell fraction, 4h 1E 63.40 0.08 76.80 0.11E-cell fraction, 8h 2E 105.20 2.78 126.80 5.64

E-cell fraction, 12h 3E 51.40 0.11 69.80 0.00E-cell fraction, 24h 4E 44.80 0.08 61.00 0.00Sperm fraction, 4h 1S 3.22 2.99 5.19 4.57Sperm fraction, 8h 2S 5.01 4.54 6.40 6.18

Sperm fraction, 12h 3S 0.83 0.85 1.13 0.87Sperm fraction, 24h 4S 1.67 1.35 2.19 1.90

Quantitation of post-coital samples(e-cell fraction)

0.00

20.00

40.00

60.00

80.00

100.00

120.00

1E 2E 3E 4E

Qua

ntity

(ng/

ul)

RPPH1 SRY

Quantitation of post-coital samples (sperm fraction)

0.00

1.00

2.00

3.00

4.00

5.00

6.00

1S 2S 3S 4S

Qua

ntity

(ng/

ul)

RPPH1SRY


Post-coital samples: E cell -

Identifiler kit profile (1 ng DNA/rxn)

1E 4 h

2E 8 h

3E 12 h

4E 24 h

2000 rfu


1 S 4h

2 S 8h

3 S 12h

4 S 24h

2000 rfuPost-coital samples: Sperm -

Identifiler kit profile (1 ng

DNA/rxn)

35 © 2007 Applied Biosystems6000 rfu

Post-coital samples: sperm fraction -

YFiler

kit profile (1 ng DNA/rxn)

1 S 4h

2 S 8h

3 S 12h

4 S 24h


Body fluid mixture samples -

Quantitation results (ng/μl)Quantitation of body fluid mixture samples

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

1M 2M 3M 4M 5M 6M 7M

Qua

ntity

(ng/

ul)

RPPH1 SRY

Sample type Sample name RPPH1 SRY Quantifiler Hu Quantifiler Y

SRY/RPPH1 measured

ratio

SRY/TERT measured

ratio Male saliva ctrl 1M 1.45 1.29 2.15 2.46 1:1.1 1:0.87

Female saliva ctrl 2M 0.54 0.00 0.81 0.00 0.000 0.000Female blood ctrl 3M 2.24 0.00 3.02 0.00 0.000 0.000

Male saliva/female saliva mix 1 4M 0.91 0.43 1.44 0.68 1:2.1 1:2.1Male saliva/female saliva mix 2 5M 1.46 0.43 2.22 0.69 1:3.4 1:3.2Male saliva/female blood mix 1 6M 3.28 1.02 5.11 1.40 1:3.2 1:3.7Male saliva/female blood mix 2 7M 6.88 1.42 9.28 1.84 1:4.8 1:5


Mixture study conclusions

●

Successful quantification of the male component in mixtures with male/female DNA ratios up to 1:1000–

Tested using 25 pg/ul

and 200 pg/ul

of male DNA.

●

Accurate measurement of the male/female ratio in a mixture sample relative to expected results

●

DNA input based on Quantifiler Duo kit results led to generation of an interpretable profile of the male component up to a 1:10

male/female mixture ratio using the Identifiler kit●

DNA input based on Quantifiler Duo kit results led to generation

of an interpretable YFiler

kit profile for the male component up to a 1:1000 male/female mixture ratio


Effect of Inhibitors

●

Evaluated the ability to detect the presence of known PCR inhibitors such as hematin and humic

acid using the

Quantifiler Duo kit●

Varied inhibitor concentrations in DNA samples and tested using the Quantifiler Duo and Identifiler kits to assess predictive performance


Humic

Acid Inhibited Samples -

IPC Signal


Humic


SRY Signal

7.5ng/µl is undetected


Humic


RPPH1 Signal

7.5ng/µl is undetected


Humic

Acid Inhibited Samples: Correlation with Identifiler Kit Profile

IPC signal with increasing humic acid concentration

272829303132333435363738394041

0ng/u

l

1ng/u

l

2ng/u

l

3ng/u

l 3.

75ng

/ul 7.

5ng/u

l 11

.25 ng

/ul 15

ng/ul

30ng

/ul

Concentration of humic acid

IPC

Ct

Ctr-Hum_0

Hum_1 ng/uL

Hum_2 ng/uL

Hum_3 ng/uL

Hum_3.75 ng/uL

Hum_7.5 ng/uL

Hum_11.2 ng/uL

Hum_15 ng/uL

Hum_30 ng/uL


Hematin Inhibited Samples: Correlation between IPC Signal and Identifiler Kit Profile

IPC signal with increasing hematin concentration

272829303132333435363738394041

0uM 2.5uM 5uM 7.5uM 10uM 12.5uM 15uM 17.5uM 20uM 40uM

Concentration of Hematin

IPC

Ct

Ctr-Hem0

Hem_2.5 uM

Hem_5.0 uM

Hem_7.5 uM

Hem_10 uM

Hem_12.5 uM

Hem_15 uM

Hem_17.5 uM

Hem_20 uM

Hem_40 uM


Inhibited Samples

●

Quantifiler Duo kit results demonstrated IPC shift in the presence of increasing amounts of inhibitor

●

Quantifiler Duo kit results correlated with STR kit performance


Degraded DNA

Degraded DNA: Concentration (ng/ul)

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

Intact 2 U 3U 4 U 5 U 6 U

Units

Conc

entra

tion

RNase P 140SRY

.RPPH1SRY

DNaseI Units


Quantification of Degraded DNA

Quantification of degraded DNA was performed using Quantifiler®

Duo (Multiplex assay) and Quantifiler®

kits.

Results with Degraded DNA Series

0.00

2.00

4.00

6.00

8.00

10.00

12.00

Intact 2u 3u 4u 5u 6u

DNA Degradation State

Qua

nt R

esul

ts (n

g/ul

)

Quantifiler HumanQuantifiler YMultiplex RPPH1Multiplex SRY


Degraded DNA: Correlation with Identifiler®

Kit

Sample Human (ng/µl)

Vol (µl) to give 1 ng

STR Peaks Detected (out of 28)

Male (ng/µl)

Vol (µl) to give 1 ng

STR Peaks Detected (out of 28)

Intact 11.68 0.09 28/28 11.73 0.09 28/28

2 u 3.86 0.26 28/28 3.87 0.26 28/28

3 u 2.61 0.38 28/28 2.51 0.40 28/28

4 u 1.35 0.74 28/28 1.18 0.85 28/28

5 u 1.04 0.96 26/28 0.91 1.10 27/28

6 u 0.23 4.28 24/28 0.18 5.48 25/28

RPPH1 SRY


Conclusions

●

The Quantifiler Duo kit is a multiplex real time PCR assay for simultaneous quantification of total human and human male DNA

●

IPC enables monitoring for the presence of PCR inhibitors●

The assay is specific for human and higher primates

●

Provides information about the mixture ratio of total human and human male DNA in a sample

●

High dynamic range of quantification from 23 pg/µl to 50 ng/µl

●

The quantification results correlate well with Identifiler®

and YFiler®

kit performance


Acknowledgements

●

Applied Markets Research and Development–

Jai Shewale, Maura Barbisin, Pius Brzoska, Robert Green, Michael Malicdem, Robert Lagace, Rixun

Fang, Cristin

O’Shea, Julio Mulero, Lori Hennessy, Chien-Wei Chang and Manohar

Furtado

●

Applied Markets Marketing–

Heidi Kijenski, Karen Cormier and Lisa Calandro

●

Applied Markets Product Development: –

Lindy Kauffman, Yixin

N Li, Sabine Short and Beth Ladin


Legal Statements

For Research, Forensic or Paternity Use Only. Not for use in diagnostic procedures.

Purchase of the Quantifiler®

Duo Kit includes an immunity from suit under patents specified in the product insert to use only the amount purchased

solely in forensic and paternity testing, and also for the purchaser’s own internal research. No other patent rights are conveyed expressly, by implication, or by estoppel. For further information contact the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

Not for re-sale.

Applera, Applied Biosystems, AB (Design), AmpFlSTR, FAM, Identifiler, NED, Quantifiler, and Y-Filer are registered trademarks, and MiniFiler is a trademark of

the Applera Corporation or its subsidiaries in the US and/or certain

other countries.

Thank You

Quantifiler®

Duo DNA Quantification Kit

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