AmpFℓ Yfiler™ Kit Update - thermofisher.com · Formation of the N+3 product is reproducible and...

AmpFℓSTR®

Yfiler™

Kit Update

Lori HennessySr. ManagerHuman Identification R&D

© 2006 Applied Biosystems

Presentation Outline

●

Characterization of the N+3 bp

peak at the DYS392 locus–

Background

–

Experimental plan–

Results

●

Identification of novel SNP in the X chromosome region homologous to the DYS456 locus–

Background

–

Experimental plan–

Results

●

Y GATA H4 Nomenclature Update

Characterization of the N+3 bp peak at the DYS392 locus


1 ng

male control DNA 007 amplified with Yfiler™

6-FAM™

VIC®

NED™

PET®


Electropherogram of the DYS392 Locus

N-39.3%

N

N+35.1%


Experimental plan

●

Clone and sequence products –

Main allele peak (N)

–

-3 bp

stutter peak (N-3)–

+3 bp

PCR product (N+3)

●

Evaluate following factors*–

Primer sequence context

–

MgCl2

, DNA concentration, allele repeat number, dye label●

Statistical analysis•

unpaired Student’s t-test or ANOVA

*Four replicates were prepared for each experiment


DNA sequence analysis of the DYS392 allele 13 and stutter products

Repeat # 1 2 3 4 5 6 7 8 9 10 11 12 13 14

N-3 stutter TAT TAT TAT TAT TAT TAT TAT TAT TAT TAT TAT TAT --- ---

N allele TAT TAT TAT TAT TAT TAT TAT TAT TAT TAT TAT TAT TAT ---

N+3 stutter TAT TAT TAT TAT TAT TAT TAT TAT TAT TAT TAT TAT TAT TAT


DYS392 Primer Set Comparison

Primer Set Size Range (bp) Reference

1 233-266 Kayser

et al 1997

2 92-125 Schoske

et al 2003

3 289-322 Butler et al 2002

4 151-184 Bosch et al 2002


Comparison of stutter percentages observed with four different primer sets

N-3Stutter

N+3Stutter

0.0

2.0

4.0

6.0

8.0

10.0

11 12 13 14 15 16

Allele Number

% S

tutte

r

0.0

2.0

4.0

6.0

8.0

10.0

11 12 13 14 15 16

Allele Number

% S

tutte

r

Primer Set 1 Primer Set 2 Primer Set 3 Primer Set 4

Six different male samples, 4 replicates, mean ± SD


Increased MgCl2

concentration affects stutter formation

0

2

4

6

8

10

12

1 .25 1 .43 1 .6 1 .77 1 .95

M g C l C o n c e n tra tio n (m M )

% S

tutte

r

N-3 s tutte rN+ 3 s tutte r

2

* **

** **

**

* P <0.01

** p <0.001


Decreased DNA template concentration affects formation of stutter

0

2

4

6

8

10

12

1ng 500 pg 250pg 125pg

DNA Concentration

% S

tutte

r N-3 stutterN+3 stutter

*

*

* *

*

* P <0.01

** p <0.001


Summary●

Demonstrated N+3 bp

product was a “positive”

stutter artifact

●

Formation of the N+3 product is reproducible and displays similar behavior to the N-3 stutter product–

Stutter products increased with increased allele repeat number and increased MgCl2

–

Stutter products increased with decreased DNA concentration–

Primer sequence context had a smaller effect on stutter percentage than the above three variables tested

●

Since both stutter products behave in a similar and reproducible fashion, the same rules that apply to the interpretation of N-3 stutter products can be applied to N+3 stutters.

Identification of a novel SNP in the X chromosome region homologous to the DYS456 locus


Background

During an extensive multi-population study to

create the Yfiler™

Haplotype database using the

AmpFSTR®

Yfiler™

PCR amplification kit,

amplification of a 71-bp fragment was observed

in some male samples analyzed.


Detection of a 71-nucleotide fragment in a male sample amplified with the AmpFSTR®Yfiler™

Kit

Male Control DNA 007

AA Male Sample #1FAM71


Primer subtraction experiment DYS456 DYS389I DYS389IIDYS390FAM71

-DYS389primer

-DYS390primer

-DYS456primer


Sequence alignment of the DYS456 locus and its homologous region on the X chromosome

DYS456 Locus GGACCTTGTGATAATGTAAGATAGATAGATA…

(on Chr. X)

Major allele T GGACCTTGTGATAATGTAAGATATATATATA…

Minor allele G GGACCTTGTGATAATGTAAGATAGATATATA…


Allele frequencies in different populations

P o p u l a t i o n N u m b e r o f c h r o m o s o m e s

T GA f r i c a n

A m e r i c a n 8 0 2 0 . 9 2 3 0 . 0 7 7

S u b - S a h a r a n A f r i c a n 5 9 0 . 8 9 8 0 . 1 0 2

C a u c a s i a n 1 1 5 6 0 . 9 9 8 0 . 0 0 2

H i s p a n i c 4 8 0 0 . 9 9 4 0 . 0 0 6

N a t i v e A m e r i c a n 1 0 6 1 0

A s i a n 3 3 0 1 0

F i l i p i n o 1 0 5 1 0

V i e t n a m e s e 1 0 3 1 0

A l l e l e


SNaPshotSingle Base Extension Reaction

ddGTPddCTPddUTPddATP

Extend and Terminate Primer

AmpliTaq®

FS

Electrophoresis

AGCCTGGTACTGACTAAGGC…..

Anneal PrimerInterrogation target

ATGACTGATTCC

AGCCTGGTACTGACTAAGGC…..ATGACTGATTCCG

HeterozygoteHomozygote


Process Flow for SNaPshot Assay

Target Amplification

Purify PCR Products

Single Base Extension Reaction

Unincorporated [F] ddNTP cleanup

Electrophoresis/Analysis


SNaPshot

primer extension assay T/G SNP Detection

Male Control DNA 007(T allele)

Female Control DNA 9947A(T, T allele)

Male #1(G allele)

Female #187(G, T allele)

2.1% frequency in females (9/428 chromosomes tested)


Expected allele frequencies in African American females

P o p u l a t i o n N u m b e r o f c h r o m o s o m e s

T GA f r i c a n

A m e r i c a n 8 0 2 0 . 9 2 3 0 . 0 7 7

S u b - S a h a r a n A f r i c a n 5 9 0 . 8 9 8 0 . 1 0 2

A l l e l e

Approx. 0.6% of African American females will be homozygous for the mutation

2pq=2x0.923x0.077=0.142q^2=0.077^2=0.006


Male: Female (1:4000) mixture studies

125 pg male DNA+500 ng female DNA


DYS456 peak heights in male/female mixture samples

9947A #187 #333 #404

0.5 ng male DNA+500 ng female DNA 1223 442 472 390



M/F mixture Female DNA Used in M/F Mixture


Summary

●

During a

multi-population study using the Yfiler multiplex PCR amplification kit, amplification of a 71-bp fragment was observed in 2.32% of the male samples analyzed (N=3141).

●

A T to G polymorphism located within a DYS456 homologous region

on the X chromosome resulted in the amplification of a 71-bp fragment.

●

This variant G was observed with the highest frequency within the African American and Sub-Saharan African populations in our study.

●

Full profiles in a mixture of male:female at 1:4000 were obtained using the Yfiler kit even in the presence of female DNA containing the

G variant.


Manuscript published in JFS March 2006 issue

Y GATA H4 Nomenclature Update


Y GATA H4 Nomenclature

●

The Yfiler kit uses NIST recommendations for the GATA- H4 locus

●

ISFG recommends using a different nomenclature depending on primer location within sequence –

GATA

H4, GATA H4.1 or GATA H4.2●

Yfiler primers amplify the GATA H4.1 region only

●

Provide clarification for Yfiler users and alignment with ISFG recommended nomenclature


GATA H4 Locus

GATA H4 Locus

GATA H4.1 GATA H4.2

Variable region Non-variable region


GATA H4 SequenceAn example of a GATA H4 allele sequence is shown here:

GATA H4 allele 10 following NIST and Yfiler kit repeat structure (TAGA repeats)

AGATAGATAGATAGATCTATAGATAGATAGGTAGGTAGGTAGATAGATAGATAGATAGATAGAT AGATAGATAGATAGAATGGATAGATTAGATGGATGAATA 1 2 3 4 5 6

7 8 9 10

GATA H4.1 allele 19 following ISFG1 ((AGAT)4 CTAT(AGAT)2 (AGGT)3 (AGAT)N )

AGATAGATAGATAGATCTATAGATAGATAGGTAGGTAGGTAGATAGATAGATAGATAGATAGATA GATAGATAGATAGAATGGATAGATTAGATGGATGAATA 1 2 3 4 5 6

1Gusmao et al. Forensic Science International, (2006) March 10, 157:pp 187-197.

7 8 9


Summary•

Differences in nomenclature are due to:

•

Inclusion of non-variable repeats preceding variable region

•

Designation of repeat structure•

Concordance between nomenclatures achieved by adding a +9 correction factor to Yfiler allele designation

•

Yfiler™

alleles 8-13

•

ISFG alleles 17-22•

A Letter to the Editor clarifying this difference was published in JFS May 2006 issue


R&D Group


Acknowledgements

•

Lisa Calandro•

Bruce Budowle

•

Our many collaborators on the Yfiler™

Haplotype Database population study

Applied Biosystems, AB (Design), AmpFSTR, GeneMapper, Identifiler, PET, SNaPshot, and VIC are registered trademarks, and Applera AmpFSTR SGM Plus, FAM, NED, and Yfiler are trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries. AmpliTaq

is a registered trademark of Roche Molecular Systems, Inc.

©2006 Applied Biosystems. All rights reserved

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AmpFℓ Yfiler™ Kit Update - thermofisher.com · Formation of the N+3 product is reproducible and...

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