Y chromosomal SNP Position Y-SNP Mutation 2715180 SRY 465 C/T - - PDF document

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Developmen Development of multiplex single base t of multiplex single base extension reaction and multiplex allele- extension reaction and multiplex allele- specific PCR assay to determine East specific PCR assay to determine East Asian Y chromosomal haplogroups Asian Y chromosomal haplogroups

Myung Jin Park1, Hwan Young Lee1,2, Na Young Kim1, Woo Ick Yang1, and Kyoung-Jin Shin1,2

1Department of Forensic Medicine and Brain Korea 21 Project for Medical Science,

Yonsei University College of Medicine

2Human Identification Research Center, Yonsei University

Y chromosomal SNP

Mutation Y-SNP Position 20175715 20175606 14018100 13981319 13005251 4138217 3496442 2888196 2881786 2794854 2715180

• 1bp

M133

• 1bp

M134

• 5bp

M175 T/C M214 T/C P31 C/G M7 G/C 47z T/C P201 G/C M324 C/T RPS4Y711 C/T SRY465

> 600 Y-SNP

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Y chromosome phylogenetic tree

A B C D E F G H I J K L M N O P Q R S T

Genome Res. 2008;18:830-38

Y chromosome consortium

Define O3

Haplogroup O

L1 retroposon insertion (LINE 1) polymorphisms was removed from this tree  Contradictory results with N7, P201, JST002611

Y haplogroup tying in forensics

 Forensic utility of Y-SNPs

 Human identification purposes (criminal, paternity, evolutionary, population studies)  Haplogroups are non-randomly distributed among populations therefore potential exists for predicting population of origin  The short PCR amplicons required for typing SNPs may result in success with degraded samples and possibly higher sensitivity

 Increasing of need for ethnicity prediction

 Globalization of crime suspects and victims  Increase of movement from East-southern population  Increase of excavation of Korean War victims and ancient remains.  Individual identification in mass disaster such as airplane crashes, tsunamis

• r terrorist attacks where people from various geographical areas are

involved

Requiring a sensitive and efficient method for the Y chromosomal haplogroup determination in East Asians

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Objects

 Development of multiplex systems for determination of East Asian Y haplogroups

 Single base extension (SBE) method with SNaPshot™ Multiplex kit

 Amplicon size < 100 bp to be suitable for the analysis of highly degraded forensic samples

 Allele specific PCR (AS-PCR) assay with fluorescent dyes

 Convenient method similar to STR typing for reference samples

 Validation of the multiplex systems

 Sensitivity test and efficiency test  Concordance test between multiplex SBE reaction and multiplex AS-PCR assay

Strategy for SNP typing

G G G C G allele 5’ 3’ 3’ 5’ A T A allele 5’ 3’ 3’ 5’ A A

G A G A G A GA

SBE reaction AS-PCR

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Materials and Methods

 DNA samples

 300 DNA samples obtained from the National Biobank of Korea.  Serially diluted DNA samples (1 ng, 500 pg, 250 pg, 125 pg, 62 pg and 31 pg) of 9948 standard DNA (Promega, Madison, MA, USA)  Artificially degraded DNA prepared by digesting 1.2 g of human genomic DNA with 0.02 U of DNase I (NEB, Ipswich, MA, USA) for 40 min  10 DNA samples extracted from 60-year-old skeletal remains

 Multiplex PCR conditions

 Multiplex SBE reactions

 Total 25 l PCR reaction: 1 ng of DNA, 2.5 l of Gole STR buffer, 2.0 U of AmpliTaq Gold polymerase and PCR primer mix  PCR cycling condition: 95°C 11 min; 94°C 20 sec, 60°C 1 min, 72°C 7 min X 33; 72°C 7 min  Total 10 l SBE reactions: 1 l of purified multiplex PCR product, SBE primer mix and a SNaPshot™ Multiplex kit (Applied Biosystems, Foster City, CA, USA)  SBE cycling condition: 96°C 10 sec, 50°C 5 sec, 60°C 30 sec X 25

 Multiplex AS-PCR assay

 Total 10 l PCR reaction: 1 ng of DNA, 1.0 l of Gole STR buffer, 2.5 U of AmpliTaq Gold polymerase and AS-PCR primer mix  AS-PCR cycling condition: 95°C 11 min; 94°C 20 sec, 60°C 1 min, 72°C 7 min X 30; 60°C 45 min

 Detection Systems

 ABI prism 310 genetic analyzer, GeneScan software 3.7, and Genemapper 3.2 software (Applied Biosystems)

Selection of Y-SNP for Multiplex SBE reactions

O3a3c O3a K

1

* M214 M133 M134 P201 M324 SRY465 P31 M175 M9 O3a3c1 O3a3b O3a3a O3a3* O3* M122 O2b1 O2b* O2a O2* O1a O1* O* NO F C DE A, B M145 RPS4Y711 M89 M119 M95 47z M159 M7

DE C O F K NO 1 2 3 a a

*

b * a

*

3 a b c 1

*

Multiplex I Multiplex II Multiplex III

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Schematic of multiplex SBE reactions

20 bp 70 bp 50 bp 30 bp 40 bp 60 bp

M145

(PCR 93 bp)

22 bp

RPS4Y711

(PCR 86 bp)

31 bp

M89

(PCR 93 bp)

39 bp

M9

(PCR 85 bp)

48 bp

M214

(PCR 99 bp)

55 bp

M175

(PCR 96 bp)

63 bp

M119

(PCR 94 bp)

25 bp

P31

(PCR 88 bp)

33 bp

M95

(PCR 79 bp)

42 bp

SRY465

(PCR 83 bp)

50 bp

47z

(PCR 87 bp)

58 bp

M122

(PCR 88 bp)

66 bp

M324

(PCR 70 bp)

23 bp

P201

(PCR 97 bp)

30 bp

M159

(PCR 99 bp)

39 bp

M7

(PCR 100 bp)

47 bp

M134

(PCR 72 bp)

55 bp

M133

(PCR 98 bp)

67 bp Multiplex I Multiplex II Multiplex III

All amplicon size ≤ 100 bp

Y haplogroup determinations using multiplex SBE reactions

DE C K NO O Multiplex I O1a O2 O2b O2b1 O3 Multiplex II Multiplex III

O3a O3a3 O3a3b O3a3c O3a3c1

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Sensitivity test

1 ng 500 pg 250 pg 125 pg 62 pg 31 pg 1 ng 500 pg 250 pg 125 pg 62 pg 31 pg 1 ng 500 pg 250 pg 125 pg 62 pg 31 pg

Multiplex I Multiplex II Multiplex III

Repetition by 5 times

All Y-SNPs were successfully called at as low concentration as 62 pg of DNA

Efficiency test

Artificially degraded DNA

M145G RPS4Y711 C M89T M9G M214C M175T M119A P31T M95C SRY465C 47zG M122T M324G P201T M159A M7C M134G M133T Treated with DNase I

The fully correct Y-SNP profiles were obtained using artificially degraded DNA with the fragment size of around 100 bp.

DNA from old skeletal remains

Y-SNP STR 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 O2b 100.0 275.2 ± 57.04 10 O3a3 100.0 84.9 ± 14.71 9 O2b 100.0 169.9 ± 10.96 8 O1a 33.3 27.8 ± 0.13 7 O3a3c 80.0 149.8 ± 11.30 6 O3a3 100.0 766.1 ± 39.03 5 O2b 100.0 106.5 ± 3.56 4 O2b 66.7 55.9 ± 5.94 3 O2 100.0 205.7 ± 2.75 2 NO 93.3 114.8 ± 10.20 1 Y haplogroup Success rate (%) Concentration (pg/l) No.

Some skeletal remain samples had been typed only at some STR loci, Y-haplogroup could be successfully determined based on the amplified Y-SNP scoring results.

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Selection of Y-SNP for AS-PCR assay

O3a3c O3a K

1

* M134 P201 SRY465 P31 M175 M9 O3a3c1 O3a3b O3a3a O3a3* O3* M122 O2b1 O2b* O2a O2* O1a O1* O* NO F C D A, B M174 RPS4Y711 M119 M95 47z M7

DE C O F K NO 1 2 3 a a

*

b * a

*

3 a b c 1

* DE N

N

O3a4

4 002611

M145 M214 M324 M133 M159 M117

Multiplex AS-PCR

M231

Schematic of multiplex AS-PCR

70 bp 90 bp 110 bp 130 bp 150 bp

D‐M174

78 C 81 wild

C‐RPS4Y711

102 wild 105 T

K‐M9

127 G 130 wild

N‐M231

153 A 156 wild

O‐M175

75 ‐5bp 80 wild

O1a‐M119

105 A 109 wild

O3‐M122

128 C 131 wild

O3a4‐002611

156 T 159 wild

O2‐P31

78 C 81 wild

O2a‐M95

102 T 105 wild

O2b‐SRY465

129 T 132 wild

O2b1‐47z

156 C 159 wild

O3a3‐P201

79 C 82 wild

O3a3b‐M7

103 wild 106 G

O3a3c‐M134

126 ‐1bp 130 wild

O3a3c1‐M117

152 ‐4bp 156 wild

FAM VIC NED PET

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Haplotyping using multiplex AS-PCR assay

N Ladder O1a O3a3c1

Future study for multiplex AS-PCR assay

 Concordant test

 A total of 300 Korean males will be tested by this assay and the results will be compared with those from the multiplex SBE reactions

 Validation test

 Sensitivity of the multiplex AS-PCR assay  Efficiency of the multiplex AS-PCR assay

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Conclusion

 Two different multiplex PCR sets, three multiplex SBE reactions and a multiplex AS-PCR assay, were developed for the identification of Y- haplogroups frequent in East Asians for analyzing forensic samples and reference samples, respectively.  Using the multiplex SBE reactions, reliable genotypes were obtained from the amount as 62 pg of DNA and highly degraded DNA from old skeletal remains.  The multiplex SBE reactions are very sensitive and optimized for analyzing

• ld degraded forensic casework samples.

 The multiplex allele-specific PCR assay was developed for simple, rapid and reliable scoring of alleles in large number of samples and will be tested concordance with the results of the multiplex SBE reactions.  This study would suggest that selective use of these multiplex sets for specific purpose is useful to obtain genotypes rapidly and effectively in forensic casework and reference samples. http://forensic.yousei.ac.kr

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