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Forensic Science International 157 (2006) 2335

www.elsevier.com/locate/forsciint

Development of multiplex PCRs for evolutionary and forensic


applications of 37 human Y chromosome SNPs
Valerio Onofri, Federica Alessandrini, Chiara Turchi,
Mauro Pesaresi, Loredana Buscemi, Adriano Tagliabracci *
Istituto di Medicina Legale, Universita Politecnica delle Marche, Policlinico Torrette, I-60020 Ancona, Italy
Received 30 November 2004; received in revised form 8 March 2005; accepted 22 March 2005
Available online 17 May 2005

Abstract

This work describes an efficient and rapid test for typing 37 single nucleotide polymorphisms (SNPs) of the non-recombining
region of Y chromosome (NRY) from a minimal amount of DNA using six PCR multiplexes. Markers were drawn following a
hierarchical strategy based on the phylogenetic tree of Y chromosome proposed by the Y Chromosome Consortium [The Y
Chromosome Consortium, A nomenclature system for the tree of human Y-chromosomal binary haplogroups, Genome Res. 12
(2002) 339348]. Two multiplexesarbitrarily named MY1 and MY2were developed to explore the basal branches of the tree
encompassing all the major clades A-R: MY1 for markers M35, M89, M172, M170, M9, M173, M45 and MY2 for markers M52,
M216, M174, M181, M201, M91, M96, M214. Four multiplexes able of typing the more superficial branches typical of most
frequent European haplogroups E3b, J2, R1 and I, were also developed and named MY-E3b (M78, M107, M224, M165, M148,
M81), MY-J2 (M158, M68, M47, M102, M137, M67), MY-R1 (M17, M269, M18, P25, SRY10831.2) and MY-I (M72, M223,
M26, M21, M161). SNP genotyping was carried out by hot-start PCR amplification with primers yielding fragments between 63
and 210 nucleotides, followed by minisequencing reaction based on dideoxy single-base extension and capillary electrophoresis of
extension products. The sequential application of these multiplexes is a robust and effective resource for typing the most frequent
European Y-SNP haplogroups, and appears to be suitable for forensic purposes and evolutionary studies.
# 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Y-SNPs; Multiplex PCR; Minisequencing; Capillary electrophoresis; Phylogeography; Forensic genetics

1. Introduction tifying stable paternal lineages and reconstructing an ances-


tral state from which to explore the history of human
The male-specific of the Y (MSY) region spans many evolution and to reconstruct family relationships by patri-
repetitive (STR) and sequencing (SNP) Y-markers, which lineage analysis. More than 200 biallele mutations were
represent a precious tool for both human evolutionary discovered by Underhill et al. [2] screening 21 populations.
studies and forensic identification purposes. The Y Chromosome Consortium genotyped 74 cell lines
Low mutation rate, paternal heritage only and absence of which revealed 245 mutational events, giving rise to 153
recombination make Y-SNPs particularly suitable for iden- NRY haplogroups. The novel nomenclature proposed by the
YCC for the single most parsimonious tree for these 153
* Corresponding author. Tel.: +39 071 596 4716; haplogroups now classifies its major clades into 18 hap-
fax: +39 071 596 4723. logroups, indicated by capital letters from A to R. In addi-
E-mail address: a.tagliabracci@univpm.it (A. Tagliabracci). tion, haplogroups belonging to the majority of clades are

0379-0738/$ see front matter # 2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.forsciint.2005.03.014
24 V. Onofri et al. / Forensic Science International 157 (2006) 2335

much more common in some regions where they first took remaining 22 are more superficial and define the most
origin [3] and, as well as their use in studies on population frequent major haplogroups in Europeans. All markers were
genetics, regional population substructure, gene flow and drawn from the cladogram of the YCC [3] except for M223
admixture, this geographical distribution of Y-SNPs, and M224. M223, defining the I1c haplogroup, was taken
together with the small size of DNA fragments which can from Cinnioglu et al. [15]. M224 was taken from Underhill
be successfully amplified (4050 bp), have forensic applica- et al. [2] and defines a haplogroup inside subclade E3b1 of
tions [4]. In casework involving unknown subjects or bio- the YCC cladogram. Loci M35, M89, M172, M170, M9,
logical traces, the regional affiliation emerging from the M173, M45, M52, M216, M174, M181, M201, M91, M96
haplogroup may narrow the search or be a useful or decisive and M214 were selected to explore the basal branches of all
factor in personal identification. haplogroups of the phylogenetic tree, arranged in two multi-
Taking into account the hierarchical structure of Y- plexes, arbitrarily named MY1 and MY2, and containing,
SNPs, if they are to be informative for forensic and respectively, the first seven and the next eight markers
evolutionary studies, they must be analyzed in two or more mentioned above (Fig. 1a and b).
steps [5], starting from the most basal branches of the The shallowest branches were explored by arranging four
phylogenetic tree to assign evidence to a major clade, and further multiplexes able to co-amplify markers belonging to
then proceeding along the relevant branches to the shal- the same clade or subclade:
lowest markers, to identify the haplogroups inside every
major clade. A hierarchical strategy like that suggested by 1. M78, M107, M224, M165, M148, M81, enclosed in a
Paracchini et al. [6] and considered in forensics by Brion hexa-plex named MY-E3b, because it is phylogenetically
[7] must be followed, which maximizes cost- and time- more superficial to mutation M35 (Fig. 1c);
effectiveness with information deriving from the lineage 2. M158, M68, M47, M102, M137, M67, investigated by a
pattern of the sample studied. Since this strategy means that second hexa-plex named MY-J2, because it can identify
several loci must be examined, in order to identify the most clade J2, which follows marker M172 in the tree
suitable ones, multiplex PCR is preferable, starting from (Fig. 1d);
the minimal amounts of DNA which can usually be recov- 3. M17, M269, M18, P25, SRY10831.2, grouped in a penta-
ered for forensic analysis. plex named MY-R1 and containing loci which explore
Many technologies are now available for SNP genotyp- clade R1, identified by mutation M173 (Fig. 1e);
ing [8,9], but most of them are not suitable for forensic 4. M72, M223, M26, M21, M161, pooled in a second penta-
purposes, because they are expensive and planned for large- plex named MY-I, because it can identify markers of
scale analysis, or require large amounts of DNA. Instead, clade I above basal marker M170 (Fig. 1f).
forensic scientists must always deal with single cases, in
which very often only minimal amounts of DNA are avail- 2.2. DNA samples
able. For this reason, we chose minisequencing genotyping,
a single-base extension of an unlabeled oligonucleotide The DNA of 97 healthy male donors was used to
primer, which provides the basis for a simple and efficient validate each multiplex PCR: 68 from Europe, 17 from
technique requiring instruments normally available in any Asia, 9 from Africa and 3 from South America. DNA was
forensic laboratory. extracted from 200 ml of whole peripheral blood using the
The aim of this study was, therefore, to set up PCR QIAamp Blood Mini Kit (Qiagen, Inc., Chatsworth, CA).
multiplexes of Y-SNPs able to genotype hierarchically the Quantitation was carried out using the QuantiBlot Human
phylogenetic tree of the Y chromosome, providing useful DNA Quantitation Kit (Applied Biosystems). One nano-
informations for forensics, employing normally available gram of DNA was used as a standard amount for all
materials, methods and technologies. The PCR multiplexes amplifications.
developed here for exploring the shallowest branches are
able to genotype the most frequent European haplogroups. 2.3. Design and validation of amplification primers
They may also be used profitably by researchers working
on human evolution and by anthropologists and human The sequences of the regions spanning the mutations to
genetists. be examined were drawn from GenBank. Multiplex PCR
primer sequences were designed by Primer Express soft-
ware (Applied Biosystems). For every multiplex, we
2. Materials and methods avoided the default setting proposed by the software, in
order to obtain the most stringent conditions possible for
2.1. SNPs selection the PCR multiplex reaction. We took care to choose Tm
values for all primers between 58 and 63 8C, with max-
Thirty-seven markers were drawn from the literature imum Tm differences of 2 8C between primer pairs and
[2,1016]. Fifteen of them are located in the basal branches 3 8C between the whole set of primers. Primer length was
of the phylogenetic tree and define the major clades AR, the chosen between 20 and 30 nucleotides, to provide good
V. Onofri et al. / Forensic Science International 157 (2006) 2335 25

Fig. 1. Cladograms and electropherograms of the Y-SNPs multiplexes object of this study: (a) MY1, (b) MY2, (c) MY-E3b, (d) MY-J2, (e) MY-
R1 and (f) MY-I.

specificity for single target sequences. The GC percentage Table 1 lists the main features of the 37 SNPs investigated
varied between 20 and 80%. Primerdimer and hairpin and the sequence of the 36 couples of primers designed in
loop formation was checked by the software, which rejects our study (M17 and M18 are very close each other and were
all primers with complementary 30 ends. amplified with the same primers). For each SNP we upload
26 V. Onofri et al. / Forensic Science International 157 (2006) 2335

Table 1
Mean features of 37 Y-SNPs and PCR amplification reactions for the six PCR-multiplexes developed in this study
Marker Polymora GenBank/dbSNP Amplicon PCR primer sequence (50 !30 ) Conc. (mM)
accession size (bp) Forward Reverse
MY1
M170 A/C rs2032597 96 gttttcatattctgtgcattatacaaattactat cattttacagtgagacacaacccac 0.24
M173 A/C rs2032624 104 aaaattttcttacaattcaagggcatt gctgcagttttcccagatcct 0.1
M45 G/A rs2032631 109 ggtgtggactttacgaaccaacct cctggacctcagaaggagcttt 0.1
M89 C/T rs2032652 110 ctgctcagcttcctggattca cactttgggtccaggatcacc 0.18
M35 G/C rs1179188 114 aactgagagggcatggtccc tgaacaactaatccatgcagactttc 0.24
M9 C/G rs3900 127 agaactgcaaagaaacggccta tgcataatgaagtaagcgctacct 0.1
M172 T/G rs2032604 136 ttttatcccccaaacccattt catgttggtttggaacagtttatcc 0.1
MY-E3b
M224 T/C AC010889 96 gttcgacatgaacacaaattgatacact tgaaaagcaagtactatgaccagctta 0.2
M107 A/G rs2032613 110 tactcatttcttaagccaacgtattaacct ccacttatgcaacgaataaaaaacactt 0.2
M148 A/G AC010889 121 tcagtcacagcaattctatcctgtcta ggtagttaccaatttttggctatttca 0.4
M81 C/T rs2032640 122 tttaagcactatcatactcagctacacatc gcgtaggtatgtagtagattgtttcttctt 0.2
M78 C/T AC010889 131 tcgacatgaacacaaattgatacactt ccttaaatctaaatattggaagcttaccat 0.2
M165 G/C AC010889 148 gatgacagaatgcgttcaccttt atgtgtaaatatttcaggtaaaaccactct 0.2
MY-I
M72 A/G rs2032637 63 ataaaagggcagaaactaccttccc tggttggtgattttctgtaatctcc 0.1
M21 A/T AC009977 113 gagaatcaaaggtcttttaagccct agcaggcagaagaatcaacaaac 0.2
M161 C/A AC003032 114 gcaatcggaagcctcaatctatac tgcaaagttaggagatttactgaatca 0.2
M26 G/A rs2032629 128 ggcttaccagtggtaaagttttattaca catgacgaaatctgcagcaaa 0.3
M223 C/T AC003032 131 cagcaagagtaagcaagaggcact aggcaagtatgccgctataaaaat 0.1
MY-J2
M67 A/T rs2032628 130 aaaggctctatcttcaagtacgtgtccta cacttgttcgtggacccctc 0.1
M68 A/G AC010889 134 gcggacaacctaaaaaggagaaaa cgtacttgaagatagagcctttccac 0.1
M158 G/A AC003032 136 tcaaaatacatgagactgcctaccct gattgtctggttgtggggaaat 0.1
M137 T/C AC010889 139 gggaacagggaagtcggtttaata gtgatcaacttctttccctcaacat 0.1
M102 G/C rs2032608 210 gaaatttattaaatgaagtatagattgaatacaag tccttaatctctaggggttttacaaa 0.1
M47 G/A AC009977 141 tggaggaagtacctgctaaattttcaa ggctgaaatcaatccaatctgtaaa 0.1
MY-R1
M18b -/insAA rs3909 124 attggggaatacctggtcataaca aatagtttggccacttaacaaaccc 0.2
M17b 4G/3G rs3908
SRY10831.2 C/T rs2534636 125 atagcaaaaaatgacacaaggcacc tgcctttcctggatatttcatataca 0.1
P25 C/CA rs150173 145 cagacacgtaagccatgtataacacc tggaccgagatacgagacaca 0.06
M269 T/C rs9786153 167 gtggattctgttacatggtatcacaa tccaaggtgctgggattacac 0.1
MY2
M52 A/C AC009977 153 ctcccacctcaacttcccagag agcaaacatttcaagagagaatgaaa 0.1
M201 G/T rs2032636 163 tatgcatttgttgagtatatgtcaaat tccaacactaagtacctattacgaaaa 0.1
M96 G/C AC010889 165 ttctccatatctgtgtaaaggcaagt ccataggtttttaatattatacctgagtg 0.1
M181 T/C rs2032599 166 gctagcaaagttggcttggg gcacactagctataagcaaaagaaat 0.1
M174 T/C rs2032602 169 aaatgtacgtttttggtttactcataatg tgcaaaaggagaaggacaaga 0.1
M91 9T/8T rs2032651 170 attgcgatgttttatttcaaaacaagatg gcgtatttttcaaaaatatatggagaa 0.1
M216 C/T rs2941566 171 aagccacttaaattccaatgga cactgctagttatgtatacctgttgaa 0.06
M214 T/C rs2032674 207 caattgtacagcacaaatatatgcctgtaaa gaggtcaagggtgtggtgag 0.1
AC010889, Homo sapiens BAC clone RP11-424G14 from Y, complete sequence; AC009977, Homo sapiens BAC clone RP11-576C2 from Y,
complete sequence; AC003032, Human Chromosome Y Cosmid 7A1 Genomic Sequence, complete sequence.
a
Ancestral and derived alleles of the polymorphism.
b
SNPs on the same amplicon.

on Primer Express software not less than 200 bp GenBank Lyophilized primers (Qiagen) were dissolved in Tris/
sequence upstream and downstream the polymorphic site, so EDTA buffer, pH 7.0, to a final concentration of 100 mM,
that the software could chose optimized primer pairs. and conserved frozen for several weeks.
V. Onofri et al. / Forensic Science International 157 (2006) 2335 27

Fig. 2. PAGE showing the PCR products of the six multiplexes.

The correct design of the primers was checked by final volume of 12.5 ml, composed of 1 PCR reaction mix,
singleplex amplification of 10 ng of standard male template, 800 mM dNTPs and 0.625 U hot-start Taq polymerase
in a final volume of 50 ml, in the following conditions: 1 (Qiagen). The MgCl2 concentration ranged from 2 (for
PCR reaction mix, 800 mM dNTPs, 1.5 mM MgCl2, 2.5 U MY1, MY2, MY-J2 and MY-R1 multiplexes) to 2.5 mM
EuroTaq polymerase (Euroclone) and 1 mM of each primer. (for MY-I and MY-E3b). PCR primers at various concentra-
The reaction consisted of denaturation at 94 8C for 5 min, tions (Table 1) were employed to assemble six multiplexes
followed by 35 denaturation cycles at 94 8C for 1 min, with loci number ranging from 5 (MY-R1 and MY-I) to 8
annealing at 56 8C for 1 min, extension at 72 8C for (MY2) for a total of 37 markers. A negative control was
1 min and a final extension at 72 8C for 10 min. Singleplex added to each amplification reaction.
amplification products were checked by 11% PAGE in Hot-start PCR involved 1 cycle at 95 8C for 15 min,
discontinuous buffer and stained with silver [17,18]. 25 followed by 35 denaturation cycles at 94 8C for 1 min,
and 100 bp ladders (Invitrogen) were used to verify the annealing at 55 8C (for MY1, MY2 and MY-J2 multiplexes)
expected size of amplicons within the six multiplexes. or 58 8C (for MY-R1, MY-E3b and MY-I) for 1 min, exten-
To check the specific amplification of the region spanning sion at 72 8C for 1 min and a final extension at 72 8C for
the selected marker, and to confirm the correct polymorphic 10 min. PCR products were detected by PAGE [17,18] in
site, sequencing of amplified fragments from the singleplex of Fig. 2.
each locus was accomplished in a GeneAmp PCR System
2400 thermal cycler using the ABI Prism BigDyeTM Termi- 2.5. Single-base extensions primers design
nator Cycle Sequencing Ready Reaction kit (Applied Bio-
systems) in a final volume of 20 ml, composed of 4 ml Inactivation of dNTPs and removal of primers were
BigDyeTM Terminator Ready Reaction mix, 1530 ng PCR performed by incubation of 1 ml of amplified product with
product, 0.25 mM primer and deionized water. The sequen- 1.5 ml of ExoSap-IT (USB) reagent for 15 min at 37 8C,
cing reaction was run for 25 cycles of 96 8C for 10 s, 50 8C for followed by enzyme inactivation at 80 8C for 15 min, or
5 s and 60 8C for 4 min. Extension products were purified by with ExoI-CIP (New England BioLabs Inc.) mix for
isopropanol precipitation and dried in a vacuum centrifuge. 60 min at 37 8C followed by 20 min at 80 8C. One micro-
They were then resuspended in 12 ml of Template Suppressor liter of the purified product were submitted to a dideoxy
Reagent (Applied Biosystems), heat-denatured and chilled on single-base extension of unlabeled oligonucleotide pri-
ice, and analyzed by capillary electrophoresis on an ABI mers using the Snapshot Multiplex kit (Applied Biosys-
Prism 310. The capillary was filled with run buffer perfor- tems) in a final volume of 5 ml. Each minisequencing
mance optimized polymer POP 6 (Applied Biosystems). primer was designed so that its 30 end was 1 bp upstream
Samples were injected in 30 s at 2 kV and separated for from the relevant polymorphism, and was 50 -tailed with
25 min at 15 kV and 50 8C. The resulting data were analyzed poly-T and poly-GACT, or neutral sequence for M17 [19],
by Sequencing Analysis software, and sequences were aligned to produce molecules with sizes ranging from 18 to 60
and compared with those present in GenBank by means of bases. This was done because extension products must
Sequence Navigator software 1.0.1. differ in length from each other by a user-selectable
margin sufficiently great to distinguish them all by elec-
2.4. Multiplex PCR amplification trophoresis (Table 2).
The same PCR reverse primer was used for locus M68,
One nanogram of DNA was submitted to amplification in since the mutation was located one nucleotide after its 30
a GeneAmp1 PCR System 9700 (AB) thermal cycler in a end.
28 V. Onofri et al. / Forensic Science International 157 (2006) 2335

Table 2
Primer sequences used for the extension reactions of the 37 markers of this study
Marker Primer Minisequencing primer sequence (50 !30 ) Conc.
length (nt) (mM)
MY1
M173 21 Fw CAATTCAAGGGCATTTAGAAC 0.2
M172 30 Rv AAGAAAATAATAATTGAAGACCTTTTAAGT 0.2
M170 35 Fw AAATTACTATTTTATTTACTTAAAAATCATTGTTC 0.2
M35 39 Rv (GACT)5CGGAGTCTCTGCCTGTGTC 0.2
M9 44 Fw (GACT)6ACGGCCTAAGATGGTTGAAT 0.2
M45 50 Fw (GACT)6AAATTGGCAGTGAAAAATTATAGATA 0.2
M89 56 Rv (T)34CAACTCAGGCAAAGTGAGAGAT 0.2
MY-E3b
M78 21 Rv TTTTGAAATATTTGGAAGGGC 0.2
M107 25 Rv GTCTCATGCTTTCTATTAGCATTCA 0.1
M224 34 Fw (T)4ACAAATTGATACACTTAACAAAGATACTTC 0.2
M165 45 Rv (T)15CCACTCTATTAGTATACCACTAATTCAATT 0.4
M148 50 Fw (T)23AGCAATTCTATCCTGTCTATCTAGGTA 0.1
M81 55 Rv (GACT)7TTGGTTTGTGTGAGTATACTCTATGAC 0.1
MY-I
M72 21 Fw GAAACTACCTTCCCAAAACCC 0.1
M223 29 Rv (GACT)GCTGAAGATGATGCAATTTATTTAC 0.1
M26 37 Rv (GACT)4TAGGCCATTCAGTGTTCTCTG 0.2
M21 44 Rv (GACT)5ACTGGAAGCTAAGTCAACTGAAAC 0.2
M161 50 Fw (T)22CTCAATCTATACAGACTTTTAGGAGGAG 0.2
MY-J2
M158 18 Rv TTGCTTCAGAAAGCCCCA 0.2
M68 26 Rv CGTACTTGAAGATAGAGCCTTTCCAC 0.2
M47 33 Fw GCTATATCATTTTACTCTGAATGTCTTAACATA 0.2
M102 40 Rv (GACT)2GCTGTTTATTCTTATTGTCTTTTCACATCTTA 0.2
M137 46 Fw (GACT)6GTGCAACCTCAACTTTGCTTTA 0.4
M67 52 Fw (GACT)6ACTCAAAATATGTGTAATTCAAAAAACA 0.4
MY-R1
M269 23 Fw GGAATGATCAGGGTTTGGTTAAT 0.2
M18 33 Fw (GACT)3GTTTGTGGTTGCTGGTTGTTA 0.2
P25 40 Rv (GACT)3CCGAGATACGAGACACAATTCTATTT 0.08
SRY10831.2 46 Fw (GACT)5CACATAGGTGAACCTTGAAAATGTTA 0.2
M17 58 Rv ACTAAACTAGGTGCCACGTCGTGAAAGTCTGACAACCAAAATTCACTTAAAAAAACCC 0.2
MY2
M52 24 Fw ATACCTATAAGAATATTGCCTGCA 0.2
M216 28 Rv CACTGCTAGTTATGTATACCTGTTGAAT 0.2
M174 35 Rv (T)16CACCCCTCACTTCTGCACT 0.2
M181 39 Fw (GACT)4GGACAACTTGATCATCTTTTTGA 0.2
M201 44 Fw (GACT)4AGATCTAATAATCCAGTATCAACTGAGG 0.2
M91 47 Rv (GACT)4GATACTACAGTAGTGAACTGATTAAAAAAAA 0.2
M96 53 Fw (GACT)6GTAACTTGGAAAACAGGTCTCTCATAATA 0.2
M214 60 Rv GA(GACT)8AGTGTGAGACACTGTCTGAAAACAAC 0.4
Primers sequences used for the extension reactions of the 37 markers of this study (poly(nt) tails in bold).

Tm, secondary structure and primerdimer formation of 2.6. Single base extension product purification
primers were evaluated using the Primer Test Document
function of Primer Express software (Applied Biosystems). A further purification step with 0.5 units of Sap (shrimp
In every reaction, extension was performed for 25 cycles at alkaline phosphatase, USB), or CIP (calf intestinal alkaline
96 8C for 10 s, 50 8C for 5 s and 60 8C for 30 s, in a phosphatase, New England BioLabs Inc.) enzyme was per-
GeneAmp PCR System 9700 (AB) thermal cycler, including formed to remove the 50 phosphoryl groups of the unin-
a reagent blank, composed of TE, pH7. corporated [F]ddNTPs for 60 min at 37 8C, followed by
V. Onofri et al. / Forensic Science International 157 (2006) 2335 29

inactivation for 15 min at 75 8C. 0.7 ml of purified product morphic sites M35, M89, M172, M170, M9, M173, M45
were mixed with 0.25 ml of internal size standard Liz120 (Fig. 1a); MY2 includes nodes M52, M216, M174, M181,
(Applied Biosystems), 9 ml of Hi-Di Formamide (Applied M201, M91, M96, M214 (Fig. 1b). These markers spread the
Biosystems), heated for 5 min at 95 8C, quenched in an ice A-R major clades and subsequent subclades, so that their
bath, and injected in an automated ABI 310 5-color sequen- combined presence or absence means that a sample can be
cer (Applied Biosystems). assigned to one of the major clades and guides the investiga-
tion along the branches towards the relevant haplogroups.
2.7. Capillary electrophoresis Four other shallower multiplexes, able to detail the European
haplogroups were developed, for use in a second step on the
The amplified product was analyzed on an ABI 310 basis of the results obtained in the first amplification. The
(Applied Biosystems), using 1 ml of the PCR product fol- four multiplexes developed in this study were set up to
lowing the manufacturers recommendations (AE 4323291 examine alternatively the most frequent European hap-
from AB). logroups nested inside any major clade identified in the first
Electrophoresis was performed for 14 min with an injec- analysis: R1, I, E3b, J2 [12,14]. The shallower multiplex
tion time of 2 s, voltage 15 kV and temperature 60 8C. MY-R1 was used when the MY1 multiplex showed the
Fluorescently labeled fragments were sized by the local derived state at loci M89, M9, M45 and M173, and allows
Southern method implemented in Genescan Analysis soft- nodes P25, SRY10831.2, M269, M18 and M17, which
ware 3.7 (Applied Biosystems). identify the R1 haplogroups, to be explored (Fig. 1c). The
The employment of five dyes, dR110, dR6G, dTAMRA, shallower multiplex MY-I was used when the MY1 multi-
dROX and LIZ used in the kit, required a specific filter plex showed the derived state of nodes M89 and M170, and
addition, E5, to process the data, and an optimized spectral explores markers M223, M26, M161, M72 and M21, which
matrix was subsequently created. identify the I haplogroups (Fig. 1d). The shallower multiplex
MY-E3b was used when the MY1 multiplex showed the
2.8. Sensitivity assays: low amount/low molecular derived state of locus M35, and explores markers M78, M81,
weight DNA experiments M148, M224, M107 and M165, which identify the E3b
haplogroups (Fig. 1e). The shallower multiplex MY-J2 was
Serial dilutions at concentrations of 10, 50, 100, used when the MY1 multiplex showed the derived state for
500 pg/ml and 1 ng/ml DNA were prepared to test the nodes M89 and M172, and explores markers M67, M102,
sensitivity of PCR amplifications. In addition, 1 ml of M158, M137, M68 and M47, which identify the J2 hap-
DNA, extracted from whole blood at a concentration of logroups (Fig. 1f). Note that this approach identifies the
20 ng/ml, was fragmented in a sonicator device (Labsonic, major haplogroups, the shallowest haplogroup inside the
Braun) using 4 mm diameter tips, in 50 W and 0.5 s cycles. subclades and, in some cases, the final haplogroup. Identi-
Sonicated DNA was submitted to PAGE, hot-start ampli- fication of the remaining single haplogroups requires
fication and minisequencing, as described in the previous exploration of the last further mutation.
sections. One nanogram of fragmented DNA was also For each sample, this hierarchical approach determines
amplified with the AmpFlSTR Identifiler PCR Amplifica- the relevant haplogroup with two amplification steps at most.
tion kit (Applied Biosystems), containing 15 autosomal For example, if a person tested for MY1 shows the derived
microsatellites and the Amelogenin marker, covering a state at loci M89 and M172, we only amplify MY-J2,
range of approximately 100 (Amelogenin) to 350 because all the other markers in the other three multiplexes
(D2S1338) nucleotides. The amplified products were ana- are certainly in the same ancestral state.
lyzed on an ABI 310 (Applied Biosystems) using 1 ml of MY2 was employed when none of MY1 loci, or only
the PCR product. M89/M9 loci, were mutated, and allowed the sample to be
assigned to the haplogroup characterizing the non-European
populations, or present at low frequency in Europeans.
3. Results and discussion
3.2. Genotyping procedure
3.1. SNP selection
In view of its use in solving forensic cases, Y-SNP
Thirty-seven binary polymorphisms including 34 transi- genotyping was performed by minisequencing, a simple
tions/transversions, two 1 bp deletions (M17 and M91) and and efficient technique requiring easily available instru-
one 2 bp insertion (M18) were selected in this study, in order ments. Moreover, since forensic scientists are frequently
to arrange six SNP multiplexes capable of exploring the forced to work with degraded or very small amounts of
most frequent European haplogroups of the Y chromosome. DNA, our efforts focused on optimizing a method yielding
Based on the known phylogeny of Y chromosome [1], two results in this extreme type of casework, selecting PCR
multiplexes, named MY1 and MY2, were set up to examine primers able to amplify amplicons less than 150200 bp
the basal branches of the tree. MY1 encompasses poly- in length.
30 V. Onofri et al. / Forensic Science International 157 (2006) 2335

3.2.1. Multiplex PCR specificity of all co-amplified loci was confirmed by check-
After preliminary tests with both Touch-down and hot- ing the expected size (Fig. 2) and sequencing of singleplex
start PCR techniques, the latter test was set up because it amplification products (data not shown).
worked better and an even larger number of amplification The optimal PCR buffer concentration, tested by increas-
fragments of all the loci composing the multiplex could be ing the MgCl2 concentrations by a factor of 0.5 mM, reached
amplified. However, this technique did yield a great number 2 mM for MY1, MY2, MY-J2 and MY-R1 and 2.5 mM for
of non-specific amplification products together with the MY-I and MY-E3b multiplexes. Beyond 2.5 mM, Taq poly-
specific ones, mainly due to the use of less expensive, merase did not work, probably due to the inhibitory effect of
non-HPLC-purified oligonucleotides. In order to minimize the high salt concentration on the PCR. This result does not
these aspecific products using non-purified oligonucleotides, fit those of Sanchez et al. [19] whose best results were
experiments were performed on primer concentrations. A obtained with a concentration of 8 mM MgCl2, but this may
reduction in the number of artifacts in the pertinent multi- be explained by our use of salt-free primers, whereas the
plex was obtained only by decreasing primer concentrations above authors used HPLC-purified primers for co-amplify-
for markers P25 and M216 to 0.06 mM and by increasing ing 35 loci simultaneously.
those of markers M26, M35 and M148 up to 0.3, 0.24 and
0.4 mM, respectively. Nevertheless, these unavoidable and 3.2.2. Multiplex minisequencing
undesirable artifacts in our working conditions did not The extension primers were designed so that they were
interfere with the successive steps of extension, and were well spaced from one other in the electropherograms, and the
not visible in the pherograms. In addition, the number of maximum length of the extension products was 60 nucleo-
PCR products, estimated by PAGE assay, was not linearly tides for M214 (Table 2). Primer concentrations were
correlated with the peak heights of the observed extension adjusted in order to obtain sufficient RFU peak heights.
products, as shown in Fig. 2, differing from reports by other Some experiments were performed to balance the peak
authors who used HPLC-purified primers [20]. heights of different extension products optimally, by increas-
The amplification primers were correctly designed, and ing or reducing the concentrations of some SNP primers
resequencing due to mispairing or self-annealing was not which showed a clear lack of balance, starting from the
necessary, except for two couples of primers which did not manufacturers recommendation of 0.2 mM final concentra-
yield the corresponding peaks, due to a synthesis problem tion for each primer. We obtained only a slight rise in peaks
during manufacture. This was solved simply by purchasing on doubling the starting concentration for loci M214, M165,
the oligonucleotide from another commercial source. All M137 and M67, and no results for the other ones. We, thus
amplicons of the loci enclosed in every multiplex were conclude that there is an unavoidable significant difference
yielded, as expected, in a range between 63 and 210 bp in primer sequence related to peak height between the
(Table 1), 28 of them falling within 150 bp. The amplicon extension products at the various loci. Furthermore, the peak

Fig. 3. Electropherograms of two different individuals showing ancestral (a) and derived state (b, see arrow) alleles of marker P25.
V. Onofri et al. / Forensic Science International 157 (2006) 2335 31

height of the extension product from locus P25 was con- and size as the expected allele, and thus require retyping of
stantly below 150 RFU and aspecific peaks were also samples. This phenomenon may be avoided by designing
present, until the poly(T) tail of the extension primer was extension primers out of PCR primers, sizing not less than
changed for a poly(GACT) tail. A lack of correlation in 2830 nt, which makes peak interpretation unambiguous
expected/measured sizes for the shortest extension products, even in cases of insufficient purification; (2) aspecific peaks
on average 45 nucleotides more, was also observed, may also be generated by primerdimer extension and we
depending on the secondary structure assumed by extension did observe this phenomenon in one case, involving the
fragments in capillary electrophoresis [20] and the influence extension primer for M269, where extension products were
of the various masses of fluorochromes on the mobility of yielded from the negative control. Strong reduction to the
shorter DNA molecules [21]. This difference progressively background noise line of the aspecific peak heights in the
disappeared when the size of the extension primers was presence of the template, which did not interfere with the
increased up to 4050 nucleotides. specific peaks, was obtained by setting the extension
Depending on the correct poly(T) tail lengths set for primer on the complementary strand; (3) the aspecificity
each primer, the expected extension products, all present in of the extension reaction may derive from the low anneal-
the electropherograms, were well spaced from one another, ing temperature necessary for the co-amplification of sev-
so that identification of the extension product from each eral loci. In our experiments, increasing the temperature up
locus was always easy and definite. Nevertheless, aspecific to 56 8C did not improve the results, as in Sanchez et al.
peaks not corresponding to the expected alleles generated [19], and the best solution was to redesign the extension
during minisequencing were observed, mainly for the primer on the complementary strand. This was done for the
following causes: (1) in most cases, the presence of aspe- minisequencing primers for markers M78, M17, M96,
cific products sizing the extension primer +1 nt is due to M201 and M214.
insufficient purification of the amplification product with Lastly, some extra peaks ranging between 27 and 39
ExoSap and annealing of unremoved PCR primers with the nucleotides, below the threshold of 150 RFU, all of them
corresponding minisequencing primers. PCR product pur- incorporating ddG, were observed in the MY-R1 multiplex.
ification seems to be essential for correct interpretation of Since, they disappeared on increasing the amount of DNA up
results. A large number of confusing peaks tend to look like to 1 ng and were also present in the blank control, we believe
2025mer alleles, sometimes with the same fluorochrome this phenomenon was due to the formation of extension

Table 3
Typing results of the 97 different geographic provenance individuals
Geographic area I stepb Markers at the Major clade or II stepc Markers at the Haplogroups Observed
of provenancea derived state subclade derived state subjects
Europe MY1, MY2 M89, M9, M45, M173 R1 MY-R1 SRY10831.2, M17 R1a1 3
P25, M269 R1b3 22
M35 E3b MY-E3b M78 E3b1* 10
M224 E3b(xE3b1, E3b2) 5
M89, M172 J2 MY-J2 J2* 1
M12 J2e 5
M67 J2f 8
M89, M170 I MY-I M223 I1c 3
M89 F*(xI, J2, K) F*(xI, J2, K) 7
M89, M9 K*(xP) K*(xP) 4
Asia MY1, MY2 M89, M9 K*(xP) K*(xP) 10
M89, M9, M45 P*(xR1) P*(xR1) 5
M89, M9, M45, M173 R1 MY-R1 SRY10831.2 R1a* 1
M174 D D 1
Africa MY1, MY2 M91 A A 4
M181 B B 2
M35 E3b MY-E3b M78 E3b1* 1
M224 E3b(xE3b1, E3b2) 2
South America MY1, MY2 M89, M9, M45 P*(xR1) P*(xR1) 3
a
Fifty-six subjects from Central Italy; 10 Polish; 1 Greek; 1 Albanese; 1 Chinese; 10 Tawainese; 1 Kazak; 1 Kirghiz; 3 Indians; 1 Iraqi; 3
Kenians; 2 Magrebins; 3 Nigerians; 1 Senegalese; 3 Amerindians from Peru.
b
MY1: M35, M89, M172, M170, M9, M173, M45; MY2: M52, M216, M174, M181, M201, M91, M96, M214.
c
MYE3b: M78, M107, M224, M165, M148, M81; MY-J2: M158, M68, M47, M12, M137, M67; MY-R1: M17, M269, M18, P25,
SRY10831.2; MY-I: M72, M223, M26, M21, M161.
32 V. Onofri et al. / Forensic Science International 157 (2006) 2335

primerdimer. In any case, the peak spectrum did not provenance: NO, P*(xR1), D for Asians, A, B, E(xE3b) for
interfere with haplotype interpretation, because no ddG Africans and P*(x R1) for Amerindians. In the second
allele was expected in the range 2739 nt length. In addition, analytical step, the emerging branch for every European
when a cut-off of 150 RFU was applied, as required for STR sample was assayed, in turn, with the relevant multiplex for
profiling, all the extra peaks were escaped. the shallowest markers, which allowed haplogrouping with
Typing was carried out both manually, by evaluating the only two analytical steps. The haplogroup frequencies
product length in Genescan, and automatically, using Gen- observed are similar to those reported in previous studies
otyper software after a multiplex-specific macro design. [10,15].
Lastly, 22 R1b types showed the contemporary presence
3.2.3. Y chromosome haplogroups of two different extension products at locus P25 (Fig. 3).
Experiments to validate the technique and strategy pro- Since contamination was excluded by STR analysis of 12
posed here were performed on 97 subjects of known geo- loci, we conclude that this result depended on paralogous
graphic area of provenance. Samples were SNP-typed twice sequence variants, already studied by Sanchez et al. [23].
with the multiplexes studied here and analysis showed that
the duplicate types were consistent. Moreover, first-step 3.3. Experiments with degraded and/or small
analysis with the MY1 and MY2 multiplexes, exploring amounts of DNA
the major clade or subclade, gave results consistent with the
geographic affiliation of the samples examined (Table 3). In Experiments were performed with decreasing amounts
particular, most of the 68 Europeans were assigned to main and low molecular-weight DNA template, to assay the
European clades or subclades R1, E3b, J2 and I; only a few sensitivity of PCR multiplex amplification. Serial experi-
samples were assigned to clades less frequent in Europeans ments with decreasing quantities of DNA template showed
and mainly characterizing Asian populations. The reason for that our six Y multiplexes work well in the range 50 pg1 ng
this genetic admixture has already been discussed [20]. of DNA. With quantities of DNA exceeding 5 ng, the
Samples from outside Europe showed, as expected, types electropherograms showed excessive peak heights and cor-
belonging to clades common in their respective region of responding increases in background noise, which interfered

Fig. 4. Complete SNPs genotyping of DNA fragmented by sonication.


V. Onofri et al. / Forensic Science International 157 (2006) 2335 33

Fig. 5. Comparison of only male (a) and malefemale mixture (1:1000) DNA (b) submitted to MY1 amplification. All of the markers were
present in (b). The other five multiplexes gave positive results too (data not shown).

with interpretation of results, although by only 1 s of elec- 4. Conclusion


trophoresis injection time, whereas no positive results or
only a few peaks were obtained under 50 pg. Thirty-seven binary polymorphisms, arranged in six PCR
Experiments to explore the suitability of the Y-SNP multiplexes containing from 5 to 8 markers and able to
multiplex with low molecular-weight DNA were performed assign the major haplogroups of the Y chromosome and the
by amplifying DNA fragments of less than approximately relevant haplogroups along the European clades, were devel-
150 bp, produced by sonication (Alessandrini et al. [22]) and oped in this study. The multiplexes were developed hier-
comparing the results with those obtained by amplifying the archically, with two more deeper multiplexes exploring the
15 autosomal microsatellites making up the AmpFISTR major clades and subclades, to discriminate between hap-
Identifiler PCR amplification kit (AB) with the same soni- logroups belonging to specific continents. The markers of
cated DNA (Fig. 4). Positive amplifications were obtained these two multiplexes were located in the strategically nodes
from 1 ng of DNA for all six Y-SNP PCR multiplexes of the phylogenetic tree and guided the choice of four other
developed in this study, whereas only pherograms with high multiplexes capable of defining European haplogroups by
noise background and questionable peaks below the thresh- proceeding along the relevant branch to the tip in only one
old of 50 RFU were obtained from the few microsatellites more step.
sizing below 150 nucleotides: D8S1179 (128168 bp), Since biallele polymorphisms show geographic distribu-
D19S433 (106140 bp), D3S1358 (114142 bp) and Ame- tion, our multiplexes may profitably be applied to studies on
logenin. human evolution, population genetics and molecular anthro-
In samples in which the amount of template was at the pology, in order to reconstruct human history. The geo-
lower limit of detection or was highly degraded, SNP locus graphic affiliation of Y-SNPs also make them attractive
drop-outs for high-molecular-weight loci were sometimes for forensic purposes, when knowledge of geographic affilia-
observed. Magnification of peak heights or peak recovery tion can help in personal identification. In mass disasters,
from these loci was achieved by a combined spin column including aircraft or boat-people accidents, when the bodies
Microcon 100 (Amicon) and enzymatic purification of PCR of victims to be identified are damaged by trauma or
amplification products before minisequencing typing ana- putrefaction and people from various geographical areas
lysis. are involved, the detection of haplogroups which are specific
In order to verify female DNA interference on the to or more frequent in certain geographical area represents a
amplification of Y-SNP multiplexes, experiments with powerful aid to forensic investigation. Similarly, SNP ana-
malefemale mixtures were also performed. Positive results lysis of evidence recovered from crime scenes may allow
were obtained although the female DNA concentration was identification of the ethnic group of the subject who left the
more than 1000 times that of the male (Fig. 5a and b). trace, narrowing the field of search and acting as a guide for
34 V. Onofri et al. / Forensic Science International 157 (2006) 2335

further investigations. Moreover, SNPs are useful tools even mosome using MALDI-TOF mass spectrometry, Nucleic
when the available DNA is highly degraded, because the Acids Res. 30 (2002) e27.
short amplicons spanning the single-base mutation poten- [7] M. Brion, Y chromosome SNP analysis using the single-base
tially allow positive amplification when STR markers, which extension: a hierarchical multiplex design, Methods Mol. Biol.
297 (2004) 229242.
are normally used in routine typing, fail. Therefore, their use
[8] A.C. Syvanen, Accessing genetic variation: genotyping single
in forensic work could also encompass cases involving nucleotide polymorphisms, Nat. Rev. Genet. 2 (2001) 930
decomposed bodies, aged human remains or old biological 942.
traces. [9] B. Sobrino, M. Brion, A. Carracedo, SNPs in forensic genetics:
In view of applications in the latter field, the simple and a review on SNP typing methodologies, Forensic Sci. Int. 154
efficient minisequencing technique, available in any forensic (2005) 181194.
laboratory, was set up as a genotyping method, and ampli- [10] P.A. Underhill, P. Shen, A.A. Lin, L. Jin, G. Passarino, W.H.
fication and sequencing procedures normally required for Yang, E. Kauffman, B. Bonne-Tamir, J. Bertranpetit, P. Fran-
forensic samples, where the template is often limited in calacci, M. Ibrahim, T. Jenkins, J.R. Kidd, S.Q. Mehdi, M.T.
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supported by a grant from the Ministero per lIstruzione, Roewer, S. Rootsi, D.C. Rubinsztein, J. Saillard, F.R. Santos,
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