J Forensic Sci, January 2014, Vol. 59, No.

1
doi: 10.1111/1556-4029.12259
PAPER Available online at: onlinelibrary.wiley.com

CRIMINALISTICS

Guangwei Hou,1 Ph.D.; Xianhua Jiang,1 M.D.; Yanyan Yang,2 M.D.; Fei Jia,1 M.D.; Qiang Li,3 M.D.;
Jinling Zhao,1 M.D.; Fei Guo,3 M.D.; and Limin Liu,3 M.D.

A 21-Locus Autosomal SNP Multiplex and its

Application in Forensic Science*

ABSTRACT: To develop a cost-effective technique for single-nucleotide polymorphism (SNP) genotyping and improve the efficiency to
analyze degraded DNA, we have established a novel multiplex system including 21-locus autosomal SNPs and amelogenin locus, which was
based on allele-specific amplification (ASA) and universal reporter primers (URP). The target amplicons for each of the 21 SNPs arranged from
63 base pair (bp) to 192 bp. The system was tested in 539 samples from three ethnic groups (Han, Mongolian, and Zhuang population) in
China, and the total power of discrimination (TPD) and cumulative probability of exclusion (CPE) were more than 0.99999999 and 0.98,
respectively. The system was further validated with forensic samples and full profiles could be achieved from degraded DNA and 63 case-type
samples. In summary, the multiplex system offers an effective technique for individual identification of forensic samples and is much more
efficient in the analysis of degraded DNA compared with standard STR typing.

KEYWORDS: forensic science, DNA typing, single-nucleotide polymorphism, multiplex amplification, allele-specific amplification/universal
reporter primers, degraded DNA, rs10003686, rs10430105, rs1022106, rs10005781, rs1000709, rs1024283, rs116187, rs1001389, rs1015570,
rs1022690, rs1003416, rs10444205, rs10437508, rs1014440, rs1007469, rs10519137, rs1018427, rs1010870, rs1004667, rs1004663, rs112603

In recent years, single-nucleotide polymorphism (SNP) has
become one of the hot spots in genetics and forensic science to its
features of short amplicons and low mutation rate. And SNP geno-
typing has been successfully applied to the field of forensic
science (1–8). There are many techniques for SNP genotyping,
such as TaqManâprobe, SNaPshot, denaturing high-performance
liquid chromatography (DHPLC), DNA microarray, and Matrix-
Assisted Laser Desorption/Ionization Time of Flight Mass
Spectrometry (MALDI-TOF MS) (9–13). However, these tech-
niques are either low-throughput, time-consuming, or expensive
and not applicable for high-throughput screening in general labo-
ratories. Thus, it is necessary to develop new techniques for SNP
genotyping.
In particular, degraded biological samples are often encoun-
tered in forensic field, and the degree of degradation can vary
significantly among different evidence samples. For moderately
degraded samples in which DNA fragments are shorter than 200
base pair (bp), it is a daunting challenge to analyze with the
widely used standard STR typing. For more severely degraded
1 This multiplex system was based on the principal of allele-spe-
Department of Forensic DNA, Criminal Science and Technology Institute
of Liaoning province, Shenyang 110032, China.
2
Department of Forensic Genetics, Wannan Medical College, Wuhu
241000, China.
3
College of Forensic Medicine, China Medical University, No.92 Beier
Road, Heping District, Shenyang 110001, China.
*Funded by a grant from the Human Resources Bureau of Lining Province
of China (LXZZ2007001).
Received 28 Nov. 2011; and in revised form 6 Aug. 2012; accepted
14 Oct. 2012.
samples, the size of residual nuclear DNA fragments may be
<100 bp, which is beyond the detection limit of routine STR
typing. It is tough to analyze these samples even with the ampli-
con-shortened STR typing (miniSTR) as it quite often leads to
partial or failed profiles (14,15). In such cases, sequencing of
mitochondrial DNA (mtDNA) is usually carried out instead.
However, even if the analysis itself is successful, the discrimina-
tion power is much lower compared with multiplex STR typing
due to restriction of the haplotypes of mtDNA. Thus, the method
is also not the first choice for degraded samples. In contrast, the
amplicons of most bi-allelic SNPs were much shorter than those
of STR typing. Therefore, SNP genotyping is likely to be more
advantageous for the analysis of degraded samples, and some
work has already been carried out to this end (16–18). For
example, 18 SNPs with the size of PCR products ranging from
40 bp to 67 bp were selected for SNP genotyping in the analysis
of degraded DNA (19). The results showed that the successful
rate of SNP genotyping was much higher than that of STR
typing for the analysis of artificially degraded DNA.
In this study, a cost-effective technique was developed with
high-level discrimination using SNP multiplex from 21 loci.
cific amplification and universal reporter primers (ASA/URP).
To test whether the multiplex system can be applied to forensic
science, we genotyped the 21 SNPs in three ethnic populations
in China. This multiplex system was then validated with sam-
ples containing low copy number samples, different tissues of
one individual and artificially degraded DNA. In addition, SNP
genotyping profiles were successfully obtained from routine
forensic cases.

© 2013 American Academy of Forensic Sciences 5

6 JOURNAL OF FORENSIC SCIENCES
Materials and Methods
Selection of SNP Loci
SNP loci in this study were selected from HapMap database
(http://www.hapmap.org). A total of 45 SNPs from the autoso-
mal chromosomes 1–22 were selected as candidate SNPs. The
criteria for SNP selection include the following: 1) far distance
(>2 Mb) between two SNP loci if on the same chromosome to
avoid linkage disequilibrium, 2) bi-allelic polymorphism, 3)
allele frequency ranges from 0.3 to 0.7. To ensure enough dis-
tance between two SNPs, only 2 loci were selected from each
autosomal chromosome with one from the end of the short arm
and the other from the end of the long arm. And the two loci
were selected to keep the distance in between as far as possible.
Amplicon Primers
The strategy of allele-specific amplification and universal repor-
ter primers (ASA/URP) with a mismatched base was combined to
build the SNP multiplex system. ASA takes the advantage of high
stringency in the polymerase chain reaction to make sure that only
one allele is amplified. To further increase the stringency of PCR
amplification, a mismatch was introduced into the 3′ terminus of
the forward primers as reported (16). The two ASA forward prim-
ers were designed to contain a 20 bp universal sequence 1 (Uni1)
or universal sequence 2 (Uni2) at the 5′ terminus. The reverse pri-
mer contained universal sequence 3 (Uni3). As described in
Fig. 1, the amplification included two phases (phase 1 and 2). In
phase 1, the allele-specific sequences in the two ASA forward
primers and one reverse primer (20 bp) were used to generate
small amount of two specific SNP amplicons ranging from 63 bp
to 192 bp. Next, the three universal sequences in the forward and
reverse primers would be used for the amplification to produce
large amount of the amplicons. In phase 2, the fluorescence-
labeled URP primers of U1, U2, and U3 with the same sequences
of Uni1, Uni2, and Uni3 were used, respectively. The amplicons
from phase1 were then labeled with fluorescence as U1 and U2
were tagged with HEX (green) or 6-FAM (blue) individually at
the 5′ terminus. All the primers for the amplicons of the 45 SNPs
were designed using Prime Primer5.0 software (PREMIER Bio-
soft International, Palo Alto, CA). Primers of five SNP loci were
excluded from the primer set because they did not pass the screen-
ing of Autodimer, Blast, or electronic PCR software. Primers for
the rest forty SNP loci were synthesized by Sangon Biotech Co.,
Ltd (China)(Table 1), and universal reporter primers were synthe-
sized by Takara Biotechnology Co., Ltd (China).(U1 primer:
HEX-5′-CGACGTGGATGTTGGGCTAT-3′; U2 primer: FAM-6-
5′-TGACTGCT GAGTGGCCAGAC-3′;U3 primer: 5′- CA-
AGGTGCTTGTGGC GCAAG-3′).
Sample Collection
A total of 539 DNA samples from bloodstains were analyzed in
the study. All bloodstains were from healthy and unrelated indi-
viduals. Among them, 203 were from Han population in Liaoning,
China; 152 were from Mongolian population in Inner Mongolia
Autonomous Region, and 184 were from Zhuang population in
Longan County, Guangxi Autonomous Region. In addition, DNA
samples extracted from different tissues of one single individual’s
corpse including heart, liver, spleen, lung, kidney, muscle, and
brain using phenol/chloride method were collected and frozen at
20°C before use. Case-type samples included 20 bloodstains, 10
muscle samples, 15 cigarette butts, eight mixed stains of vaginal
fluid/semen, and 10 cartilages of routine cases within 1 year. The
bloodstains, cigarette butts, and mixed stains of vaginal fluid/
semen were stored at room temperature. The muscle and cartilage
samples were frozen at 20°C. One probative evidence sample of
muscle tissue and a comparison sample from toothbrush of one
murder case were also collected for testing.
DNA Extraction and Quantification
DNA from population samples was extracted using the
organic method (20). DNA samples were diluted to 1 ng/lL for
typing low template DNA and 10 ng/lL for artificial degradation
analysis. DNA samples were frozen at 20°C and thawed prior
to use. For case-type samples, DNA from 8 mixed stains of
vaginal fluid/semen was extracted as described (21). DNA from
the rest 55 samples was extracted using Prepfilerâ Forensic
DNA Extraction Kit. For probative evidence sample, DNA from
the muscle tissue and cells left on the toothbrush in the murder
case was extracted using Prepfilerâ Forensic DNA Extraction
Kit. For DNA from different tissues of one single individual’s
corpse, two microliter of each DNA sample was diluted to
200 lL and 1 lL was used in each reaction.
SNP Multiplex Amplification
Each 25 lL multiplex amplification reaction contained oligo-
nucleotide primers with different concentrations (data not shown),
200 lM dNTP mixture, 1xPCR Buffer, 1.5 mM MgCl2 (Takara
Biotechnology Co. Ltd, Dalian, China), 4 units of AmpliTaq Gold
(Applied Biosystems, Foster City, CA), 0.4 lg/lL bovine serum
albumin (Takara Biotechnology Co., Ltd), and 1 ng DNA
template. Amplifications were carried out in 0.2 mL tubes on
GeneAmp PCR system 9700 (Applied Biosystems) with the fol-
lowing conditions: 95°C for 11 min, 6 cycles of 30 sec at 94°C,
15 sec at 60°C, 15 sec at 72°C, 15 sec at 60°C, 15 sec at 72°C,
15 sec at 60°C, 30 sec at 72°C, 29 cycles of 30 sec at 94°C,
105 sec at 76°C, 3 cycles of consist of 60 sec at 94°C, 30 sec at
60°C, 60 sec at 76°C followed by a 45 min extension at 60°C.
Separation of PCR Products Using Capillary Electrophoresis (CE)
One microliter of each PCR product and 10 lL mixture of
GeneScanTM Size Standards ROX-500 (Applied Biosystems):
HI-DITM Formamide (Applied Biosystems) (ratio 1:37) was
added to each well of a 96-well micro-liter plate. PCR products
were separated in POPTM-4 gel on an ABI 3130XL CE sequencer
controlled by Collection software version 1.1 (ABI) according to
the manufacturer’s protocol. Electrophoresis was run for 40 min
at 1.5 KV after 10 sec injection. PCR products with lower
amounts of starting DNA template (<0.5 ng) were injected for a
longer time period (15 sec) to increase peak heights.
Data Analysis
Electrophoresis maps were analyzed with GeneMaperTM3.1
software. The ROX 500 size standard was used to determine the
size (bp) of peaks detected. Genotypes of each SNP locus were
determined according to the numbers of fragments and colors of
fluorescent labels. Fluorescence balance of peak height from
SNP loci is calculated as the following: Peak height ratios
(PHR) of heterozygous alleles are ratios between the lower peak
height and the higher (smaller peak/higher peak 9 100%) at one
heterozygous locus.
 HOU ET AL. . 21 SNP MULTIPLEX APPLICATION 7

TABLE 1––Primer sequences and SNP information.

Forward Primer (5′-3′)

Allele1 Target Size Uni1 (CGACGTGGATGTTGGGCTAT) Reverse Primer (5′-3′)
Locus Chr. (green)/ Allele2 (blue) (bp) Uni2 (TGACTGCTGAGTGGCCAGAC) Uni3 (CAAGGTGCTTGTGGCGCAAG)
Amelogenin X/Y Amelo-X 57 Uni1- CCAGATGTTTCTCAAGTGGTCCTG Uni3- TGCTTAAACTGGGAAGCTGITGGT
Amelo-Y Uni2- AAAGTGGTTTCTCAAGTGGTCCCA
rs10003686 4q35 rs10003686-A 63 Uni1- CAGAGGAGTGCTTCCCtAA Uni3- ATTTGTTGGGTTTTGTCTGGAT
rs10003686-G Uni2- CAGAGGAGTGCTTCCCtAG
rs10430105 1p34 rs10430105-A 67 Uni1-TAGGTGGGAAAGCAGAACgCA Uni3-GAAGATAGAAGCAATCAGGGTAGA
rs10430105-G Uni2-TAGGTGGGAAAGCAGAACgCG
rs1022106 6q22 rs1022106-C 83 Uni1- TAACCCTCCTGTTTGTCAcCC Uni3- AAGTGTCAGTTTTATTGTGTTGCC
rs1022106-G Uni2- TAACCCTCCTGTTTGTCAcCG
rs10005781 4q23 rs10005781-T 91 Uni1- GCTTGATTAAGGCCAGaGT Uni3- GTGCAGGTGGGGTGGAGT
rs10005781-C Uni2- GCTTGATTAAGGCCAGaGC
rs1000709 9q32 rs1000709-G 95 Uni1- GAGTGTCCTTCTCTGTGtCG Uni3- TCTGACTCCCTAAGCACTACATC
rs1000709-A Uni2- GAGTGTCCTTCTCTGTGtCA
rs1024283 20q13.1 rs1024283-C 100 Uni1-GCACTGTCCTTAGCACTGAtAC Uni3-CCAAAGCACTTATTTCCACTTG
rs1024283-G Uni2-GCACTGTCCTTAGCACTGAtAG
rs116187 17q24 rs116187-G 102 Uni1-TGTTGGGAAGGCACTTTaAG Uni3-TCGGATGCTGGAATGGG
rs116187-A Uni2-TGTTGGGAAGGCACTTTaAA
rs1001389 9p24 rs1001389 -A 107 Uni1- GACCTCTAAGTGTTTTGGTGcCA Uni3- ACAATGCTATTTAAGTCCCTGG
rs1001389 -C Uni2- GACCTCTAAGTGTTTTGGTGcCC
rs1015570 7q34 rs1015570-C 116 Uni1- TTTTGAGCAGGGCAGAAaTC Uni3- ATCCCATGAGCAAGAAGCC
rs1015570-A Uni2- TTTTGAGCAGGGCAGAAaTA
rs1022690 6q23 rs1022690-A 119 Uni1- CCATCTTTGGAAACATCTAtCA Uni3- AAGCAACAGAAAATGGACTAAGC
rs1022690-G Uni2- CCATCTTTGGAAACATCTAtCG
rs1003416 9q34 rs1003416-G 124 Uni1- CATCATCAGTCCTAATCAtCG Uni3- CATACTTTCCATCACCCCAGA
rs1003416-A Uni2- CATCATCAGTCCTAATCAtCA
rs10444205 10p15 rs10444205 -T 127 Uni1-TCTACAGAGACACCACCTTCtTT Uni3-GTTGTACCCTAGTGGATCAGTCA
rs10444205 -C Uni2-TCTACAGAGACACCACCTTCtTC
rs10437508 10q23 rs10437508-A 130 Uni1-GAAGGATCACATTGGTGTcCCA Uni3-GCCAGTGTGTACTCAAAAGAATC
rs10437508-G Uni2-GAAGGATCACATTGGTGTcCCG
rs1014440 19q12 rs1014440-C 133 Uni1-TCTCTGGCACTTGGTGTAaGC Uni3-AGTGTCCATCCATGCCTCTT
rs1014440-T Uni2-TCTCTGGCACTTGGTGTAaGT
rs1007469 12q24.2 rs1007469-A 142 Uni1- CCTAGACACCACCCTGAGgAA Uni3-CACAACCTACGCCAGCC
rs1007469-G Uni2- CCTAGACACCACCCTGAGgAG
rs10519137 15q22 rs10519137-G 147 Uni1-ATGATCCCCAGATGACCAgAG Uni3- TGGAGAGTGGAGACTGTTTGC
rs10519137-C Uni2-ATGATCCCCAGATGACCAgAC
rs1018427 20p12 rs1018427-T 151 Uni1-TGTAAGCACCTGAATCACTaGT Uni3-CTCCTCCAGGTGCCAGATT
rs1018427-C Uni2-TGTAAGCACCTGAATCACTaGC
rs1010870 20p13 rs1010870-T 156 Uni1-GTGGCAACCCCAACACgGT Uni3-GATGCCCTTCAGCTCTGGA
rs1010870-C Uni2-GTGGCAACCCCAACACgGC
rs1004667 14q24 rs1004667-T 164 Uni1-TGAGATCCCACGCAGtAT Uni3-CACCCAAATCTATCATCCTCTG
rs1004667-C UniI2-TGAGATCCCACGCAGtAC
rs1004663 21q22 rs1004663-C 167 Uni1-CCACAATGGCTTACCGAcAC Uni3-ACTTGACACAGCATTCCAAGG
rs1004663-G Uni2-CCACAATGGCTTACCGAcAG
rs112603 22p13 rs112603-A 181 Uni1-ACACCACCCCTCGTATGcGA Uni3-GACCTGGGTGAGCTTTCTTC
rs112603-T Uni2-ACACCACCCCTCGTATGcGT
rs1004344 10q26 rs1004344-G 192 Uni1- AACCCTGGTAGTCTCACAcCG Uni3- CCCTT AAAAC AGCACCCTGA
rs1004344-T Uni2- AACCCTGGTAGTCTCACAcCT
rs10442625 1p36.3 rs10442625-G 62 Uni1- TGAACTATTTGGAAGTGGtAG Uni3- TGGAGGTGAGGTTCATTGG
rs10442625-A Uni2- TGAACTATTTGGAAGTGGtAA
rs11874900 18q21 rs11874900-A 71 Uni1- CTTCCCCTCAACTTCtGA Uni3- CCCACCAGGAAGCATGAC
rs11874900-G Uni2- CTTCCCCTCAACTTCtGG
rs1011947 18q21 rs1011947-C 80 Uni1- GAGCCCTTTCTTGTCtCC Uni3- CTGTGACACCGTGTG
rs1011947-T Uni2- GAGCCCTTTCTTGTCtCT
rs1001098 2q36 rs1001098-C 74 Uni1-TCGATTTGGATTTTGATtAC Uni3- ATCATATTCCTCCCCAGAAT
rs1001098-T Uni2-TCGATTTGGATTTTGATtAT
rs10519045 15q21 rs10519045-C 69 Uni1- CCTGGCAGAAGTCTGACAAGaAC Uni3- ATAGCAGAGG GAGCCATAAAAG
rs10519045-T Uni2- CCTGGCAGAAGTCTGACAAGaAT
rs1000361 3q24 rs1000361-T 75 Uni1-TACCCACTTATTTTTCGCCtAT Uni3-AGACAGGCAAGCACCAAGGT
rs1000361-C Uni2-TACCCACTTATTTTTCGCCtAC
rs1005988 3p25 rs1005988-T 79 Uni1- CTTCCCAATATGCTTCCcTT Uni3- TGCTGTTTCTTCATTTCACC
rs1005988-G Uni2- CTTCCCAATATGCTTCCcTG
rs10518820 15q15 rs10518820-T 78 Uni1- ATCTTAGGAAGTGAACAAGGaTT Uni3- ACATC CCAGG CACTGACG
rs10518820-G Uni2- ATCTTAGGAAGTGAACAAGGaTG
rs1012509 6q22 rs1012509-C 93 Uni1- CTCATAAACAAGCAACCtCC Uni3- GTATCAAAAATGGGAAGTAAGC
rs1012509-T Uni2- CTCATAAACAAGCAACCtCT
rs1018920 7q21 rs1018920-G 109 Uni1- GGAGGAAGAACTGGAGTtGG Uni3- CATCTTACTCCCTCCAACAACA
rs1018920-C Uni2- GGAGGAAGAACTGGAGTtGC
rs1043003 10p15 rs1043003-A 108 Uni1- GTTTGTAACACTCCCCTTtCA Uni3- ATTTC TTCTAAGGGCATCAC AG
rs1043003-G Uni2- GTTTGTAACACTCCCCTTtCG
rs1006892 21q22 rs1006892-T 134 Uni1- TTAAAGCACACCCTACTCCgGT Uni3- GAGGC TTGAA TTGTG TCCC
rs1006892-C Uni2- TTAAAGCACACCCTACTCCgGC
8 JOURNAL OF FORENSIC SCIENCES

TABLE 1—Continued.

Forward Primer (5′-3′)

Allele1 Target Size Uni1 (CGACGTGGATGTTGGGCTAT) Reverse Primer (5′-3′)
Locus Chr. (green)/ Allele2 (blue) (bp) Uni2 (TGACTGCTGAGTGGCCAGAC) Uni3 (CAAGGTGCTTGTGGCGCAAG)
rs10154012 20q11.2 rs10154012-T 136 Uni1- CACCTCCACCCTTCCAaAT Uni3- GTGGTAATTTGTTGCAGCGTC
rs10154012-C Uni2- CACCTCCACCCTTCCAaAC
rs10866572 5p15.3 rs10866572-A 135 Uni1- GATGGAAACGAGGAAGTTATcGA Uni3- GCCAGATGTGCTAGGTTACCA
rs10866572-G Uni2- GATGGAAACGAGGAAGTTATcGG
rs1004344 10q26 rs1004344-G 151 Uni1- AACCCTGGTAGTCTCACAcCG Uni3- CCCTT AAAAC AGCACCCTGA
rs1004344-T Uni2- AACCCTGGTAGTCTCACAcCT
rs10403489 19p13.3 rs10403489-G 157 Uni1- GCCCTCATGGAGTGTCaGG Uni3- GGGTTTCTCC ACGTT GGTC
rs10403489-A Uni2- GCCCTCATGGAGTGTCaGA
rs11875115 18q12 rs11875115-A 160 Uni1- TCTCTTTAGGACTTGCCTGaGA Uni3- ATCAAAGCTGGATTGGGG
rs11875115-G Uni2- TCTCTTTAGGACTTGCCTGaGG
rs10492650 13q34 rs10492650-A 164 Uni1-CAGACAATGCCACCTAATGcGA Uni3-TTCCTTGCTGCCAATATAAGTC
rs10492650-C Uni2- CAGACAATGCCACCTAATGcGC
rs11874093 18p11.3 rs11874093-T 177 Uni1- TGTTTATGCCTTATCCGTGcCT Uni3- AACCA GGAGGCAGAGACTACC
rs11874093-C Uni2- TGTTTATGCCTTATCCGTGcCC
Each forward primer contains universal sequence1 (uni1) or sequence2 (uni2), and each reverse primers contains universal sequence 3 (uni 3).
The bold letter indicates the SNP locus.

FIG. 1––Diagrammatical representation of the ASA/URP principle for SNP genotyping.

Statistics
The frequency of alleles and genotypes of 21 SNPs was calcu-
lated by direct counting. Departures from Hardy–Weinberg equi-
librium (HWE) were analyzed using chi-square method. Test of
linkage disequilibrium was carried out using SHEsis platform on
the internet to calculate the values of D’ and r2 (22). The forensic
parameters of 21 SNPs in each ethnic group including polymor-
phism information content (PIC), heterozygosity (H), power of
discrimination (PD), and probability of exclusion (PE) were calcu-
lated based on the genotyping results of the three populations (23).
Results and Discussion
Establishment of Amplification with 21 SNP Multiplex Using
Fluorescence-Labeled Universal Primers
Primer sets of forty autosomal SNP loci were screened using
single locus and multiplex amplification, and primers of nineteen
SNP loci were excluded. The rest primers of twenty-one SNP loci
were used in the multiplex system together with the amelogenin
locus. The SNP loci in the multiplex system were listed in Table 1.
The 21 autosomal SNP loci and the amelogenin locus were
amplified simultaneously in a single tube using the multiplex sys-
tem. After capillary electrophoresis of PCR products, each homo-
zygous locus showed a single peak with blue or green color and
heterozygous locus showed two peaks with the same size and two
colors. The capillary electrophoresis map from one sample was
shown in Fig. 2. The SNP genotypes from six samples were veri-
fied by direct sequencing of all loci. The consistency of genotypes
determined by the multiplex system established in this study with
direct sequencing results proved the reliability of this system.
Frequency Distributions of 21 SNPs in Three Populations of
China and Peak Height Ratios (PHR) of Heterozygous Alleles
A total of 539 bloodstains including 203 from Han, 152 from
Mongolian, and 184 from Zhuang populations in China were
 HOU ET AL. . 21 SNP MULTIPLEX APPLICATION 9

FIG. 2––Capillary electrophoresis map of 21 SNPs and amelogenin loci of one sample after PCR with fluorescence labeled multiplex.

TABLE 2––Allele frequencies of 21 SNPs loci from three Chinese populations.

Mongolian
Population Zhuang Population
Han Population(n = 203) (n = 152) (n = 184)
Allele1 (green)/ HWE Test HWE Test HWE Test
SNP Locus Allele2 (blue) Allele1 Allele2 P(df = 1) Allele1 Allele2 P(df = 1) Allele1 Allele2 P(df = 1)
rs10003686 A/G 0.5739 0.4261 0.974 0.6875 0.3125 0.757 0.7880 0.2120 0.324
rs10430105 A/G 0.3941 066059 0.890 0.3157 0.6843 0.748 0.3396 0.6604 0.168
rs1022106 C/G 0.4113 0.5887 0.500 0.4408 0.5592 0.864 0.2446 0.7554 0.684
rs10005781 T/C 0.5764 0.4236 0.905 0.5164 0.4836 0.252 0.4701 0.5299 0.625
rs1000709 G/A 0.3227 0.6773 0.574 0.3158 0.6842 0.964 0.3424 0.6576 0.637
rs1024283 C/G 0.5148 0.4852 0.816 0.5329 0.4671 0.708 0.4103 0.5897 0.774
rs116187 G/A 0.5172 0.4828 0.408 0.5132 0.4868 0.330 0.6766 0.3234 0.944
rs1001389 A/C 0.4039 0.5961 0.750 0.3783 0.6217 0.198 0.4946 0.5054 0.920
rs101570 C/A 0.5541 0.4459 0.296 0.6151 0.3849 0.233 0.6576 0.3424 0.634
rs1022690 A/G 0.4163 0.5837 0.413 0.3190 0.6810 0.561 0.3859 0.6141 0.411
rs1003416 G/A 0.5049 0.4951 0.624 0.5066 0.4934 0.746 0.5190 0.4810 0.647
rs10444205 T/C 0.3719 0.6281 0.536 0.3685 0.6315 0.574 0.3480 0.6520 0.222
rs1043508 A/G 0.3326 0.6674 0.679 0.3355 0.6645 0.445 0.2989 0.7011 0.364
rs1014440 C/T 0.4582 0.5418 0.500 0.4408 0.5592 0.415 0.3098 0.6902 0.416
rs1007469 A/G 0.4237 0.5763 0.203 0.4309 0.5691 0.686 0.4701 0.5299 0.487
rs10519137 G/C 0.6157 0.3843 0.383 0.6842 0.3158 0.699 0.6983 0.3016 0.546
rs1018427 T/C 0.6009 0.3991 0.498 0.5790 0.4210 0.760 0.4972 0.5028 0.305
rs1010870 T/C 0.7833 0.2167 0.520 0.4836 0.5164 0.621 0.5679 0.4321 0.318
rs1000667 T/C 0.3794 0.6206 0.245 0.4605 0.5395 0.467 0.4158 0.5842 0.396
rs1004663 C/G 0.4779 0.5221 0.456 0.4506 0.5494 0.481 0.4620 0.5380 0.505
rs112603 A/T 0.3768 0.6232 0.323 0.3586 0.6414 0.413 0.2908 0.7092 0.215

genotyped using the SNP multiplex. HWE test showed no signif-
icant deviation from expectation (p > 0.05) for all 21 SNPs
(Table 2).The genotypes of 21 SNPs from Zhuang population
were analyzed for linkage disequilibrium. All the r2 values were
<0.067, indicating that no significant association between alleles
at different loci.
Peak height ratios (PHR) of heterozygous alleles were ana-
lyzed from 61 of 203 bloodstains from Han population. At opti-
mal DNA template levels (0.5–1.0 ng), the values of PHR were
high in nearly all 21 SNPs with the lowest 52.82% observed for
rs1003416 (Table 3).
Forensic Parameters of 21 SNPs in Three Populations of China
The forensic parameters of the 21 SNPs from each ethnic
group were calculated (Tables 4–6). The results showed that the
selected 21 SNPs were highly polymorphic among Han in Lia-
oning, Mongolian in Inner Mongolia, and Zhuang in Guangxi.
The total power of discrimination (TPD) and cumulative proba-
bility of exclusion (CPE) of 21 SNPs from each ethnic group
were all more than 0.99999999 and 0.98, respectively.
Typing of Low Template DNA
To test whether the multiplex system can be used at low
amount of templates, amplifications were carried out with differ-
ent amount of DNA templates from one reference sample
(16 pg, 32 pg, 62 pg, 125 pg, 250 pg, 500 pg, and 1 ng). As
showed in Fig. 3, complete profiles could be achieved even
when the amount of DNA template was as low as 125 pg. How-
ever, allele dropouts were observed in some loci when the
amount of DNA template was <62 pg.
10 JOURNAL OF FORENSIC SCIENCES

TABLE 3––Peak height ratios of heterozygous alleles of 21 SNP loci TABLE 5––Forensic parameters of 21 SNPs loci from Mongolian population
collected from 61 samples in ABI 3130XL capillary electrophoresis with of Inner Mongolia autonomous region in China.
10 sec injection.
Loci H PIC Pm PD PE
Peak Height Ratios of Heterozygous Alleles*
SNP Locus 0.5–1.0 ng rs10003686 0.4325 0.3374 0.4209 0.5791 0.1687
rs10430105 0.4349 0.3387 0.4126 0.5874 0.1694
rs10003686 55.70  21.13 rs1022106 0.4963 0.3715 0.3820 0.6180 0.1857
rs10430105 56.93  21.14 rs10005781 0.5028 0.3747 0.4017 0.5983 0.1874
rs1022106 69.74  18.70 rs1000709 0.4351 0.3389 0.4165 0.5835 0.1693
rs10005781 63.65  20.64 rs1024283 0.5012 0.3741 0.3839 0.6161 0.1869
rs1000709 66.76  19.10 rs116187 0.4631 0.3542 0.3866 0.6134 0.1771
rs1024283 64.13  22.70 rs1001389 0.4735 0.3598 0.4150 0.5850 0.1799
rs116187 56.29  14.24 rs101570 0.4766 0.3614 0.3731 0.6269 0.1807
rs1001389 66.79  17.06 rs1022690 0.4375 0.3403 0.4085 0.5915 0.1700
rs101570 69.04  19.06 rs1003416 0.4837 0.3651 0.4012 0.5988 0.1825
rs1022690 64.32  20.10 rs10444205 0.5026 0.3748 0.3899 0.6101 0.1873
rs1003416 52.82  21.08 rs1043508 0.4668 0.3563 0.3869 0.6131 0.1781
rs10444205 63.07  21.08 rs1014440 0.4350 0.3388 0.4164 0.5836 0.1694
rs1043508 58.68  22.97 rs1007469 0.4938 0.3703 0.3878 0.6123 0.1851
rs1014440 73.89  19.67 rs10519137 0.4350 0.3388 0.4158 0.5842 0.1694
rs1007469 54.89  21.29 rs1018427 0.4909 0.3688 0.3874 0.6126 0.1843
rs10519137 63.02  21.40 rs1010870 0.5028 0.3747 0.3859 0.6141 0.1874
rs1018427 63.45  25.69 rs1000667 0.5002 0.3734 0.3923 0.6077 0.1867
rs1010870 65.96  20.04 rs1004663 0.4611 0.3532 0.3808 0.6192 0.1766
rs1000667 55.71  15.13 rs112603 0.3675 0.2985 0.4686 0.5314 0.1491
rs1004663 64.50  19.84 Total / / 3.3608E-09 0.999999996 0.984646
rs112603 64.30  24.01
H, PIC, Pm, PD and PE stand for heterozygosity, polymorphism informa-
*Peak height ratios of heterozygous alleles = smaller peak/higher tion content, probability of match, power of discrimination and probability of
peak 9100 (%). exclusion.

TABLE 4––Forensic parameters of 21 SNPs loci from Han population of TABLE 6––Forensic parameters of 21 SNPs loci from Zhuang population of
Liaoning in China. Guangxi province of China.

Loci H PIC Pm PD PE Loci H PIC Pm DP PE

rs10003686 0.4916 0.3696 0.3800 0.6200 0.1847 rs10003686 0.3360 0.2784 0.5002 0.4998 0.1391
s10430105 0.4800 0.3637 0.3850 0.6150 0.1817 rs10430105 0.4457 0.3403 0.4236 0.5764 0.1754
rs1022106 0.4867 0.3670 0.3945 0.6055 0.1835 rs1022106 0.3986 0.3377 0.4672 0.5328 0.1419
rs10005781 0.4907 0.3691 0.3750 0.6250 0.1845 rs10005781 0.5010 0.3743 0.3853 0.6147 0.1870
rs1000709 0.4356 0.3395 0.4233 0.5767 0.1698 rs1000709 0.4529 0.3491 0.3984 0.6016 0.1744
rs1024283 0.5021 0.3749 0.3792 0.6208 0.1874 rs1024283 0.4867 0.3670 0.3883 0.6117 0.1834
rs116187 0.5020 0.3749 0.3931 0.6069 0.1873 rs116187 0.4401 0.3420 0.4128 0.5872 0.1709
rs1001389 0.4839 0.3656 0.3898 0.6102 0.1828 rs1001389 0.5028 0.3751 0.3750 0.6250 0.1875
rs101570 0.4967 0.3722 0.3626 0.6374 0.1860 rs101570 0.4529 0.3491 0.3984 0.6016 0.1744
rs1022690 0.4884 0.3679 0.3707 0.6293 0.1839 rs1022690 0.4766 0.3618 0.3782 0.6218 0.1808
rs1003416 0.5025 0.3751 0.3840 0.6160 0.1875 rs1003416 0.5021 0.3748 0.3842 0.6158 0.1873
rs10444205 0.4696 0.3582 0.4019 0.5981 0.1790 rs10444205 0.4563 0.3508 0.3891 0.6109 0.1754
rs1043508 0.4462 0.3455 0.4208 0.5792 0.1727 rs1043508 0.4214 0.3313 0.4192 0.5808 0.1656
rs1014440 0.4991 0.3734 0.3661 0.6339 0.1866 rs1014440 0.4308 0.3375 0.4127 0.5873 0.1678
rs1007469 0.4909 0.3693 0.4042 0.5958 0.1845 rs1007469 0.5010 0.3743 0.3642 0.6358 0.1870
rs10519137 0.4756 0.3614 0.4030 0.5970 0.1806 rs10519137 0.4237 0.3327 0.4291 0.5709 0.1663
rs1018427 0.4821 0.3647 0.3967 0.6033 0.1823 rs1018427 0.5028 0.3751 0.3581 0.6419 0.1875
rs1010870 0.3412 0.2819 0.4944 0.5056 0.1409 rs1010870 0.4935 0.3705 0.3988 0.6012 0.1851
rs1000667 0.4732 0.3600 0.4105 0.5895 0.1800 rs1000667 0.4886 0.3679 0.3977 0.6023 0.1839
rs1004663 0.5016 0.3747 0.3635 0.6365 0.1872 rs1004663 0.4999 0.3737 0.3894 0.6106 0.1867
rs112603 0.4720 0.3594 0.3802 0.6198 0.1797 rs112603 0.4148 0.3275 0.4234 0.5766 0.1637
Total / / 3.08233E-09 0.999999997 0.984769502 Total / / 5.20649E-09 0.999999995 0.98236771
H, PIC, Pm, PD and PE stand for heterozygosity, polymorphism informa- H, PIC, Pm, PD and PE stand for heterozygosity, polymorphism informa-
tion content, probability of match, power of discrimination and probability of tion content, probability of match, power of discrimination and probability of
exclusion. exclusion.

Genotypes of 21 SNPs among different tissues from the same

Consistency Among Tissues
To determine whether the genotypes of 21 SNPs remain the
same from tissue to tissue of a single individual, DNA samples
extracted from heart, liver, spleen, lung, kidney, muscle, and brain
of one corpse were analyzed for the 21 SNPs using the multiplex
system. The samples were also analyzed for 15 STRs using
AmpFlSTRâ SinofilerTM PCR Amplification Kit according to the
manufacturer’s instructions. Full SNP and STR profiles were
obtained from DNA samples of seven different tissues, respectively.
donor were identical. Similarly, types of the 15 STRs were also
the same from different tissues. These results indicated that regard-
less of the tissue tested, each individual is characterized by a single
SNP genotype for all 21 SNP loci (data not shown).
Typing of Artificially Degraded DNA
To determine the detection threshold of degraded samples
with the multiplex system, DNA degradation experiment was
 HOU ET AL. . 21 SNP MULTIPLEX APPLICATION 11

FIG. 3––Electrophoresis profiles after amplification with different amount of DNA template. The amount of DNA template used in each amplification was
marked on each profile.

FIG. 4––Genotyping map of 21 SNPs after DNA templates treated with DNase I (A: Fresh DNA without DNase I treatment; B: 5 min treatment with
DNase I; C: 30 min treatment with DNase I).

conducted using DNase I treatment and the results were com-
pared with STR analysis. More specifically, 5 ng of DNA was
artificially degraded with 10 U of DNase I for 5 min and
30 min, respectively, and the degraded DNA samples were
analyzed using our SNP multiplex system with the same fresh
DNA as a reference (Fig. 4). As a comparison, these DNase
I-treated DNA samples were also amplified with AmpFlSTRâ
SinofilerTM PCR Kit for STRs (Fig. 5). With our method, after
degradation of 5 min, genotypes of all 21 SNPs were observed
with the intensity of fluorescent signals comparable with that of
the fresh sample (200RFU) (Fig. 4A,B). In contrast, the intensity
of fluorescent signals of most STR loci amplified with Amp-
FlSTRâ SinofilerTM PCR Kit was about 300RFU, which was
apparently much lower than that of the untreated sample
(2000RFU) (Fig. 5A,B). In addition, losing alleles at two loci
D12S391 and D13S317 was observed (Fig. 5B). After degrada-
tion of 30 min, genotypes of 17 of the 21 SNPs were observed
using our method, but there was complete locus dropout at
rs1018427, rs1010870, rs1004663, and rs112603 (Fig. 4C). The
intensity of fluorescent signals for 17 SNPs detected was only
moderately reduced compared with that of the fresh sample. In
sharp contrast, no typing results were detected using
AmpFlSTRâ SinofilerTM PCR Kit (Fig. 5C). These data indicated
that our method is superior to the AmpFlSTRâ SinofilerTM PCR
Kit in terms of successful detection rate, which was consistent
with the genotyping results of 18 SNPs in a previous study (19).
12 JOURNAL OF FORENSIC SCIENCES

FIG. 5––STR profiles after amplification of DNase I treated DNA templates using the AmpFlSTRâ SinofilerTM PCR Kit (A: Fresh DNA without DNase I treat-
ment; B: 5 min treatment with DNase I; C: 30 min treatment with DNase I).

FIG. 6––Comparison of genotyping maps of 21 SNPs after amplification on DNA from different sources of one case (A: DNA from Cigarette stub; B: DNA
from sperms of the mixed stain).

FIG. 7––STR profiles after amplification of DNA from one probative evidence sample of muscle tissue using the AmpFlSTRâ SinofilerTM PCR Kit..
FIG. 8––Genotyping map of the 21 SNP multiplex system of DNA from one probative evidence sample of muscle tissue.
Case-Type Samples
Individual identification test of the 21 SNPs was performed
using 63 biological samples from 25 routine cases within 1 year.
Full profiles of 21 SNPs were achieved from all samples. SNP
genotyping was consistent with the results of STR analysis using
AmpFlSTRâ SinofilerTM PCR Kit. SNP profiles of two samples
from one case were showed in Fig. 6. The genotypes of 21 SNPs
from the cigarette stub and sperms of the mixed stain were identi-
cal. These results indicate that the 21 SNPs multiplex system are
suitable for identification of biological samples in forensic cases.
Probative Evidence Samples
To further test whether the 21 SNPs system works for
degraded DNA, 1 postmortem muscle from a murder case was
analyzed. In more detail, on July 7, concerns were raised about
the disappearance of a 55-year-old man that had not been seen
for about a week in summer. The police officers searched the
man’s house that located in a small village near Shenyang city,
but there were not any valuable clues. Further search of the
whole village led to the finding of a black plastic bag containing
1 decayed muscle in the field about two miles away from the
old man’s house. After DNA extraction from the muscle, the
AmpFlSTRâ SinofilerTM Kit was first used to analyze the DNA
sample and only partial profiles of 15 STRs were detected, with
allele dropouts observed at D7S820, CSF1PO, and D18S51 due
to large amplicons (Fig. 7).To get more information from the
DNA sample, the 21 SNPs multiplex system was used and a full
profile of 21 SNPs was achieved from the DNA sample (Fig. 8).
HOU ET AL. . 21 SNP MULTIPLEX APPLICATION 13
Finally, the muscle was determined to be from the man’s body
as the same profile was generated on the DNA from the man’s
toothbrush. When the case was resolved and the suspect was
captured, he confessed that the victim was killed and mutilated,
and the body parts were discarded everywhere around the vil-
lage. This case further proved that the SNP genotyping was not
interfered by the degradation of large fragments and is more
likely to obtain whole profiles compared with STR typing.
In summary, a 21 SNP multiplex system was developed in the
study. This system provided a reliable technique for individual
identification of nondegraded and degraded DNA samples in the
forensic field.
Acknowledgment
We thank the staff at College of Forensic Science, China
Medical University for technical help.
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Additional information and reprint requests:
Limin Liu, M.D.
College of Forensic Medicine
China Medical University
No.92 Beier Road, Heping District
Shenyang, Liaoning 110001
China
E-mail: lmliu@mail.cmu.edu.cn