Typing of 67 SNP Loci on X Chromosome by PCR and MALDI-TOF MS

Introduction

Single nucleotide polymorphisms (SNP) typing on X chromosome could effectively complement the analysis of autosomal, Y-chromosomal or mitochondrial markers very efficiently. The markers may be easily amplified in samples with low-quality and degraded DNA.

In some deficiency kinship cases, e.g. relationship between paternal grandmother and granddaughter or relationship between half-sibling sisters who have the same father, X-chromosome markers are valuable and necessary, as reviewed by Pereira et al (2011). Additionally, for solving special father-daughter duos or mother-daughter-father trios, in which only 1~2 loci (out of 19~39) autosomal STRï¼ˆShort Tandem Repeatï¼‰loci were exclusionary, X-SNP loci with genetic stability may be used as supplementary markers to autosomal STR loci to further confirm or exclude paternity or maternity.

Therefore, we planned this study. 67 SNPs on X chromosome were amplified in four multiplex PCR systems followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS, mentioned by Storm N et al 2003 and Tost et al 2005) in Chinese Han population samples. Hardy-Weinberg equilibrium was analyzed and forensic genetic parameters were calculated.

Materials and Methods

Sample

295 blood samples were collected from unrelated Chinese Han individuals (215 males and 80 females) under informed consent. DNA was extracted from whole blood by using BLOOD DNA EXTRACTION kit according to manufacturer’s instructions (Sangon Biotech, Shanghai, China).

Primer Design

67 SNP markers (Table 1) on X chromosome with high minimal allele frequency (MAF>0.4) were selected from HapMap (www.hapmap.org) and NCBI SNP database (http://www.ncbi.nlm.nih.gov/SNP). The locus to locus space was about 3~20Mb. 67 groups of primers were designed via MassARRAY Assay Design software (Sequenom Inc.). Each group has three primes, including a pair of PCR primes and a single base extension prime (Table 2). Primers were synthesized by Sangon Biotech (Shanghai, China).

Table 1: Primers for Amplification and Extension Reactions of 67 X-SNPs

SNP_ID	Forward primer(5’→3’) for PCR	Reverse primer (5’→3’) for PCR	Amplicons (bp)	Primer for extension reactions	molecular weightï¼ˆDï¼‰
Reaction â… (17-plex)
rs203648	ACGTTGGATGTAGCTCCTCTGAGTCTTGTC	ACGTTGGATGTGAAAAGCATGCCCTCTGCG	95	CCACAGGCCCCCACAGC	5070.3
rs1166756	ACGTTGGATGGCTCAGGCAAGCACATGTTT	ACGTTGGATGGTTCTAGAGGGCAACATAGC	101	GTCATCGTCAATGCAGG	5210.4
rs611711	ACGTTGGATGGTTTTCTAGGAAAGAACAAG	ACGTTGGATGCCTCATCCTGAAAACATCGC	100	AAGAACAAGAGAACCAGT	5558.7
rs7876774	ACGTTGGATGAGAGGAGTCTGTCTGATCTG	ACGTTGGATGAGGTCATCAGGAAAGTCATC	98	ATGGGACGAGTGTGTCTA	5594.6
rs1544545	ACGTTGGATGTTATCCCCATTCAGACACAC	ACGTTGGATGGACTCTGTTAGGGTTGATTG	102	CCAAGTCTCCCACTATTTA	5682.7
rs5905043	ACGTTGGATGTTGAGCCTGGGATGTTAGAC	ACGTTGGATGTACTCAGCATACTTCTCCCC	96	TTCCCATTGAGAGAAACCT	5771.8
rs1229078	ACGTTGGATGCAAACCTGTAACACATGACC	ACGTTGGATGGTAAGATTGTTTAAATTTAGG	97	TGACCAAAGTAACCATTGAG	6134
rs5932595	ACGTTGGATGAAGCTTGCTTCTGCCCATTC	ACGTTGGATGTTTGCTGAGCATTATGGCCC	100	ATGATGGAACAGAAATCAGA	6207.1
rs17264553	ACGTTGGATGTCAAAGACAGCCAGTAAGCC	ACGTTGGATGTTGAGAATTGCAGCCATCAG	95	CAGCCAGTAAGCCTACCCAAC	6329.1
rs2070289	ACGTTGGATGATCCTCACGATGGCAGTAGT	ACGTTGGATGATACAGGAAGGGCGGGAGTA	98	GTAGTGCTCCAAATATGCTAAG	6758.4
rs1874111	ACGTTGGATGCCTTTCAGATTACTGAGTAG	ACGTTGGATGTGCTCCAGATAAGCCACTTG	100	GTACAAGAAATTAAGAGTGGAA	6864.5
rs5968332	ACGTTGGATGCCACCCACACATTATTAATGC	ACGTTGGATGGAGGCAGGTTTCCGATTTTG	100	CCACACATTATTAATGCTTATCA	6941.6
rs5931302	ACGTTGGATGTGCACATTCCAGGCAAGGTC	ACGTTGGATGAATAGGGACGGAAAAGTTGC	94	TCCAGGCAAGGTCCATGCTAAAAA	7354.8
rs4442270	ACGTTGGATGAAAGAAATGACCCAGTGGAG	ACGTTGGATGGTCTGTCATTTCGATTCAGG	82	AATGACCCAGTGGAGAATTATGAC	7409.8
rs5923750	ACGTTGGATGGGAAGAGAGAATTTGGGAAG	ACGTTGGATGTCTTTCAGGATTCAAAAGGC	99	TTTACTCTGCCATTTTCACTGACAT	7532.9
rs5917032	ACGTTGGATGTCACCACTGTGCTATCCTTC	ACGTTGGATGCCTCCATTAGGGCTTTTCCA	99	TAATGCATAACTGCAATCTAATCATG	7937.2
rs6639398	ACGTTGGATGGCTTATACCAGTCTCATTGC	ACGTTGGATGAATGTATCAGGACAGAGAGAC	84	ggaTCATTGCACTAATTGAAATATTG	8008.2
Reaction â…¡ (16-plex)
rs2209420	ACGTTGGATGCTCATTCATGTGGGAGCTTT	ACGTTGGATGACCCTTCCCAGCCTCTTGTA	99	TGATCTCGTGGAGGCAA	5250.4
rs5986750	ACGTTGGATGAGGATTTGCAGGCCCAATAG	ACGTTGGATGGAACTCTTCAGGAGCGTATC	97	AGACACTCCACAGGTGTA	5492.6
rs5989571	ACGTTGGATGGTACAGGATGTGGGAAAGTG	ACGTTGGATGGAGAACTGATGCTCCACATA	105	TGTGGGAAAGTGCGTATG	5634.7
rs7471388	ACGTTGGATGGTGTGTCTGCTTTTGCCTTC	ACGTTGGATGTCCTGTGGATTCTGAATGTG	89	TCATTACTGACTGAAGACC	5771.8
rs149860	ACGTTGGATGCCAGTGCCCTATTCTGCAGT	ACGTTGGATGAAAGGAGATGTAAGAGTGGG	97	CCTATTCTGCAGTGACTGAG	6108
rs5954988	ACGTTGGATGTTTCAAACCTTGCCCCCATC	ACGTTGGATGGGCAACAATAGACACTGATG	82	CCCATCCATTCTTTCCCACTC	6188
rs471205	ACGTTGGATGCTTCCACGAAGGACAAACTG	ACGTTGGATGGACTTGGTCTCAGACATCAG	100	TCCCTCTGCTGTTATAGCCTT	6314.1
rs2214174	ACGTTGGATGTAAATCCTCTGTGTTTTCC	ACGTTGGATGTCATGTCTGAAAGCTGTTGC	99	CTCTGTGTTTTCCAATGAAAAT	6699.4
rs2768595	ACGTTGGATGGAGGGATTGAATCTCTAGTC	ACGTTGGATGTGGTCTATATCCATCAAGTC	96	TTGAATCTCTAGTCTAAGATGA	6748.4
rs6639989	ACGTTGGATGGTTATGAGGCTTTGAAAAGG	ACGTTGGATGAGGACATCGATGATAATCTG	98	TGAGGCTTTGAAAAGGAAATCG	6847.5
rs6418251	ACGTTGGATGAGCTGGAAAATCATTTGGAG	ACGTTGGATGGAATGTTACAGTGAAAGAGC	88	TGGAGATTTACAGAGGGGATTAG	7207.7
rs9781645	ACGTTGGATGGTAAGGCTGAGCTTTAGAGG	ACGTTGGATGGAAAGTTTGTCCTTCATCAG	93	GGGCCACAGTCCATTCCAATAGCA	7306.8
rs2694717	ACGTTGGATGTGACTAACGAGTGCCTTAGC	ACGTTGGATGGGACACAGAACATCATTCTC	99	CTTAGCATAATGTGGAGATGATGA	7455.9
rs5912619	ACGTTGGATGTTAGTTCTGCTCTCATGTTG	ACGTTGGATGACAAGAACAAGCCAAACCC	85	CTCTCATGTTGATTATTACTTTTCTT	7858.1
rs1468160	ACGTTGGATGCTAAAATCTCCATGATTCAG	ACGTTGGATGGCAATCTACAAGTTTTGGTC	96	ATATTATTGTTATGATCTCCTTTTTA	7906.2
rs17280621	ACGTTGGATGTGAGCAGTCAGATGGAGTTG	ACGTTGGATGAGAAGGGACCTGCTATAGTG	89	GAGCAGTCAGATGGAGTTGAGATTCC	8075.3
Reaction â…¢ (17-plex)
rs7060326	ACGTTGGATGGACTGCCATGTCTTATTTCC	ACGTTGGATGTAAATCAGTGCCTTCCCTGC	90	TTCCTTGGCACCTCACA	5081.3
rs2808742	ACGTTGGATGGGGAAGTAATGGGTCATAAG	ACGTTGGATGCCCTCTTATAGTCTTAAGTC	102	AATGGAGCACTGGTAGT	5274.4
rs5920670	ACGTTGGATGTGAAGAATTCTTGGGTCTGC	ACGTTGGATGAGGAAATAAGAGTACGCTCC	89	CTCCCAGCCAGGTCAGCC	5405.5
rs4826623	ACGTTGGATGCAGCTTCTACTTCACTAGCC	ACGTTGGATGGAAAAACAGCTCATTTTGG	92	GAAAGTCCTGAGTGCTCTA	5827.8
rs594031	ACGTTGGATGTCTTCTCAAGGTCTCAGAGC	ACGTTGGATGACATGGGCTTAGATATGAGG	94	AGAGCTAATAAGTGACTGC	5860.8
rs6631828	ACGTTGGATGGTTGAAATGGTAGTGTTCAG	ACGTTGGATGTTTCAGACCATGCATCTAGG	90	AGTGTTCAGTTACAAGAAACC	6438.2
rs6646036	ACGTTGGATGCATAAATCAGCTTCCATATC	ACGTTGGATGGATGTGAGCTAAGGGTAAGG	95	CTTCCATATCTTGATCACTAAA	6628.3
rs3924423	ACGTTGGATGGTGAGTTTTTAGTGGCTCCC	ACGTTGGATGCCTTCCATCTTCTCTAGGTC	87	CTCCCGACAGAGCAATTTATGG	6719.4
rs6611148	ACGTTGGATGGGATCTATATACTAAAAACC	ACGTTGGATGCTATCTCCTTTGATTTCTTTC	98	TATACTAAAAACCAGAAAACAGA	7050.7
rs6633608	ACGTTGGATGCAGCTAGTTCACCAAGTTGC	ACGTTGGATGAAATTTCACATCCTGGTGAG	96	TTGCATTCTTTCTATTCATTCCTT	7200.7
rs5964206	ACGTTGGATGTGGCAACAAGATTTCCTGTG	ACGTTGGATGAAAGAGCAGCAGTGCTGTAG	99	TGTGTAATTTTTTTTCAAATTGGA	7377.8
rs12849634	ACGTTGGATGTGAGACCTCAGGATGACAGT	ACGTTGGATGACAGCCTCATACTGCAAGTC	98	TGACAGTGAATACAACTGAAATGT	7408.9
rs973212	ACGTTGGATGTATTCTCTCTTAGGCACTGG	ACGTTGGATGCTTAGTCACATCAAAGGAGC	95	TCTCTCTTAGGCACTGGTATACAAC	7591.9
rs6620798	ACGTTGGATGGCCAGCATGAGAGACAGAGA	ACGTTGGATGCATGATAGAGTTCTCATAAG	99	CCAGCATGAGAGACAGAGAGTGATG	7789.1
rs5916781	ACGTTGGATGGAAAGAAAGGCATATTTCCTG	ACGTTGGATGACTTGAAGCTATGAACTGTG	93	ttcCCTGTTTTTATTTACACAAATGA	7885.2
rs12840669	ACGTTGGATGCTGTACACTTAAATTGGCAC	ACGTTGGATGCAAATGAACTTTATTGGAG	81	gACTTAAATTGGCACATTTTATTATA	7958.2
rs4276834	ACGTTGGATGCATTCAAAATGCTAACAGAGG	ACGTTGGATGCCACCCAAGGACAGCTATTC	97	ATGCTAACAGAGGAAAATATTTCTTA	8001.2
Reaction â…£ (17-plex)
rs5933388	ACGTTGGATGATCACCCCAAGAAGAAACCC	ACGTTGGATGGGAGGGGTTGGAAAAATATG	83	GAAGAAACCCAGGACCC	5182.4
rs6418330	ACGTTGGATGGTTTCATGCACTCTTTCCAC	ACGTTGGATGATGGGATCACTGAATACTTC	95	ACTCTTTCCACTAGCATC	5369.5
rs5927322	ACGTTGGATGTCAAAATATATCCTGGCAGC	ACGTTGGATGCTCTGGGAATGACCTAAATC	97	CCTGGCAGCAAAGATATA	5516.6
rs10855633	ACGTTGGATGTCTTTTAGATACATGGCCAG	ACGTTGGATGGCAATTTGGAGATTTCTCA	100	ATGGCCAGTAATAGGATC	5547.6
rs5986751	ACGTTGGATGATCTGCCAAGACTTCACTGC	ACGTTGGATGCAGAAAGCCTGTTGTGATCC	100	TTTTGCTTCCCTCACAGTG	5720.7
rs2519557	ACGTTGGATGTGACAACTATAAGACCCATC	ACGTTGGATGTGGCTGATTTCGCTGACTTG	99	GACCCATCTATTAGAGAGCC	6086
rs6527549	ACGTTGGATGGAGAAATACAGGTGTTTCGTG	ACGTTGGATGCCTGCCTAACTGTCTGTAAA	100	ggGGTGTTTCGTGTGTTGAC	6226
rs6649211	ACGTTGGATGGAGTTAAAAATTCTCCCATCC	ACGTTGGATGCTGTGACAGTTGGTAAGGTG	84	ttgATTCTCCCATCCACAATA	6300.1
rs5928614	ACGTTGGATGTTTGTGTCGTCTCTGTTGTG	ACGTTGGATGTGAAGGAGAAAATGTAATG	96	TTGTGTAAACTCTTTAGACAA	6419.2
rs183277	ACGTTGGATGGAAGGAACGTCAGTGGTCAG	ACGTTGGATGCTGCCACCTATATGATCCAG	102	AACGTCAGTGGTCAGCTTTGT	6452.2
rs7880460	ACGTTGGATGCCACATCCTTGTCAACACAC	ACGTTGGATGAGTTCATAGCCACTTGGTTG	83	TCAACACACATTATTATCTTTTT	6922.5
rs5955927	ACGTTGGATGGCACAGCGCCTGATATAAAG	ACGTTGGATGGCTTAAATATGTATCTCTTCC	99	AGTAGTTTCTCAATAGCATTTTG	7043.6
rs5908324	ACGTTGGATGTTAATGGGCTGAAACAGAAC	ACGTTGGATGTTTTAAGCCTCTTTTTAAAC	98	GAAACAGAACAAATTTTATTTCAC	7327.8
rs5906919	ACGTTGGATGGCTCTACTCACGTGCCTTTT	ACGTTGGATGCATTTCGGTTAGAGTTTAGTC	97	CCTTTTATAAAGTTTAGGTCTGAAC	7646
rs5980274	ACGTTGGATGGTGAATCTGAATGTTCTGC	ACGTTGGATGTGGTATTGCTCGTACATGG	100	CTTTATGTATCAATGTTCCTATCATA	7885.2
rs5926442	ACGTTGGATGGACTTTCTTGCTAAAAAGTT	ACGTTGGATGCTGTAAAAACAAAATTTCTC	100	cacCTTTCTTGCTAAAAAGTTTATCAC	8168.3
rs5956616	ACGTTGGATGCTCAAATTCTATTTGTAAGC	ACGTTGGATGTGGATTTTAGGCTTCCTGAC	82	ccCTATTTGTAAGCATATACTCAATAC	8177.3

SNP Typing

SNP genotyping was performed using the MassARRAY MALDI-TOF MS platform (Sequenom Inc.). Briefly, three 17-plex and one 16-plex amplification reactions were processed following standard protocols for iPLEX chemistry. The reaction products were used as templates for the primer extension reactions. All reactions were performed in 384 microtiter plates (Sequenom, Inc.). PCR amplification and primer extension reaction were carried out on a GeneAmp PCR System 9700 (Applied Biosystems, Norwalk, CT), and no-template controls were carried along in every plate to exclude contaminations. A panel of genomic DNA samples genotyped by the assay was sequenced simultaneously for quality control.

Multiplex PCR

SNP genotyping was performed using the MassARRAY MALDI-TOF MS platform (Sequenom Inc.). PCRs (final volume, 5 Î¼L) contained 1Î¼L the desired primers at their optimized concentrations, 0.625Î¼L PCR buffer(10×) (Qiagen GmbH), 0.325Î¼L 25 mmol/L MgCl2, 1Î¼L dNTP (2.5mmol/L) (Tatara Inc.), 0.1Î¼L of HotStarTaq polymerase (5U/Î¼L), 1Î¼L of DNA , and 0.95Î¼L H2O. A tag (5’-ACGTTGGATG) was included in the primer sequence. PCR conditions were 94 °C for 15 min followed by 45 cycles at 94 °C for 20 s, 56 °C for 30 s, and 72 °C for 1 min; and finally 72 °C for 3 min.

SAP Dephosphorylation

After amplification, the products of four reactions (i.e. 17-plex, 16-plex, 17-plex and 17-plex) were treated with shrimp alkaline phosphatase (SAP) to remove excess dNTPs. This reaction contained 0.17Î¼L SAP buffer (10×), 0.3Î¼L SAP (1U/Î¼L), 1.53Î¼L H2O (all from Sequenom Inc.). The reaction conditions were 37 °C for 40 min, followed by 85 °C for 15 min.

Primer Extension Reactions

The PCR products were then used as templates for the primer extension reactions. This reaction (final volume was 9Î¼L) contained 0.94Î¼L extension primers at optimized concentrations,0.1Î¼L iPlex termination mix, 0.2Î¼L iPlex buffer (10×), 0.0205Î¼L iPlex enzyme and 0.7395Î¼L H2O (all from Sequenom Inc). Extension reactions were performed at 94 °C for 30 s followed by 40 cycles at 94 °C for 5 s and 5 cycles of 52 °C for 5 s, 80 °C for 5 s; and finally 72 °C for 3 min. The extension products were treated with a cationic exchange resin (AG® 50W-X8 Resin; Bio-Rad Laboratories, Inc.) for 30 min to remove salts.

MALDI-TOF MS

The products were spotted onto the MassARRAY SpectroCHIP with an auto-spot arm (Sequenom, Inc.) and then the target plate was inserted into the MALDI-TOF mass spectrometer of MassARRAY compact System (Sequenom, Inc.). The mass range of the MS instrument was set at 3920—12023 Da. SNP loci was genotyped by MassArray Typer Analyzer software version 4.0 (Sequenom, Inc.).

Statistical Analysis

Tests of Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium were performed using the Power marker v3.25 program. The formula for calculation of expected heterozygosity (H) and polymorphism information contents (PIC) were introduced by Nei and Roychoudhury (1974) The formula for calculation of discrimination power and exclusion probability were published by Szibor et al (2003).

Results and Discussion

Validation

The procedure to type SNPs used in this study was convenient with multiplexes where 67 X-SNP markers were amplified in four reactions. Validating carried out on control DNA 9947A and DNA extracts prepared from blood samples with known X-SNP profiles from MALDI-TOF MS assay by sequencing revealed fully consistent and reproducible results.

The MALDI-TOF MS technique requires the initial concentration of DNA is not less than 50ng/µL. If the amount of DNA is limited, it may be necessary to use other platform such as SNaPshot (Bouakaze et al 2007 and Wei et al 2012) or to perform a whole genome amplification of the sample previously.

Testing of Linkage Disequilibrium and Calculation for ‘Forensic Statistics’

Population data of 67 X-SNPs were obtained. The exact test of 67 X-SNPs showed no deviation from Hardy-Weinberg equilibrium (p>0.05) except for rs12849634 (p=0.0007).

Two out of the other 66 X-SNP loci (i.e.rs1229078, rs1544545) were found to be low informative (MAF<0.3).Tight LD were observed at six groups ï¼ˆrs5986750- rs5986751, rs183277 – rs7060326, rs6611148 – rs4826623, rs5923750 – rs1166756, rs6620798 – rs5917032, rs2519557 – rs6649211ï¼‰and slight LD were found at two groups (rs4276834 – rs5928614ï¼›rs5968332 – rs6418330). 52 X-SNP loci showing independent inheritance and high polymorphisms were finally selected out (Table 2). Based on the population data (Table 2), the combined values of Discrimination Power in female population and male populations are as follows: CDPf=0.9999999999999999999 and CDPm=0.99999999999999931. The combined values of Exclusion Probabilities in trio cases and duo case were are as follows: CPEtrios=0.99999999996 and CPEduos=0.9999995. The results revealed that the SNP panel on X chromosome yield the equivalent power in forensic identification as 15 STR markers currently used.

Table 2: Forensic efficiency of 52 X-SNPs

No	X-SNP	allele	frequency	H	PIC	DP_female	DP_male	PE_trios	PE_duos
1	rs6639398	A/G	0.50/0.50	0.5017	0.3750	0.6250	0.5000	0.3750	0.2500
2	rs6631828	C/T	0.50/0.50	0.5017	0.3750	0.6250	0.5000	0.3750	0.2500
3	rs5917032	C/G	0.50/0.50	0.5017	0.3750	0.6250	0.5000	0.3750	0.2500
4	rs5912619	A/G	0.51/0.49	0.5016	0.3750	0.6250	0.4999	0.3750	0.2500
5	rs2070289	A/G	0.49/0.51	0.5016	0.3750	0.6250	0.4999	0.3750	0.2500
6	rs3924423	C/T	0.51/0.49	0.5015	0.3749	0.6249	0.4998	0.3749	0.2499
7	rs9781645	C/T	0.51/0.49	0.5014	0.3748	0.6248	0.4997	0.3748	0.2498
8	rs183277	G/A	0.51/0.49	0.5013	0.3748	0.6248	0.4996	0.3748	0.2498
9	rs2209420	A/C	0.51/0.49	0.5013	0.3748	0.6248	0.4996	0.3748	0.2498
10	rs5933388	C/T	0.51/0.49	0.5013	0.3748	0.6248	0.4996	0.3748	0.2498
11	rs5908324	A/G	0.48/0.52	0.5010	0.3746	0.6246	0.4993	0.3746	0.2496
12	rs5980274	C/T	0.52/0.48	0.5004	0.3744	0.6244	0.4987	0.3744	0.2494
13	rs6639989	G/T	0.53/0.47	0.4998	0.3741	0.6240	0.4981	0.3741	0.2490
14	rs5954988	C/T	0.54/0.46	0.4990	0.3737	0.6237	0.4973	0.3737	0.2487
15	rs1874111	A/T	0.46/0.54	0.4989	0.3734	0.6236	0.4972	0.3734	0.2486
16	rs2808742	A/G	0.54/0.464	0.4987	0.3735	0.6235	0.4970	0.3735	0.2485
17	rs5931302	C/G	0.54/0.46	0.4983	0.3733	0.6233	0.4966	0.3733	0.2483
18	rs6611148	G/T	0.46/0.54	0.4982	0.3731	0.6231	0.4963	0.3731	0.2481
19	rs5986750	A/G	0.55/0.45	0.4973	0.3728	0.6228	0.4956	0.3728	0.2478
20	rs5964206	C/G	0.45/0.55	0.4957	0.3718	0.6220	0.4940	0.3718	0.2470
21	rs6646036	C/T	0.45/0.55	0.4957	0.3718	0.6220	0.4940	0.3718	0.2470
22	rs7876774	C/T	0.56/0.44	0.4950	0.3717	0.6216	0.4934	0.3717	0.2467
23	rs2694717	A/C	0.44/0.56	0.4945	0.3714	0.6213	0.4928	0.3714	0.2464
24	rs5920670	A/G	0.44/0.56	0.4945	0.3714	0.6213	0.4928	0.3714	0.2464
25	rs594031	C/T	0.44/0.56	0.4938	0.3710	0.6210	0.4921	0.3710	0.2461
26	rs5906919	A/G	0.57/0.43	0.4931	0.3708	0.6206	0.4915	0.3708	0.2457
27	rs471205	A/G	0.43/0.57	0.4931	0.3708	0.6206	0.4915	0.3708	0.2457
28	rs7471388	C/G	0.43/0.57	0.4931	0.3707	0.6206	0.4915	0.3707	0.2457
29	rs5928614	A/G	0.57/0.43	0.4926	0.3702	0.6203	0.4909	0.3702	0.2454
30	rs1166756	C/G	0.43/0.57	0.4924	0.3703	0.6202	0.4908	0.3703	0.2454
31	rs5926442	C/T	0.43/0.57	0.4907	0.3694	0.6193	0.4889	0.3694	0.2445
32	rs2214174	A/G	0.42/0.58	0.4902	0.3692	0.6191	0.4885	0.3692	0.2443
33	rs6418330	A/G	0.42/0.58	0.4901	0.3692	0.6190	0.4884	0.3692	0.2442
34	rs149860	C/G	0.58/0.42	0.4901	0.3692	0.6190	0.4884	0.3692	0.2442
35	rs5905043	C/T	0.42/0.58	0.4901	0.3693	0.6190	0.4884	0.3693	0.2442
36	rs6649211	A/C	0.42/0.57	0.4900	0.3691	0.6190	0.4883	0.3691	0.2442
37	rs5989571	A/T	0.58/0.42	0.4887	0.3682	0.6182	0.4870	0.3682	0.2435
38	rs17280621	A/G	0.58/0.42	0.4887	0.3684	0.6182	0.4870	0.3684	0.2435
39	rs5956616	A/G	0.42/0.58	0.4875	0.3678	0.6176	0.4859	0.3678	0.2429
40	rs203648	A/G	0.58/0.42	0.4875	0.3678	0.6176	0.4859	0.3678	0.2429
41	rs2768595	A/T	0.58/0.42	0.4874	0.3678	0.6176	0.4857	0.3678	0.2429
42	rs6633608	A/T	0.40/0.60	0.4837	0.3661	0.6156	0.4821	0.3661	0.2410
43	rs1468160	A/G	0.60/0.40	0.4829	0.3655	0.6151	0.4813	0.3655	0.2406
44	rs5955927	C/T	0.60/0.40	0.4827	0.3653	0.6150	0.4811	0.3653	0.2405
45	rs7880460	G/T	0.61/0.39	0.4792	0.3638	0.6130	0.4776	0.3638	0.2388
46	rs12840669	C/T	0.62/0.38	0.4752	0.3614	0.6107	0.4736	0.3614	0.2368
47	rs6418251	A/C	0.62/0.38	0.4734	0.3605	0.6097	0.4718	0.3605	0.2359
48	rs973212	C/T	0.62/0.38	0.4718	0.3597	0.6088	0.4702	0.3597	0.2351
49	rs5916781	A/G	0.62/0.38	0.4710	0.3592	0.6083	0.4694	0.3592	0.2347
50	rs5927322	A/G	0.64/0.36	0.4624	0.3549	0.6031	0.4608	0.3549	0.2304
51	rs5932595	G/T	0.34/0.66	0.4512	0.3486	0.5960	0.4496	0.3486	0.2248
52	rs17264553	G/T	0.34/0.66	0.4501	0.3480	0.5953	0.4486	0.3480	0.2243

Comparison of Population Genetic Data

Of the 67 X-SNP loci, 10 loci (i.e. rs1229078, rs1544545, rs4442270, rs1874111, rs5968332, rs1166756, rs12849634, rs5932595, rs203648 and rs611711) has been reported the power of discrimination and power of exclusion among Spanish population from coastal area and Pas valley by Zarrabeitia (2007). Analysis of pairwise genetic data between the Spanish population and the Chinese Han groups revealed strong similarities at seven X-SNP loci (i.e. rs4442270, rs1874111, rs5968332, rs1166756, rs5932595, rs203648 and rs611711), but highly significant differences at three X-SNP loci (i.e. rs1229078, rs1544545 and rs12849634). For example, the discrimination power of rs12849634 in Chinese Han female population and male populations were 0.0616 and 0.0316 respectively, but the values were as high as 0.6 and 0.5 respectively in the Spanish population. As for the other two loci, rs1229078 and rs1544545, their polymorphisms in the Spanish population were also higher than that in Chinese Han population.Therefore, it is necessary to investigate the polymorphisms in particular race in order to apply the X-SNP markers to the race.

The results in this study showed that the panel consisting of 52 X-SNP markers was high informative in Chinese Han population. It gives reasonable discrimination and paternity exclusion power that is sufficient for forensic identification.

Acknowledgements

This work was supported by grants from Shanghai Natural Science Foundation and National Institute Scientific Program (Nos.08ZR1419800 and GY0805).

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Typing of 67 SNP Loci on X Chromosome by PCR and MALDI-TOF MS

Research in Genetics

Li Li, Yan Liu and Yuan Lin

Shanghai Key Laboratory of Forensic Medicine, Institute of Forensic Science, Ministry of Justice, China, Shanghai, China

Academic Editor: Jian Tie

Cite this Article as:
Li Li, Yan Liu and Yuan Lin (2015), "Typing of 67 SNP Loci on X Chromosome by PCR and MALDI-TOF MS," Research in Genetics, Vol. 2015 (2015), Article ID 374688, DOI: 10.5171/2015.374688

Copyright © 2015. Li Li, Yan Liu and Yuan Lin. Distributed under Creative Commons CC-BY 4.0

Abstract

Typing of 67 SNP Loci on X Chromosome by PCR and MALDI-TOF MS

Li Li, Yan Liu and Yuan Lin

Shanghai Key Laboratory of Forensic Medicine, Institute of Forensic Science, Ministry of Justice, China, Shanghai, China

Academic Editor: Jian Tie

Cite this Article as: Li Li, Yan Liu and Yuan Lin (2015), "Typing of 67 SNP Loci on X Chromosome by PCR and MALDI-TOF MS," Research in Genetics, Vol. 2015 (2015), Article ID 374688, DOI: 10.5171/2015.374688

Copyright © 2015. Li Li, Yan Liu and Yuan Lin. Distributed under Creative Commons CC-BY 4.0

Abstract

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Cite this Article as:
Li Li, Yan Liu and Yuan Lin (2015), "Typing of 67 SNP Loci on X Chromosome by PCR and MALDI-TOF MS," Research in Genetics, Vol. 2015 (2015), Article ID 374688, DOI: 10.5171/2015.374688