Application of SNaPshot Technology in Semen-Specific cSNP Genetic Marker

doi:10.12116/j.issn.1004-5619.2021.510804

Abstract

Abstract:

Objective To explore the feasibility of genetic marker detection of semen-specific coding region single nucleotide polymorphism （cSNP） based on SNaPshot technology in semen stains and mixed body fluid identification. Methods Genomic DNA （gDNA） and total RNA were extracted from 16 semen stains and 11 mixtures composed of semen and venous blood, and the total RNA was reverse transcribed into complementary DNA （cDNA）. The cSNP genetic markers were screened on the validated semen-specific mRNA coding genes. The cSNP multiplex detection system based on SNaPshot technology was established, and samples were genotyped by capillary electrophoresis （CE）. Results A multiplex detection system containing 5 semen-specific cSNPs was successfully established. In 16 semen samples, except the cSNP located in the TGM4 gene showed allele loss in cDNA detection results, the gDNA and cDNA typing results of other cSNPs were highly consistent. When detecting semen-venous blood mixtures, the results of cSNP typing detected were consistent with the genotype of semen donor and were not interfered by the genotype of venous blood donor. Conclusion The method of semen-specific cSNPs detection by SNaPshot technology method can be applied to the genotyping of semen （stains） and provide information for determining the origin of semen in mixed body fluids （stains）.

Key words: forensic genetics, body fluids identification, mixture analysis, coding region single nucleotide polymorphism, SNaPshot method, semen

CLC Number:

Rui-yang TAO, Shou-yu WANG, Chun-yan YUAN, Ruo-cheng XIA, Cheng-tao LI. Application of SNaPshot Technology in Semen-Specific cSNP Genetic Marker[J]. Journal of Forensic Medicine, 2023, 39(5): 465-470.

Figures/Tables 4

References 20

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混合斑样本编号	精液		静脉血		V_精液∶V_静脉血
混合斑样本编号	供体	体积/μL	供体	体积/μL	V_精液∶V_静脉血
A1	M01	25	M06	75	1∶3
A2	M01	10	M06	90	1∶9
A3	M01	2	M06	98	1∶49
B1	M03	10	M01	90	1∶9
B2	M03	5	M01	95	1∶19
B3	M03	2	M01	98	1∶49
C1	M06	50	M03	50	1∶1
C2	M06	25	M03	75	1∶3
C3	M06	10	M03	90	1∶9
C4	M06	5	M03	95	1∶19
C5	M06	2	M03	98	1∶49

混合斑样本编号	精液		静脉血		V_精液∶V_静脉血
混合斑样本编号	供体	体积/μL	供体	体积/μL	V_精液∶V_静脉血
A1	M01	25	M06	75	1∶3
A2	M01	10	M06	90	1∶9
A3	M01	2	M06	98	1∶49
B1	M03	10	M01	90	1∶9
B2	M03	5	M01	95	1∶19
B3	M03	2	M01	98	1∶49
C1	M06	50	M03	50	1∶1
C2	M06	25	M03	75	1∶3
C3	M06	10	M03	90	1∶9
C4	M06	5	M03	95	1∶19
C5	M06	2	M03	98	1∶49

cSNP	cDNA		gDNA
cSNP	扩增子长度/ bp	引物序列（5'→3'）	扩增子长度/bp	引物序列（5'→3'）
rs9876921	156	F：TCATCTGGTTGGTGAAGATGGTG R：CAGAGGAGCCTTCTGGATACTTG	217	F：TCATCTGGTTGGTGAAGATGGTG R：CAATCTTGACTATTTTCATGTTCTGGG
rs1995640	199	F：GTGTGCTATGATGAGCTTTGAGAA R：CAACGCTCTCAGCACTGTAGTC	150	F：CGTGCTCATTGGGAATTGGAC R：GTAGTCAGGATCCCAGCAAACA
rs1058274	253	F：TGCTCGGGTGATTCTGGGG R：ACAAAGGTCAGTAGAACTGGGGA	224	F：CTGTAATGGTGTGCTTCAAGGTATC R：ACAAAGGTCAGTAGAACTGGGGA
rs2301366	247	F：GGGACAAAAAGGTGGATCAAAAGG R：TTGACTTCCACCTAGATGTCGTTG	83	F：TTCTATTCAATACACATATCATGTAGATGCC R：TTGACTTCCACCTAGATGTCGTTG
rs737008	90	F：AGTTTGGTGGATGTGCTATTTTGT R：CACGGAGGAGAGCCATGAGG	220	F：AGTTTGGTGGATGTGCTATTTTGT R：GACAAAGAAGTCGCAGACGAAG

cSNP	cDNA		gDNA
cSNP	扩增子长度/ bp	引物序列（5'→3'）	扩增子长度/bp	引物序列（5'→3'）
rs9876921	156	F：TCATCTGGTTGGTGAAGATGGTG R：CAGAGGAGCCTTCTGGATACTTG	217	F：TCATCTGGTTGGTGAAGATGGTG R：CAATCTTGACTATTTTCATGTTCTGGG
rs1995640	199	F：GTGTGCTATGATGAGCTTTGAGAA R：CAACGCTCTCAGCACTGTAGTC	150	F：CGTGCTCATTGGGAATTGGAC R：GTAGTCAGGATCCCAGCAAACA
rs1058274	253	F：TGCTCGGGTGATTCTGGGG R：ACAAAGGTCAGTAGAACTGGGGA	224	F：CTGTAATGGTGTGCTTCAAGGTATC R：ACAAAGGTCAGTAGAACTGGGGA
rs2301366	247	F：GGGACAAAAAGGTGGATCAAAAGG R：TTGACTTCCACCTAGATGTCGTTG	83	F：TTCTATTCAATACACATATCATGTAGATGCC R：TTGACTTCCACCTAGATGTCGTTG
rs737008	90	F：AGTTTGGTGGATGTGCTATTTTGT R：CACGGAGGAGAGCCATGAGG	220	F：AGTTTGGTGGATGTGCTATTTTGT R：GACAAAGAAGTCGCAGACGAAG

cSNP	基因	突变区域	突变类型	等位基因	最小等位基因频率（dbSNP数据库）
rs9876921	TGM4	编码区	错义突变	A/G	0.38
rs1995640	TGM4	编码区	同义突变	A/G	0.43
rs1058274	KLK3	3'非翻译区	-	A/G	0.20
rs2301366	SEMG1	编码区	错义突变	A/T	0.28
rs737008	PRM1	编码区	无义突变	G/T	0.49