Development and Forensic Application of EX20+30Y Kit

doi:10.12116/j.issn.1004-5619.2020.500509

Abstract

Abstract: Objective

To develop a multiplex PCR amplification system （EX20+30Y for short） of 19 autosomes, 30 Y-STR loci plus the gender indicator, and evaluate its forensic application value.

Methods

With the six-color fluorescence labeling technology, a multiplex amplification system of 19 autosomal STR loci and 30 Y-STR loci plus the gender indicator was constructed. Blood samples from 210 unrelated individuals, 69 daily case samples and standard samples 9948 and 9947A were collected for loci detection and analysis. The EX20+30Y multiplex amplification system was evaluated by its sensitivity, mixed sample detection ability, species specificity, balance, direct amplification ability, sample applicability and anti-inhibition ability.

Results

Multiplex amplification of blood samples from 210 unrelated individuals by the detection system obtained accurate genotyping results. The detection sensitivity of standard samples was 0.125 ng and the species specificity was high. The 69 samples from daily cases were genotyped correctly. Moreover, standard sample 9948 could be accurately genotyped even if the samples contained a certain concentration of inhibitors.

Conclusion

The multiplex amplification system established in this study can conduct combined examination of 19 autosomes, 30 Y-STR loci plus the gender indicator with accurate genotyping and high sensitivity. It has a good forensic application prospect.

Key words: forensic genetics, short tandem repeat, autosome, Y chromosome, kit

CLC Number:

DF795.2

Xu-ming ZHOU, Deng-xing GAO, Fa-yuan LI. Development and Forensic Application of EX20+30Y Kit[J]. Journal of Forensic Medicine, 2021, 37(5): 639-645.

Figures/Tables 7

Tab. 1 Site information of 19 autosome and 30 Y-STR loci in EX20+30Y Kit

基因座	等位基因范围	荧光标记^1）	核心重复模式	染色体位置	GenBank 登录号^2）	引物体系浓度/（μmol·L^-1）
DYS391	6~13	6-FAM	[TCTA]	Yq11.21	AC011302：11	0.343
D3S1358	11~20	6-FAM	[AGAT]	3p21.31	AC099539	0.093
D13S317	6~15	6-FAM	[GATA]	13q22~31.1	AL353628.2	0.076
D7S820	6~15	6-FAM	[GATA]	7q11.21~21.11	AC004848	0.102
D16S539	5~15	6-FAM	[GATA]	16q24.1	AC024591.3	0.236
Penta E	5~25	6-FAM	[AAAGA]	15q26.2	AC027004	0.500
DYS635	17~27	6-FAM	[TCTA] [TGTA]	Yq11.21	AC004772：23	0.480
DYS385a/b	9~28	6-FAM	[GAAA]	Yq11.222	AC022486：11	0.124
DYF387S1a/b	29~45	6-FAM	[AAAG] [GTAG] [GAAG] [AAAG] [GAAG] [AAAG] [GAAG] [AAAG]	Yq11.223/Yq11.23	AC025246	0.165
DYS456	13~19	HEX	[AGAT]	Yp11.2	AC010106：15	0.166
TPOX	6~14	HEX	[AATG]	2p25.3	M68651	0.345
TH01	4~13.3	HEX	[AATG]	11p15.5	D00269	0.528
D2S1338	14~27	HEX	[TGCC] [TTCC]	2q35	AC010136	0.400
D6S1043	9~21.3	HEX	[AGAT]	6q15	G08539：11	0.194
FGA	15~50.2	HEX	[TTTC]₃TTTTTTCT [CTTT]CTCC[TTCC]₂	4q28	M64982	0.159
DYS438	7~14	HEX	[TTTTC]	Yq11.21	AC002531：10	0.235
DYS448	15~23	HEX	[AGAGAT]	Yq11.223	AC025227：22	0.165
DYS570	9~27	HEX	[GATA]	Yp11.2	AC012068	0.126
DYS437	12~17	SUM	[TCTA]	Yq11.21	AC002992	0.107
D19S433	9~17.2	SUM	[AAGG] [AAAG] [AAGG] [TAGG] [AAGG]	19q12	AC008507.6	0.255
vWA	12~22	SUM	[AGAT]	12p13.31	M25858	0.380
DYS19	12~19	SUM	[TAGA]	Yp11.2	AC017019：15	0.066
CSF1PO	7~15	SUM	[AGAT]	5q33.1	X14720	0.040
D18S51	9~28	SUM	[GAAA]	18q21.33	AP001534	0.080
DYS627	10~28	SUM	[AGAA]N₁₆[AGAG] [AAAG]N₈₁[AAGG]	Yp11.2	AC009491	0.200
DYS576	12~23	SUM	[AAAG]	Yp11.2	AC010104	0.100
DYS449	21~39	SUM	[TTTC]	Yp11.2	AC051663	0.240
DYS460	5~14	LYN	ATCT	Yq11.222	AC009235	0.050
D8S1179	8~19	LYN	[TATC]	8q24.13	AF216671	0.200
D5S818	7~15	LYN	[AGAT]	5q21-31	AC008512	0.267
D21S11	25~37	LYN	[TCTA] [TCTG]	21q11.2-q21	AP000433	0.170
D12S391	15~28	LYN	[AGAT]_8~17[AGAC]_6~10[AGAT]_0~1	Chr12； 12.341 Mb	AF0769651	0.290
Penta D	2.2，5~17	LYN	[AAAGA]	21q22.3	AP001752	0.180
DYS458	12~23	LYN	[GAAA]	Yp11.2	AC010902：16	0.135
DYS533	7~15	LYN	[ATCT]	Yq11.221	AC053516	0.321
DYS518	30~49.2	LYN	[AAAG] [GAAG] [AAAG] [GGAG] [AAAG]	Yq11.221	AC010972	0.360
Amelogenin	X，Y	PUR	-	X：p22.1-22.3 Y：p11.2	NG_012040.1	0.119
DYS393	9~16	PUR	[AGAT]	Yp11.2	AC006152：12	0.166
DYS389Ⅰ	11~16	PUR	[TCTG] [TCTA]	Yq11.21	AC004617：12	0.180
DYS439	7~15	PUR	[AGAT]	Yq11.21	AC002992：13	0.190
DYS389Ⅱ	25~33	PUR	[TCTG] [TCTA] [TCTG] [TCTA]	Yq11.21	AC004617：29	0.263
DYS392	7~17	PUR	[TAT]	Yq11.223	AC011745：13	0.198
Y-GATA-H4	7~15	PUR	[TAGA]	Yq11.1	AC011751：12	0.216
DYS390	19~27	PUR	[TCTA] [TCTG]	Yq11.221	AC011289：24	0.160
DYS481	16~32	PUR	[CTT]	Yp11.2	AC016991.5：22	0.143
DYS596	12~18	PUR	[GGA] [GTA] [GGA] [GAA] [GGAGAA]	Yp11.2	AC016991	0.384
DYS527a/b	16~29	PUR	[GGAA]	Yq11.223	AC016752	0.270

Tab. 1 Site information of 19 autosome and 30 Y-STR loci in EX20+30Y Kit

基因座	等位基因范围	荧光标记^1）	核心重复模式	染色体位置	GenBank 登录号^2）	引物体系浓度/（μmol·L^-1）
DYS391	6~13	6-FAM	[TCTA]	Yq11.21	AC011302：11	0.343
D3S1358	11~20	6-FAM	[AGAT]	3p21.31	AC099539	0.093
D13S317	6~15	6-FAM	[GATA]	13q22~31.1	AL353628.2	0.076
D7S820	6~15	6-FAM	[GATA]	7q11.21~21.11	AC004848	0.102
D16S539	5~15	6-FAM	[GATA]	16q24.1	AC024591.3	0.236
Penta E	5~25	6-FAM	[AAAGA]	15q26.2	AC027004	0.500
DYS635	17~27	6-FAM	[TCTA] [TGTA]	Yq11.21	AC004772：23	0.480
DYS385a/b	9~28	6-FAM	[GAAA]	Yq11.222	AC022486：11	0.124
DYF387S1a/b	29~45	6-FAM	[AAAG] [GTAG] [GAAG] [AAAG] [GAAG] [AAAG] [GAAG] [AAAG]	Yq11.223/Yq11.23	AC025246	0.165
DYS456	13~19	HEX	[AGAT]	Yp11.2	AC010106：15	0.166
TPOX	6~14	HEX	[AATG]	2p25.3	M68651	0.345
TH01	4~13.3	HEX	[AATG]	11p15.5	D00269	0.528
D2S1338	14~27	HEX	[TGCC] [TTCC]	2q35	AC010136	0.400
D6S1043	9~21.3	HEX	[AGAT]	6q15	G08539：11	0.194
FGA	15~50.2	HEX	[TTTC]₃TTTTTTCT [CTTT]CTCC[TTCC]₂	4q28	M64982	0.159
DYS438	7~14	HEX	[TTTTC]	Yq11.21	AC002531：10	0.235
DYS448	15~23	HEX	[AGAGAT]	Yq11.223	AC025227：22	0.165
DYS570	9~27	HEX	[GATA]	Yp11.2	AC012068	0.126
DYS437	12~17	SUM	[TCTA]	Yq11.21	AC002992	0.107
D19S433	9~17.2	SUM	[AAGG] [AAAG] [AAGG] [TAGG] [AAGG]	19q12	AC008507.6	0.255
vWA	12~22	SUM	[AGAT]	12p13.31	M25858	0.380
DYS19	12~19	SUM	[TAGA]	Yp11.2	AC017019：15	0.066
CSF1PO	7~15	SUM	[AGAT]	5q33.1	X14720	0.040
D18S51	9~28	SUM	[GAAA]	18q21.33	AP001534	0.080
DYS627	10~28	SUM	[AGAA]N₁₆[AGAG] [AAAG]N₈₁[AAGG]	Yp11.2	AC009491	0.200
DYS576	12~23	SUM	[AAAG]	Yp11.2	AC010104	0.100
DYS449	21~39	SUM	[TTTC]	Yp11.2	AC051663	0.240
DYS460	5~14	LYN	ATCT	Yq11.222	AC009235	0.050
D8S1179	8~19	LYN	[TATC]	8q24.13	AF216671	0.200
D5S818	7~15	LYN	[AGAT]	5q21-31	AC008512	0.267
D21S11	25~37	LYN	[TCTA] [TCTG]	21q11.2-q21	AP000433	0.170
D12S391	15~28	LYN	[AGAT]_8~17[AGAC]_6~10[AGAT]_0~1	Chr12； 12.341 Mb	AF0769651	0.290
Penta D	2.2，5~17	LYN	[AAAGA]	21q22.3	AP001752	0.180
DYS458	12~23	LYN	[GAAA]	Yp11.2	AC010902：16	0.135
DYS533	7~15	LYN	[ATCT]	Yq11.221	AC053516	0.321
DYS518	30~49.2	LYN	[AAAG] [GAAG] [AAAG] [GGAG] [AAAG]	Yq11.221	AC010972	0.360
Amelogenin	X，Y	PUR	-	X：p22.1-22.3 Y：p11.2	NG_012040.1	0.119
DYS393	9~16	PUR	[AGAT]	Yp11.2	AC006152：12	0.166
DYS389Ⅰ	11~16	PUR	[TCTG] [TCTA]	Yq11.21	AC004617：12	0.180
DYS439	7~15	PUR	[AGAT]	Yq11.21	AC002992：13	0.190
DYS389Ⅱ	25~33	PUR	[TCTG] [TCTA] [TCTG] [TCTA]	Yq11.21	AC004617：29	0.263
DYS392	7~17	PUR	[TAT]	Yq11.223	AC011745：13	0.198
Y-GATA-H4	7~15	PUR	[TAGA]	Yq11.1	AC011751：12	0.216
DYS390	19~27	PUR	[TCTA] [TCTG]	Yq11.221	AC011289：24	0.160
DYS481	16~32	PUR	[CTT]	Yp11.2	AC016991.5：22	0.143
DYS596	12~18	PUR	[GGA] [GTA] [GGA] [GAA] [GGAGAA]	Yp11.2	AC016991	0.384
DYS527a/b	16~29	PUR	[GGAA]	Yq11.223	AC016752	0.270

Fig. 1 Loci distribution in EX20+30Y Kit

Tab. 2 Y-STR peak number in the mixed samples with different concentrations

V₉₉₄₈∶V_9947A	标准品9948质量/pg	标准品9947A质量/pg	3次测量平均值/（ $x ˉ$ ±s，个）
1∶1	500	500	30±0
1∶4	200	800	30±0
1∶9	100	900	27±2
1∶19	50	950	19±2

Tab. 2 Y-STR peak number in the mixed samples with different concentrations

V₉₉₄₈∶V_9947A	标准品9948质量/pg	标准品9947A质量/pg	3次测量平均值/（ $x ˉ$ ±s，个）
1∶1	500	500	30±0
1∶4	200	800	30±0
1∶9	100	900	27±2
1∶19	50	950	19±2

Fig. 2 Example of the blood sample typing result map

Tab. 3 Loci detection rate of different concentrations of inhibitors

抑制剂	浓度/ （μmol·L^-1）	检出率/ %	抑制剂	浓度/ （μmol·L^-1）	检出率/ %	抑制剂	浓度/ （μmol·L^-1）	检出率/ %
血红素	20	100	腐殖酸	20	100	Ca²⁺	0.1	100
	50	100		50	82		0.2	100
	80	78		80	36		0.5	100
	100	38		100	28		0.7	100
	150	0		150	0		1	94
血红蛋白	50	100	靛蓝	4	100	EDTA	0.5	100
	80	100		5	100		0.8	100
	100	94		8	100		1	100
	150	46		10	100		1.2	100
	200	0		12	94		1.5	84

Fig. 3 Electrophoretogram of standard sample 9948 tested by 3500xL Gene Analyzer and EX20+30Y Kit

Fig. 4 Electrophoretogram of standard sample 9948 tested by 3730xl Gene Analyzer and EX20+30Y Kit

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