Fluorescent Multiplex Amplification of Three X-STR Loci

遗 传 学 报

Acta Genetica Sinica, December

ISSN 0379-4172

2006, 33 (12)：1053–1059

Fluorescent Multiplex Amplification of Three X-STR Loci LIU Qiu-Ling1, LÜ De-Jian1, ZHU Jia-Zhen1, LU Hui-Ling1,①, LUO Yan-Min2, FANG Qun2 1. Faculty of Forensic Medicine, Zhongshan Medical College, Sun Yat-sen University, Guangzhou 510089, China; 2. Fetal Medicine Center, Department of Obstetrics and Gynecology, First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080,China Abstract: This study was carried out to evaluate the value of three X-STR loci (DXS6803, DXS981and DXS6809) in forensic application and thereby investigate their polymorphism. The primer for each locus was labeled with fluorochrome 6-FAM. A fluorescent multiplex PCR for simultaneously amplifying three X-STR loci was set up. The PCR products that were obtained were analyzed using capillary electrophoresis and ABI PRISM 3100 Genetic Analyzer, with GENESCAN Analysis Software. When 340 male and 195 female individuals of Han population in China were tested, 13, 12, and 11 alleles were observed for DXS6803, DXS981 and DXS6809, respectively. One hundred and eighty three haplotypes were detected in the male individuals. The haplotype diversity reached 0.9926. The results show that the three loci of the multiplex system provide significant information on polymorphism for forensic identification and paternity testing, particularly for complicated paternity deficient cases. Key words: X-STR; fluorescent multiplex PCR; capillary electrophoresis; polymorphism

Many autosomal and Y-chromosomal DNA polymorphisms have been evaluated for forensic use and have been widely applied for stain analysis and kinship determination. Several Gene print® Fluorescent STR multiplex systems have been produced, e.g., Powerplex® 2.1 system, Powerplex™ 16 system, Powerplex® Y system, etc. Recently, more X-chromosomal STR markers (Chr-X STRs) have been recognized for forensic and genetic analysis[1 7]. Chr-X -

STRs are very useful in paternity deficient cases, such as determination of paternity of alleged half-sisters versus the father in the absence of the mother. Chr-X STR is potentially complementary to other genetic markers (autosomal, Y-chromosomal, and mitochondrial). To develop reliable Chr-X STR multiplex systems for forensic case studies and to increase the pool of relevant data for allele distribution and frequency in Chr-X STR, a convenient procedure was developed by the authors to amplify a triplex systems with Chr-X STR loci (DXS6803, DXS981, and DXS6809). The polymorphisms of the three loci and haplotypes

Received: 2006-03-07; Accepted: 2006-06-12 ① Corresponding author. E-mail: [email protected]

of male individuals are tested.

1 1. 1

Materials and Methods Sample preparation and DNA extraction

Samples were obtained from 535 unrelated Han population in China, including 340 males and 195 females. Genomic DNA was extracted using Chelex100 methods. 1. 2

PCR amplification

1. 2. 1

Primer sequences

PCR amplification was performed using the following primer sequences (http://www.gdb.org). DXS6803 F:5′-6-FAM-GAAATGTGCTTTGACAGGAA3′; R:5′-CAAAAAGGGACATATGCTACTT-3′; DXS981 F:5′-6-FAM-TCAGAGGAAAAGAAGTAGACATACT-3′;

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遗传学报

R:5′-TTCTCTCCACTTTTCAGAGTCA-3′; DXS6809 F:5′-6-FAM-TGAACCTTCCTAGCTCAGGA-3′; R:5′-TCTGGAGAATCCAATTTTGC-3′. 1. 2. 2

PCR amplification reaction

Amplification was carried out in a 25 μL PCR reaction volume containing 10-20 ng DNA, 200 μmol/L for each dNTP, with 1.5 mmol/L MgCl2 (ABI, USA), 1×buffer (ABI, USA), and 1.0 U Taq plus DNA polymerase (Beijing, China). The primer was 7.5 pmol, 5.8 pmol, and 5.0 pmol for DXS6803, DXS981, and DXS6809，respectively. Samples were amplified in the Primus Thermal Cycles (MWGBIOTECH AG, Germany) under the following conditions: initial denaturation (94℃ for 5 min), followed by 30 cycles of denaturation (94℃ for 45 s), annealing (60℃ for 45 s), and extension (72℃ for 45 s). A final extension was performed at 72℃ for 30 min. 1. 2. 3

Sample electrophoresis and data analysis

PCR products were resolved and detected by capillary electrophoresis using ABI PRISM 3100 Genetic Analyzer with denaturing polymer POP4 (Perkin-Elmer). Fragment sizing was supported using the Genescan™-500 LIZ™ size standards. Allele typing was based on in-house allelic ladder. K562 (Promega, Madison, WI) was used as the control DNA, which served to calibrate the allelic ladder. 1. 3

Statistical analysis Haplotype and allelic frequencies were tested

using ARLEQUIN 1.1 (http://lgb.unige.ch/arlequin). The possibility of linkage disequilibrium was also estimated using it. The power of discrimination in females (PDF) and males (PDM), the haplotype diversity, PEtrio and PEmotherless were individually calculated using the formula[8].

2

Results The three markers were amplified as described

Acta Genetica Sinica

Vol.33 No.12 2006

in Section 1.2.2 with satisfactory results (Fig. 1). Repeated analysis of random DNA samples gave consistent results. The K562 control DNA that can be employed for calibrating allelic ladder showed the allele 9, 13.3, and 34 for DXS6803, DXS981, and DXS6809, respectively. Table 1 shows the allelic frequencies of loci (DXS6803, DXS981, and DXS6809), which were calculated separately for females and males, and other statistical information. Table 2 shows the haplotype frequencies of DXS6803, DXS981, and DXS6809 in 340 unrelated males from Han population. One hundred and eighty three different haplotypes were found. The haplotype diversity reached 0.9926.

3

Discussion

This article described a convenient procedure for amplifying three X-linked STRs (DXS6803, DXS981, and DXS6809) in a single reaction. No stutter bands were detected. About 20 ng DNA was routinely used, although 6 ng DNA was sufficient for allele typing. Besides, Chr-X STR had the potential to efficiently complement the analysis of other genetic markers. The X-linked markers may be particularly useful in cases of deficiency paternity testing, when a child is female. Man transmits the same X chromosome to all his daughters. Thus, all of them should share at least one allele at every locus. The case whether presumed half-sister alleles share the genetic father alleles could be tested using analysis with X-linked markers, without parents being tested. Moreover, the X-STRs were also useful to conform a paternal grandmothergranddaughter relation, because granddaughter(s) shares at least one identical allele in each X-STR loci to their grandmother[7]. So X-STRs were recently recognized as very important tools in forensic application, particularly in special cases. The study of X-linked markers may help solve otherwise impossible cases[9]. Three X-linked markers are located on the long arm of X-chromosome; DXS6803 and DXS981 were located at the Xq11-13 [5,10] , and DXS6809 was

LIU Qiu-Ling et al.: Fluorescent Multiplex Amplification of Three X-STR Loci

Fig. 1

1055

The electrophoretogram of triplex Chr-X STR system (DXS6803, DXS981, and DXS6809)

located at the Xq21.33[11]. According to different size of amplified fragments of three X-STR markers, the primers of DXS6803, DXS981, and DXS6809 were labeled with one fluorescent 6-FAM (blue). They are simultaneously amplified in a single reaction. The products were analyzed by capillary electrophoresis and ABI PRISM 3100 Genetic Analyzer. Sixteen

PCR samples can be tested in half an hour using a DNA scanner with sixteen capillary. Thirteen alleles were identified at locus DXS6803, ranging from allele 7 to 14, including incomplete repeats. The most common alleles are 11.3. DXS981 has twelve alleles ranging from allele 11 to 17 and including incomplete repeats, the most common

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Table 1

遗传学报

Acta Genetica Sinica

Vol.33 No.12 2006

Allele frequencies of DXS6803, DXS981, and DXS6809 in Chinese population DXS6803

DXS981

Allele

DXS6809

Allele Male

Female

Total

7

0.0059

0.0103

0.0082

7.3

0.0029

0.0051

9

0.0147

9.3

Allele Male

Female

Total

Male

Female

Total

11

0.0176

0.0077

0.0123

28

0.0029

0.0051

0.0041

0.0041

11.3

0.0029

0.0026

0.0027

29

0.0118

0.0077

0.0096

0.0180

0.0164

12

0.0382

0.0487

0.0438

30

0.0235

0.0231

0.0233

0.0059

0.0026

0.0041

12.3

0.0824

0.0846

0.0836

31

0.1382

0.1308

0.1342

10

0.1353

0.1410

0.1384

13

0.1441

0.1564

0.1507

32

0.1706

0.1718

0.1712

10.3

0.0706

0.0641

0.0671

13.3

0.1971

0.1769

0.1863

33

0.2412

0.2641

0.2534

11

0.1382

0.1282

0.1329

14

0.2706

0.2949

0.2836

34

0.2265

0.2359

0.2315

11.3

0.4559

0.4282

0.4411

14.3

0.0765

0.0667

0.0712

35

0.1265

0.1026

0.1137

12

0.0618

0.0923

0.0781

15

0.1029

0.0974

0.1000

36

0.0441

0.0462

0.0452

12.3

0.0706

0.0795

0.0753

15.3

0.0265

0.0333

0.0301

37

0.0059

0.0077

0.0068

13

0.0235

0.0231

0.0233

16

0.0324

0.0282

0.0301

38

0.0088

0.0051

0.0069

13.3

0.0059

0.0026

0.0041

17

0.0088

0.0026

0.0055

14

0.0088

0.0051

0.0069

PD

0.7400

0.5551

0.8403

0.6747

0.8236

0.6385

PEtrio

0.7900

0.8446

0.8275

PEmotherless

0.5948

0.7072

0.6759

PD: power of discrimination; PEtrio: power of paternity exclusion in standard trios for X-STR; PEmotherless: power of paternity exclusion in motherless cases for X-STR.

alleles being 14. Eleven alleles, ranging from 28 to 38, were found in Chinese population at locus DXS6809. No significant differences were observed between males and females at the three loci. Hardy-Weinberg equilibrium (HWE) was performed on female samples, and the genotype distributions did not deviate from HWE at the DXS981 and DXS6809 loci. But genotype distributions of the DXS6803 deviate from HWE. Thus, there are rare allele 7, 7.3, 9, and 9.3. When these alleles were combined, deviation from HWE was not found (P > 0.05). Male samples were investigated by haplotype analysis and for linkage disequilibrium. One hundred and eighty three haplotypes were found. The frequency of the most abundant haplotype (11.3/14/34) still does not exceed

4%, and more than 34.4% of haplotypes were unique. In this study, linkage disequilibrium among loci was not detected by the exact test. Investigation in 223 family trios in which the child is female and in 193 families with mother and son, revealed no mutations at the DXS981 and DXS6809. Two mutations were found at the DXS6803. Mutation ratio is 0.3%. The triplex ChrX-STR systems for simultaneous analysis of loci (DXS6803, DXS981, and DXS6809) have been developed and may be used to create database of population for forensic analysis. This may be particularly useful for complicated paternity deficient cases.

LIU Qiu-Ling et al.: Fluorescent Multiplex Amplification of Three X-STR Loci

Table 2

1057

Haplotype frequencies of DXS6803, DXS981, and DXS6809 in male of Han population in China (n=340)

No.

Haplotype

Frequency

No.

Haplotype

Frequency

No.

Haplotype

Frequency

1

11.3/14/34

0.0353

31

11/13.3/34

0.0088

61

12.3/13.3/31

0.0059

2

11.3/13/33

0.0324

32

11.3/15/32

0.0088

62

11/14/35

0.0059

3

11.3/13.3/34

0.0294

33

10/14/33

0.0088

63

9/13.3/36

0.0059

4

11.3/14/33

0.0235

34

10/14/34

0.0088

64

14/14.3/34

0.0059

5

11.3/13.3/33

0.0235

35

11.3/15.3/36

0.0059

65

12.3/13/33

0.0059

6

11.3/15/31

0.0206

36

10/12/33

0.0059

66

9.3/12.3/33

0.0059

7

11.3/13.3/31

0.0206

37

11/14/34

0.0059

67

7.3/13/34

0.0029

8

11/14/33

0.0176

38

10.3/13.3/33

0.0059

68

11/13/31

0.0029

9

11.3/14/32

0.0147

39

10/13/32

0.0059

69

12/15/32

0.0029

10

11.3/12.3/33

0.0147

40

11.3/15/33

0.0059

70

10/14/29

0.0029

11

10/14.3/31

0.0118

41

13/14/33

0.0059

71

12/12.3/34

0.0029

12

11.3/15/34

0.0118

42

10/16/34

0.0059

72

12/14.3/33

0.0029

13

11.3/14/31

0.0118

43

11.3/14.3/34

0.0059

73

10/15/37

0.0029

14

11/13.3/32

0.0118

44

11.3/13/34

0.0059

74

7/16/34

0.0029

15

11.3/13/35

0.0118

45

11/14/31

0.0059

75

11.3/14/38

0.0029

16

11.3/14/35

0.0118

46

11.3/14.3/35

0.0059

76

12.3/16/36

0.0029

17

11.3/12.3/34

0.0118

47

10.3/14/32

0.0059

77

11.3/12.3/35

0.0029

18

12.3/14/32

0.0118

48

11/14.3/35

0.0059

78

10.3/13.3/31

0.0029

19

11.3/13.3/32

0.0088

49

10/13.3/32

0.0059

79

12/14.3/32

0.0029

20

11.3/13/32

0.0088

50

11.3/13.3/30

0.0059

80

10/15/38

0.0029

21

12.3/14/34

0.0088

51

12/14.3/34

0.0059

81

11.3/13/38

0.0029

22

11/13/34

0.0088

52

10/13/33

0.0059

82

10/12/35

0.0029

23

10/13/34

0.0088

53

11.3/14.3/33

0.0059

83

10.3/12/33

0.0029

24

11/14/32

0.0088

54

12/15.3/35

0.0059

84

11/15/32

0.0029

25

11/12.3/33

0.0088

55

11.3/13.3/35

0.0059

85

11.3/15.3/32

0.0029

26

10.3/14/31

0.0088

56

11.3/12.3/32

0.0059

86

11/13.3/35

0.0029

27

11.3/13/31

0.0088

57

11.3/15/36

0.0059

87

11/13/35

0.0029

28

11.3/12/32

0.0088

58

12/14/35

0.0059

88

10.3/13/36

0.0029

29

10/13.3/35

0.0088

59

12.3/14/33

0.0059

89

11/11/34

0.0029

30

10.3/14/35

0.0088

60

11.3/11/34

0.0059

90

11/12/35

0.0029

1058

遗传学报

Acta Genetica Sinica

Vol.33 No.12 2006

(Table 2 continued) No.

Haplotype

Frequency

No.

Haplotype

Frequency

No.

Haplotype

Frequency

91

10/14.3/33

0.0029

122

12/13.3/35

0.0029

153

10.3/12/32

0.0029

92

11.3/13.3/36

0.0029

123

10.3/15/34

0.0029

154

10.3/14/36

0.0029

93

11/15/35

0.0029

124

12/12.3/32

0.0029

155

11.3/12/31

0.0029

94

11.3/15/30

0.0029

125

11/13.3/33

0.0029

156

10.3/16/32

0.0029

95

10/16/33

0.0029

126

12/14/32

0.0029

157

12.3/13.3/33

0.0029

96

12/14/31

0.0029

127

11.3/13.3/29

0.0029

158

12/16/32

0.0029

97

10.3/13/34

0.0029

128

10/13.3/37

0.0029

159

11.3/15.3/33

0.0029

98

10/13.3/29

0.0029

129

12.3/13.3/36

0.0029

160

12.3/14/31

0.0029

99

13/13.3/33

0.0029

130

11/14/29

0.0029

161

10.3/14/33

0.0029

100

10.3/14.3/35

0.0029

131

11.3/17/30

0.0029

162

11/12.3/35

0.0029

101

13/15/30

0.0029

132

13/15/33

0.0029

163

11.3/15.3/31

0.0029

102

10/14/32

0.0029

133

11/12/32

0.0029

164

12.3/13/36

0.0029

103

10/17/32

0.0029

134

11.3/12/35

0.0029

165

11.3/14/30

0.0029

104

10/14.3/35

0.0029

135

10/14/35

0.0029

166

11.3/12.3/31

0.0029

105

12/15/35

0.0029

136

11.3/11/33

0.0029

167

12.3/13/35

0.0029

106

11.3/14/36

0.0029

137

11.3/14.3/31

0.0029

168

10/12/31

0.0029

107

11/15/34

0.0029

138

11.3/16/34

0.0029

169

11.3/14/28

0.0029

108

11.3/16/31

0.0029

139

11/15/31

0.0029

170

13.3/13/30

0.0029

109

12.3/14.3/31

0.0029

140

10.3/15/33

0.0029

171

10/13.3/34

0.0029

110

12.3/16/31

0.0029

141

10.3/13/33

0.0029

172

12.3/13.3/32

0.0029

111

11/13/32

0.0029

142

13/14/35

0.0029

173

10/17/30

0.0029

112

11.3/16/33

0.0029

143

10.3/12.3/32

0.0029

174

10/12.3/31

0.0029

113

12.3/13/34

0.0029

144

11.3/13/36

0.0029

175

11.3/11/31

0.0029

114

11/14.3/32

0.0029

145

12/13/33

0.0029

176

13.3/12.3/35

0.0029

115

10/12.3/34

0.0029

146

12/13.3/32

0.0029

177

11.3/15/35

0.0029

116

12.3/15.3/33

0.0029

147

10/14.3/34

0.0029

178

9/14/32

0.0029

117

10.3/13.3/34

0.0029

148

12/12.3/33

0.0029

179

11/14.3/33

0.0029

118

11/11/32

0.0029

149

13/14/32

0.0029

180

7/13.3/34

0.0029

119

10/15/33

0.0029

150

11.3/15.3/34

0.0029

181

9/13.3/34

0.0029

120

12/15/34

0.0029

151

10/12.3/33

0.0029

182

14/14/32

0.0029

121

12/15/36

0.0029

152

9/12.3/32

0.0029

183

13/11.3/34

0.0029

Haplotype diversity

0.9926

LIU Qiu-Ling et al.: Fluorescent Multiplex Amplification of Three X-STR Loci

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Forensic Sci Int, 2005, 154: 173-180. [6] Szibor R, Krawczak M, Hering S, Edelmann J, Kuhlisch E, Krause D. Use of X-linked makers for forensic purposes. Int J Legal Med, 2003, 117: 67-74. [7] Shin S H, Yu J S, Park S W, Min G S, Chung K W. Genetic analysis of 18 X-linked short tandem repeat markers in Korean population. Forensic Sci Int, 2005, 147: 35-41. [8] Wu Xin-Yao. Advanced Forensic Medicine.Henan: Zhengzhou

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297-299 (in Chinese). [9] Zarrabeitia M T, Amigo T, Sanudo C, Zarrabeitia A, Gonzalez-Lamuno D, Riancho J A. A new pentaplex system to study short tandem repeat markers of forensic interest on X chromosome. Forensic Sci Int, 2002, 129: 85-89. [10] Edelmann J, Szibor R. The X-linked STRs DXS7130 and DXS6803. Forensic Sci Int, 2003, 136: 73-75. [11] Edelmann J, Deichsel D, Plate I, Kaser M, Szibor R. Validation of the X-chromosomal STR DXS6809. Int J Legal Med, 2003, 117: 241-244.

3 个 X-STR 基因座荧光标记复合扩增 刘秋玲1，吕德坚1，祝家镇1，陆惠玲1，罗艳敏2，方 群2 1. 中山大学中山医学院法医物证教研室，广州 510089； 2. 中山大学附属一院妇产科胎儿医学中心，广州 510080 摘 要: 为研究 DXS6803、DXS981 和 DXS6809 3 个基因座多态性及其在法医学中的应用，建立 X 染色体基因座(DXS6803、 DXS981 和 DXS6809)的荧光复合扩增体系。用荧光标记引物 PCR 技术复合扩增 3 个基因座，并用 ABI PRISM 3100 毛细 管电泳及其软件进行基因分型。结果在中国汉族 340 名无关男性个体及 195 名无关女性个体中，DXS6803、DXS981 和 DXS6809 三个基因座分别发现了 13、12、11 个等位基因，男性个体共检出 183 种单倍型, 单倍型多样性为 0.9926。结果 表明这 3 个基因座有较高的多态性信息，在个体识别和亲权鉴定(特别是在缺失双亲的特殊检案)中有重要的应用价值。 关键词:

X-STR；荧光复合扩增；毛细管电泳；多态性

作者简介: 刘秋玲(1974-)，女，广东兴宁人，医学在职硕士，主管技师，专业方向：法医遗传学。E-mail: [email protected]