letter Aa2000nAtureAmericaInc..http:/iGenetics.nature.com PCR. We used the RepeatMasker2 http://ftp.genome. Table 1. Distribution of Y-chromosome haploty pes by geographic population group ashington. edu) to identify uman repeat DNA sequence 10111213售4 71819221222324252622830132333533183940 We designed primers to amplify nique sequences and repeat ele- ments other than line as confirmed by a negative female control, yield amplicons 300-500 bp in length. The description of the 167 Y mark ersaregivenintaBleA(http: netics.nature.com/supplementary nfo/). All primers had a uniform nealing rature. which allowed a single PCR protocol to be d an initial denatu for 10 1 1s211181111171132111421727113111151z AmpliTaq Gold, 14 cycles of denatu- ration at 94C for 20 s, primer anealing at 63-56C using 0.5C decrements and extension at 72C °cfor20s,56" C for 1 min,72°for I min and a final 5-min extension at 1 72C. Each 50-ul PCR reaction con- merase,10 mM Tris-HCL pH8.3, 50 Pakistan 955号:8 mM KCl, 2.5 mM MgCl2, 0. 1 mM each of the four deoxyribonu- cleotide triphosphates, 0. 2 HM each of forward/reverse primers and 50 ng genomic DNA. PCR yields were 151410241111511011155231102113311711614112221216110 on ethidium bromide stained ■8384B858890919995100101@131041010510710101101111111311411511T uid chromatog ified PCR products at 2z898 y.equimolar ratio with a reference the mixture to a 3 min, 95.C dena- CAsh+ 30363 turing step followed by gradual reannealing from 95-65C over 30 min. We loaded 10 ul of each mix- ture onto a DNASep column d the ampli were eluted in 0. 1 M triethylamm ium acetate, pH 7, with a linear acetonitrile gradient at a flow rate of cognized het- two or more I Table 2.Defining features of haplogroups No. mutations per mutations from Total by computer simu- haplogroup minus root to individual lation(availableathttp://insertion.Haplogroup mutation(s) per haplogroup 6.1±0. DNA sequencing. We purified poly- Iml 10 0.4±0.24 morphic and reference PCR sam- oles with QIAquick spin columns rands to determine the location d chemical nature of any poly- VIl 9.3±0.35 8.9±0.68 M175 9.2±0.1 sence of M173 encing reaction contained Totals 8.59±0.20 ified PCR product, 4 ul d Mean and standard errorletter 360 nature genetics • volume 26 • november 2000 PCR. We used the RepeatMasker2 program (http://ftp.genome. washington.edu) to identify human repeat DNA sequences. We designed primers to amplify unique sequences and repeat ele￾ments other than LINE as confirmed by a negative female control, yield￾ing amplicons 300–500 bp in length. The description of the 167 Y mark￾ers are given in Table A (http:// genetics.nature.com/supplementary _info/). All primers had a uniform annealing temperature, which allowed a single PCR protocol to be used. It comprised an initial denatu￾ration at 95 °C for 10 min to activate AmpliTaq Gold, 14 cycles of denatu￾ration at 94 °C for 20 s, primer annealing at 63–56 °C using 0.5 °C decrements and extension at 72 °C for 1 min, followed by 20 cycles at 94 °C for 20 s, 56 °C for 1 min, 72 °C for 1 min and a final 5-min extension at 72 °C. Each 50-µl PCR reaction con￾tained 1 U AmpliTaq Gold poly￾merase, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 0.1 mM each of the four deoxyribonu￾cleotide triphosphates, 0.2 µM each of forward/reverse primers and 50 ng genomic DNA. PCR yields were determined semi-quantitatively on ethidium bromide stained agarose gels. Denaturing high-performance liq￾uid chromatography analysis. We mixed unpurified PCR products at an equimolar ratio with a reference Y chromosome and then subjected the mixture to a 3 min, 95 °C dena￾turing step followed by gradual reannealing from 95–65 °C over 30 min. We loaded 10 µl of each mix￾ture onto a DNASep column (Transgenomic), and the amplicons were eluted in 0.1 M triethylammo￾nium acetate, pH 7, with a linear acetonitrile gradient at a flow rate of 0.9 ml/min2. We recognized het￾eroduplex mismatches by the appearance of two or more peaks in the elution profiles under appropri￾ate temperature conditions, which were optimized by computer simu￾lation (available at http://insertion. stanford.edu/melt.html). DNA sequencing. We purified poly￾morphic and reference PCR sam￾ples with QIAquick spin columns (Qiagen). We sequenced both strands to determine the location and chemical nature of any poly￾morphic sites, using the amplimers as sequencing primers and ABI Dye-terminator cycle sequencing reagents (PE Biosystems). Each cycle sequencing reaction contained 6 µl purified PCR product, 4 µl dye 17 1 5 1 2 1 7 2 6 5 1 3 1 4 1 3 15 16 2 20 6 1 3 1 1 1 1 7 13 2 1 12 2 11 1 1 1 7 1 1 1 20 3 11 5 1 11 7 4 3 7 28 1 3 2 8 1 1 1 1 1 4 1 2 1 12 1 21 1 2 2 1 1 1 2 1 6 23 1 14 1 5 1 1 3 3 3 19 2 1 1 18 1 1 1 1 1 71 1 3 2 17 12 14 2 17 2 7 1 1 36 11 1 16 1 2 4 4 1 3 14 11 8 1 21 9 11 1 2 2 1 1 12 2 8 10 16 2 1 12 4 1 1 2 1 1 17 6 9 1 4 3 3 2 1 1 1 4 7 11 1 13 12 1 1 151 111 2 21 1 121241 4 18 1 2 1 1 11 4 3 1 1 11 1 1 5 1 4 10 24 1 1 1 15 1 10 1 1 1 5 5 23 1 10 2 1 1 3 3 1 1 7 1 1 68 1 4 1 1 22 2 12 16 1 40 1 88 1 44 1 5 28 37 39 53 3 1 29 3 60 2 22 2 7 5 26 45 2 2 24 2 5 2 2 12 1 10 1 30 3 6 3 12 6 184 1 2 8 2 6 1 28 2 4 88 2 3 3 11 2 7 38 1 23 311 1 20 5 46 1 74 1 161 1 18 7 2541 23 12 7 1 1 5 5 83 4 106 5 52 1 2 7 17 3 2 12 7 12 2 2 5 4 1 3 1 2 2 7 5 89 2 1 1 73 3 6 12 1 23 6 83 4 1062 10 2 6 2 1 Haplotype # Sudan Ethiopia Mali Morocco C. Africa Khoisan S. Africa Europe Sardinia Basque Mid-east C. Asia + Siberia Pakistan + India Hunza Japan China Taiwan Cambo + Laos New Guinea Australia America Total Haplotype # Sudan Ethiopia Mali Morocco C. Africa Khoisan S. Africa Europe Sardinia Basque Mid-east C. Asia + Siberia Pakistan + India Hunza Japan China Taiwan Cambo + Laos New Guinea Australia America Total Table 1 • Distribution of Y-chromosome haplotypes by geographic population group Haplotype# Sudan Ethiopia Mali Morocco C. Africa Khoisan S. Africa Europe Sardinia Basque Mid-east C. Asia + Siberia Pakistan + India Hunza Japan China Taiwan Cambo + Laos New Guinea Australia America Total 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 98 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 Total 81 Table 2 • Defining features of haplogroups Avg. no. of No. mutations per Most recent mutations from Total no. haplogroup minus defining root to individual of defining No. haplotypes Haplogroup mutation haplotypes* individuals mutation(s) per haplogroup I M91 6.1±0.95 52 20 8 II M60 6.1±0.41 52 12 10 III M96 10.4±0.24 218 27 21 IV M174 10.5±0.96 9 7 4 V M130 6.6±0.60 40 8 5 VI M89 & 7.4±0.25 163 25 23 absence of M9 VII M175 9.3±0.35 137 18 15 VIII M9 & absence 8.9±0.68 67 16 11 of M175 and M45 IX M173 10.2±0.20 195 13 13 X M74 & 9.2±0.1 129 6 6 absence of M173 Totals – 8.59±0.20 1062 152 116 *Mean and standard error. © 2000 Nature America Inc. • http://genetics.nature.com © 2000 Nature America Inc. • http://genetics.nature.com