Homozygosity Mapping Using Pooled DNA

Val C. Sheffield1

1 University of Iowa, Iowa City, Iowa
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 1.11
DOI:  10.1002/0471142905.hg0111s13
Online Posting Date:  May, 2001
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Abstract

Genomic screening is a common and popular method for localizing human disease genes. Genotyping each individual sample can be a laborintensive and expensive process. This unit describes the technique of pooling DNA from many related affected individuals, genotyping the resulting pools, and scoring the results. This technique can greatly reduce the effort required to screen for genetic linkage and is particularly useful when applied to inbred populations. The unit includes a table showing a pool of human STRP markers developed by the Cooperative Human Linkage Center.

     
 
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Table of Contents

  • Key Concepts
  • Basic Protocol 1: Amplification and Analysis of Pooled DNA
  • Reagents and solution
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Amplification and Analysis of Pooled DNA

  Materials
  • Individual DNA samples from affected and unaffected (control) individuals
  • TE buffer, pH 7.4 ( appendix 2D)
  • Primers for short tandem repeat polymorphic markers (STRPs; Research Genetics; see Table 1.11.1)
    Table 1.1.1   Materials   STRP Markers for Genotyping Pooled DNA a   STRP Markers for Genotyping Pooled DNA

    Chromosome Marker Locus Size range (bp)
    1 ATA20F08 D1S1622 252‐270
    1 GATA29A05 D2S1368 179‐207
    1 GGAA3A07 D1S1612 98‐127
    1 GATA64G10 D1S1669 384‐392
    1 GATA27E01 D1S1597 162‐178
    1 GATA4A09 D1S547 286‐306
    1 GATA61A06 D1S1665 221‐237
    1 GATA109 D1S1728 159‐167
    1 GGAA23C07 D1S1678 297‐309
    1 GGAA20F08 D1S1675 225‐253
    1 GATA6A05 D1S551 173‐185
    1 GATA72H07 D1S2134 259‐295
    1 GATA12A07 D1S534 197‐211
    1 ATA29D04 D1S1631 131‐155
    1 GATA65H06 D1S2130 246‐266
    1 GGAA22G10 D1S1677 191‐219
    1 ATA2E04 D1S1588 118‐135
    1 GATA25B02 D1S1595 255‐280
    1 ATA4E02 D1S1589 201‐214
    1 GGAA5F09 D1S1679 142‐163
    1 GATA50F12 D1S1663 409‐417
    1 GATA48B01 D1S1660 226‐246
    1 GATA44E05 D1S1656 128‐168
    1 GGAT2A07 D1S552 244‐256
    1 GATA50F11 D1S1609 177‐205
    1 GATA87F04 D1S2141 236‐264
    1 GATA4H09 D1S549 164‐184
    1 ATA29C07 D1S3462 256‐271
    1 GATA7C01 D1S518 188‐212
    2 GATA72G11 D2S1780 312‐336
    2 GATA8F07 D2S405 240‐256
    2 GAAT1A5 D2S423 112‐124
    2 GATA12H10 D2S427 250‐258
    2 GATA71G04 D2S1777 195‐207
    2 GGAA20G10 D2S1400 114‐122
    2 ATA4F03 D2S1356 237‐246
    2 GATA69E12 D2S1394 161‐177
    2 ATA27D04 D2S1352 114‐129
    2 GATA88G05 D2S1790 281‐325
    2 GATA5G02 D2S436 178‐190
    2 GATA65C03 D2S1391 113‐137
    2 GATA71D01 D2S1776 288‐312
    2 GATA8H05 D2S442 200‐204
    2 GATA8F03 D2S441 134‐150
    2 GATA4G12 D2S434 262‐274
    2 GATA23D03 D2S1363 171‐191
    2 GATA52A04 D2S1384 140‐156
    2 GATA26B04 D2S1326 238‐266
    2 GATA4E11 D2S410 164‐178
    2 GATA30E06 D1S1649 108‐136
    3 GGAA4B09 D3S2403 252‐264
    3 GATA22G12 D3S2387 180‐202
    3 ATA10H11 D3S2409 118‐127
    3 GGAT2G03 D3S2406 314‐348
    3 GATA88H04 D3S1050 228‐248
    3 GATA27C08 D3S2432 144‐156
    3 GATA3H01 D3S1763 264‐272
    3 GATA73D01 D3S3038 188‐232
    3 GGAA16E09 D3S2465 312‐328
    3 ATA28H11 D3S4009 240‐250
    3 GATA32D12 D3S2436 164‐180
    3 GATA6G12 D3S2398 270‐286
    3 GATA84B12 D3S3045 177‐201
    3 ATA22E01 D3S2418 97‐115
    3 GATA6F06 D3S1766 210‐218
    3 GATA68F07 D3S2460 144‐172
    3 GATA22F11 D3S2427 218‐238
    3 GATA3C02 D3S1744 138‐154
    4 GATA25B06 D4S2408 265‐275
    4 GATA7D01 D4S1627 183‐198
    4 GATA22G05 D4S2366 121‐145
    4 GATA27G03 D4S2368 307‐328
    4 GATA28F03 D4S3248 238‐254
    4 GATA90B10 D4S2639 165‐193
    4 GATA42H02 D4S2417 254‐274
    4 GATA11E09 D4S1644 194‐202
    4 ATA27C07 D4S2397 133‐145
    4 GATA107 D4S1625 186‐202
    4 GATA24H01 D4S2367 137‐145
    4 ATA26B08 D4S2394 241‐259
    4 GATA10G07 D4S3243 161‐169
    4 GATA62A12 D4S2623 205‐242
    4 GATA2F11 D4S1647 131‐151
    4 GGAA19H07 D4S2431 234‐246
    4 GATA8A05 D4S1629 139‐151
    5 GATA84E11 D5S2505 261‐297
    5 GATA3A04 D5S807 166‐202
    5 GATA31H11 D5S1492 119‐123
    5 GGAA11C04 D5S1473 246‐262
    5 GATA7C06 D5S1470 177‐199
    5 GATA52A12 D5S1501 99‐113
    5 GATA68A03 D5S2501 310‐338
    5 GATA62A04 D5S1505 238‐270
    5 GATA32A11 D5S2494 153‐185
    5 GATA3E10 D5S817 260‐272
    5 GATA6E05 D5S820 194‐202
    5 GATA2H09 D5S816 222‐250
    5 GATA67D03 D5S2500 149‐181
    5 GATA69H12 D5S1719 86‐110
    5 ATA23A10 D5S1480 218‐239
    5 GATA7H10 D5S1471 156‐172
    5 GATA3H06 D5S1462 186‐198
    5 ATA4D10 D5S1453 142‐163
    6 GATA29C09 D6S1960 272‐292
    6 GTAT1H06 D6S1019 224‐234
    6 GGAT3H10 D6S1017 153‐173
    6 ATA28B11 D6S1031 250‐265
    6 GATA89B07 D6S1281 176‐208
    6 GATA112F02 D6S1270 114‐138
    6 GATA68H04 D6S1056 242‐270
    6 ATC4D09 D6S1006 194‐200
    6 ATA11D10 D6S1021 142‐157
    6 GATA22G09 D6S1007 288‐296
    6 GATA3H05 D6S477 210‐230
    6 GATA31 D6S474 151‐167
    6 GATA23F08 D6S1040 271‐283
    6 GATA52D05 D6S1050 176‐204
    6 ATA22G07 D6S1027 117‐138
    6 GATA64D02 D6S1053 291‐319
    6 GGAA8D08 D6S503 246‐262
    6 GATA85F06 D6S1280 170‐190
    7 GGAA3F06 D7S1808 240‐254
    7 GATA41G07 D7S1802 180‐200
    7 GATA61G06 D7S2201 104‐120
    7 GATA73D10 D7S2204 214‐268
    7 GATA13G11 D7S817 157‐167
    7 GATA5D08 D7S821 238‐270
    7 GATA31A10 D7S2846 172‐196
    7 GATA4H10 D7S1805 196‐212
    7 GATA45E12 D7S1826 144‐164
    7 GATA43C11 D7S1804 244‐280
    7 GATA32C12 D7S1824 166‐190
    8 ATA3A02 D8S1099 250‐259
    8 ATC2D12 D8S1100 192‐195
    8 GATA23D06 D8S1106 138‐148
    8 GGAA23E06 D8S1990 368‐388
    8 GATA21C12 D8S1128 242‐262
    8 GATA7G07 D8S1179 168‐190
    8 GGAA8G07 D8S1113 217‐241
    8 GGAA20C10 D8S1477 140‐180
    8 GATA72C10 D8S1145 262‐290
    8 GATA25C10 D8S1130 137‐157
    8 GATA12B06 D9S920 276‐288
    8 ATA19G07 D8S1119 175‐196
    8 GAAT1A4 b 140‐160
    8 GATA41A01 D8S1136 245‐261
    8 GATA26E03 D8S1132 143‐159
    8 GATA8G10 D8S1110 259‐275
    8 GATA6B02 D8S592 148‐156
    9 GATA62F03 D4S2624 c 284‐296
    9 GATA7D12 D9S301 204‐230
    9 GATA71E08 D9S1118 142‐178
    9 GGAA22E01 D9S938 383‐419
    9 GATA4D10 D9S302 256‐304
    9 GATA21F05 D9S689 252‐268
    9 GATA27A11 D9S925 171‐194
    9 ATA18A07 D9S910 106‐133
    9 GATA48D07 D9S930 277‐301
    9 GATA64G07 D9S934 206‐230
    10 GATA6E06 D10S674 226‐242
    10 ATA31G11 D10S1412 153‐168
    10 GATA70E11 D10S1423 219‐243
    10 GATA73E11 D10S1426 157‐169
    10 ATA20B07 D10S1220 231‐243
    10 ATA24F10 D10S1225 181‐193
    10 ATA29C03 D10S1230 120‐135
    10 ATA24A05 D10S1223 272‐293
    10 GGAA2F11 D10S677 198‐222
    10 GGAA5D10 D10S1213 99‐119
    10 GATA48G07 D10S1237 400‐432
    10 GATA88F09 D10S1435 260‐276
    10 GATA64A09 D10S1239 162‐186
    10 GGAT1A4 D10S2327 204‐224
    10 GAAT5F06 D10S2325 135‐143
    11 ATA33B03 D11S2362 212‐233
    11 GATA48E02 D11S1981 147‐175
    11 GGAA7G08 D11S1986 188‐240
    11 GATA23F06 D11S1999 109‐134
    11 GGAA5C04 D11S1985 240‐280
    11 ATA34E08 b 157‐169
    11 GATA64D03 D11S4464 228‐252
    11 GATA6B09 D11S1392 203‐211
    11 GATA23E06 D11S1998 149‐157
    11 GATA30G01 D11S2002 223‐251
    11 GGAA17G05 D11S1984 177‐197
    12 GATA7F09 D12S374 276‐292
    12 GATA4H03 D12S372 174‐190
    12 ATA27A06 D12S1042 119‐140
    12 GATA6C01 D12S373 209‐225
    12 GATA26D02 D12S1052 153‐164
    12 ATA25F09 D12S2070 89‐107
    12 GATA32F05 D12S2078 254‐278
    12 GGAT2G06 D12S398 117‐137
    12 GATA63D12 D12S1064 172‐196
    12 GATA85A04 D12S1300 112‐136
    12 GATA11H08 D12S391 221‐241
    12 GATA13D05 D12S392 139‐148
    12 GATA4H01 D12S395 222‐238
    12 GATA3F02 D12S375 165‐189
    13 GATA64F08 D13S800 293‐309
    13 GGAA29H03 D13S1493 212‐252
    13 GATA51B02 D13S796 149‐169
    13 GATA43H03 D13S793 249‐269
    13 ATA26D07 D13S779 182‐200
    13 GATA23C03 D13S787 253‐261
    13 GATA7G10 D13S317 178‐198
    14 GATA74E02 D14S742 395‐415
    14 GATA31B09 D14S1280 292‐304
    14 ATA25E04 D14S597 178‐193
    14 GGAA10C09 D14S587 254‐274
    14 GATA4B04 D14S306 194‐208
    14 GATA5H04 D14S297 96‐112
    14 GATA51E09 D14S610 360‐376
    14 ATA19H08 D14S592 227‐239
    14 GGAA4A12 D14S588 117‐129
    14 GATA30A03 D14S606 265‐277
    14 GGAA21G11 D14S617 143‐175
    15 ATA24A08 D15S652 288‐306
    15 GATA63A03 D15S659 174‐210
    15 GATA22F01 D15S657 336‐348
    15 GATA27A03 D15S642 197‐213
    15 GATA73F01 D15S816 130‐150
    15 ATA24D04 D15S653 296‐314
    15 GATA50G06 D15S643 207‐223
    15 GATA81F03 D15S817 149‐173
    16 GGAA3G05 D16S753 250‐270
    16 ATA3A07 D16S748 187‐208
    16 GATA81D12 D16S2624 137‐153
    16 GGAA23C09 D16S771 247‐271
    16 GATA22F09 D16S3253 177‐184
    16 GATA71H05 D16S769 263‐275
    16 GATA62G05 D16S2619 143‐163
    17 GTAT1A05 D17S1308 304‐316
    17 GAAT2C03 D17S1298 246‐254
    17 GATA25A04 D17S1299 190‐202
    17 GGAA9D03 D17S1294 252‐264
    17 GATA10H07 D17S969 130‐138
    17 GGAA7D11 D17S1293 260‐290
    17 GATA8C04 D17S974 209‐217
    17 GATA28D11 D17S1301 151‐163
    17 GATA31B11 D17S2059 243‐268
    17 GATA49C09 D17S1290 172‐208
    18 GATA6D09 D18S851 250‐266
    18 ATA23G05 D18S858 196‐208
    18 GATA13 D18S535 131‐155
    18 GATA26C03 D18S1270 280‐292
    18 GATA11A06 D18S542 182‐194
    18 GATA64H04 D18S877 120‐144
    18 GATA10A09 D18S541 269‐283
    18 ATA1H06 D18S844 184‐196
    19 GATA66B04 D19S714 225‐257
    19 GATA29B01 D19S589 162‐182
    19 GAAA1B03 D19S601 210‐224
    19 GGAA2A03 D19S433 190‐210
    19 GATA21G05 D19S1034 216‐232
    20 GATA47F05 D20S481 220‐248
    20 ATA21E04 D20S473 180‐186
    20 GGAA7E02 D20S470 260‐302
    20 GATA46C01 D20S1085 179‐193
    20 GATA81E09 D20S604 125‐158
    20 GATA29F06 D20S477 242‐262
    21 GATA70B08 D21S1446 210‐225
    21 GATA11C12 D21S1432 128‐144
    20 GATA42A03 D20S478 252‐276
    22 GATA6F05 D22S685 172‐197
    22 GGAT3C10 D22S445 110‐130
    22 GATA21F03 D22S689 202‐230
    21 GGAA3C07 D21S1437 119‐143
    7 GATA3F01 D7S820 210‐222
    2 GATA11H10 D2S1360 140‐176
    14 UT562 D14S118 248‐274
    2 GATA86E02 D2S1788 183‐207
    8 UT721 D8S373 194‐218
    2 GATA27A12 D2S1328 156‐164
    7 GATA4E04 D7S1830 212‐220
    2 GGAA20G04 D2S1399 134‐170
    5 GATA11A11 D5S1456 194‐206
    2 ATA27H09 D2S1353 141‐165
    6 GATA32B03 D6S1009 239‐278
    3 GATA68D03 D3S2459 177‐205
    3 GATA92B06 D3S3053 226‐238
    3 GATA8B05 D3S1768 194‐198
    4 GATA5B02 D4S1652 137‐149
    9 ATA25F10 D9S915 195‐216
    4 ATA2A03 D4S2361 152‐158
    6 ATA1F08 D6S1003 298‐316
    3 GATA4A10 D3S1764 228‐248
    7 GATA23F05 D7S1799 174‐182
    11 GATA28D01 D11S2000 204‐244
    14 GATA51F04 D14S611 157‐173
    16 GATA73G05 D16S2622 75‐87
    12 PAH PAH 229‐257
    7 GATA44F09 D7S2847 176‐204
    6 GATA81B01 D6S1277 282‐314
    9 GATA21A06 D9S921 175‐229
    17 GATA64B04 D17S1303 225‐245
    7 GATA24D12 D7S1818 183‐199
    11 ATA27C09 D11S2359 206‐221
    16 GATA11C06 D16S539 152‐158
    12 ATA29A06 D12S1045 80‐104
    7 GATA112F07 D7S2195 237‐297
    9 GATA89A11 D9S1122 191‐211
    13 GATA6B07 D13S325 202‐218
    21 ATA27F01 D21S1440 158‐167
    12 GATA5A09 D12S1090 218‐258
    14 GATA91G06 D14S749 160‐184
    19 GGAT2H06 b 211‐223
    18 ACT1A01 D18S843 180‐189
    19 GATA23B01 D19S586 232‐244
    21 GATA49E01 D21S1435 163‐187
    7 GATA119B03 D7S3047 170‐186
    1 GATA22D12 D1S1594 118‐131
    9 GATA12C06 D9S319 162‐179
    2 GAAT3C11 D2S2949 and D2S1359 128‐136
    14 ATA4B10 D14S580 205‐223
    4 GAAT1D1 D4S2362 144‐152
    9 ATA3H11 D8S569 198‐204
    6 ATA6B05 D6S1034 125‐147
    7 GATA30D09 D7S3058 210‐242
    6 ATA6C09 D6S1035 135‐153
    10 ATA10G07 D10S1218 213‐231
    9 GAAT1C06 D9S905 297‐301
    8 GATA31A06 D8S1989 224‐244
    11 GATA72A01 D11S2367 145‐161
    10 GGAA23C05 D10S1248 241‐265
    13 GGAA22G01 D13S895 161‐181
    15 GTAT1B2 D15S231 110‐114
    15 ATA24A06 D15S651 232‐244
    16 GATA86C08 D16S2625 171‐191
    5 ATA20G07 D5S2488 228‐246
    16 GAAT1E9 D16S749 136‐144
    17 ATA1H07 D17S1288 163‐197
    16 GAAT2B10 D167750 115‐119
    20 ACT1A04 D20S468 195‐209
    17 ATC6A06 D22S686 118‐130
    22 GGAA10F06 D22S686 180‐208
    18 ATA7D07 D18S1357 127‐138
    19 GATA66B01 D19S594 212‐220
    20 GATA86F01 D20S606 147‐163
    19 GATA84G04 b 340‐380
    9 GATA11A07 D9S318 270‐290
    7 GATA87D11 D7S2212 197‐217
    19 ATA9A06 b 223‐229
    10 GATA87G01 D10S1432 165‐181
    1 GATA68D01 b 243‐251
    11 GATA90D07 D11S2371 190‐214
    15 GATA88H02 D15S822 255‐303
    12 GATA73H09 D12S1294 177‐209
    3 GGAA22B10 D3S3054 378‐398
    20 GATA72E11 D20S602 276‐288
    18 GATA88A12 D18S976 172‐200
    22 ATA2G02 D22S1266 245‐260
    21 GATA8G04 D21S1270 186‐194
    3 GATA52H09 D3S2454 248‐260
    22 GATA11B12 D22S683 168‐200

     aMarkers were developed by the Cooperative Human Linkage Center and are grouped into sets than can be coamplified.
     bNo D number has been assigned.
     cThis marker has been mapped to chromosome 9 using radiation hybrids.
  • recipe10× stock amplification buffer (see recipe)
  • 2.0 mM 4dNTP mixture ( appendix 2D)
  • 5 U/µl Taq DNA polymerase
  • Light mineral oil
  • Formamide loading buffer ( appendix 2D)
  • Rain‐X (UNELKO) or Sigmacote (Sigma)
  • recipeBinding solution (see recipe)
  • 96‐well microtiter plate for use in thermal cycler, or 0.5‐ml microcentrifuge tubes
  • Thermal cycler, preferably accommodating 96‐well microtiter plates
  • Equipment for denaturing polyacrylamide gel electrophoresis: e.g., Life Sciences Model S2 sequencing‐gel apparatus
  • Water bath or heat block, 95°C
  • Additional reagents for quantifying DNA by absorption spectroscopy ( appendix 3D), PCR amplification of SSLPs (STRPs; unit 2.5), denaturing polyacrylamide gel electrophoresis ( appendix 3F), and silver staining of gels (unit 2.5)
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Literature Cited

Literature Cited
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