Human Molecular Genetics, Vol. 1, No. 1 67
© 7992 Oxford University Press
Dinucleotide repeat polymorphism at the GABAA receptor 03 (GABRB3) locus in the Angelman/Prader - Willi region (AS/PWS) of chromosome 15 Apiwat Mutirangura, Susan A.Ledbetter, Akira Kuwano, A.Craig Chinault and David H.Ledbetter Institute for Molecular Genetics, Baylor College of Medicine, Houston, TX 77030, USA
Primer Sequences: CA strand: 5'-CTCTTGTTCCTGTTGCTTTCAATACAC-3' GT strand: 5'-CACTGTGCTAGTAGATTCAGCTC-3' Length of amplified fragment: 181 to 201 bp. Polymorphism: Estimated from 80 chromosomes of 40 unrelated individuals. Allele Basepairs Frequency Allele Basepairs Frequency Al 201 0.050 A7 189 0.013 A2 199 0.050 A8 187 0.013 A3 197 0.138 A9 185 0.088 A4 195 0.113 A10 183 0.088 A5 193 0.088 All 181 0.325 A6 191 0.038 % Heterozygosity = 82.5% PIC = 0.83. Chromosomal Localization: Assigned to the AS/PWS region at 15ql 1 —13. The localization of the YAC was confirmed by fluorescence in situ hybridization. The dinucleotide repeat was confirmed to be on chromosome 15 by PCR of somatic cell hybrids. PCR Conditions: PCR was performed in a total volume of 20 y\ containing 25 ng genomic DNA, 0.5 /iM of each primer, 50 mM KCL, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 0.01% (w/v) gelatin, 250 yM each dNTP, and 0.5 unit Taq polymerase (Perkin-Elmer/Cetus). Initial denaturation was at 95 °C for 4 min, followed by 26 cycles of 94°C/1 min, 55°C/2 min, 72°C/2 min, and a final extension of 7 min at 72°C. Acknowledgements: Supported by grants from the Beneficia Foundation and NIH (HD20619 and HG00210). References: 1) Wagstaff,J. etal. (1991) Am. J. Hum. Genet. 49, 330-337. 2) Ymer.S. etal. (1989) EMBO J. 8, 1665-1670. 3) Green.E.D. and Olson.M.V. (1990) Proc. Natl. Acad. Sci USA 87, 1213-1217. 4) Feener.C.A. etal. (1991) Am. J. Hum. Genet. 48, 621-627.
V.Sharma and M.Litt Departments of Biochemistry and Molecular Biology and Molecular and Medical Genetics, Oregon Health Sciences University, Portland, OR 97201, USA
Source and Description: Cosmid ICRFcl02BO6134 was from a flow sorted chromosome 21 cosmid library. DNA sequences flanking a (TCTA)4 (TCTG)6 (TCTA)3TA(TCTA)3TC(ATCT)io repeat within an Alu I subclone (VS17TB2) of this cosmid (GenBank accession no. M84567) were used to design PCR primers. PCR Primers: VS17T #3: 5'-GTGAGTCAATTCCCCAAG-3' VS17T #4: 5'-GTTGTATTAGTCAATGTTCTCC-3' Polymorphism: Allelic fragments were resolved on 4.5% denaturing acrylamide gels. Allele El E2 E3 E4 E5 E6
Length(nt)
Frequency
264
.006
248 240
.013 .117
236 232
.026 .065
228
.097
Allele E7 E8 E9 E10 Ell E12
Length(nt) 224 220 212 204
1% 172
Frequency .032 .247 .221 .156 .013 .006
Alleles found in four CEPH parents were: 133101 == A3, A9; 133102 = A4, A5; 137701 = A7, A8; 137702 = A9, A l l . Allele frequencies were measured in 72 unrelated European Caucasians. The heterozygosity was 90% and the PIC was 0.82. Chromosomal Localization and Mendelian Inheritance: Cosmid ICRFcl02B06134 was identified by hybridization with the D21S11 probe pPW236B. Linkage analysis with the D2IS 11 EcoRI RFLP in 5 CEPH families (50 informative meioses) gave a maximum LOD score of 14.1 at d = 0. Mendelian inheritance was observed in all cases. PCR Conditions: PCR was performed in a total volume of 12.5 jtl using standard buffer and dNTP concentrations according to (1) except that 0.25 mM spermidine was included and the products were not radioactively labelled. 'Touchdown' amplification (2) in a Perkin-Elmer/Cetus System 9600™ thermocycler was performed, using an initial annealing temperature of 62° for 1 min which was decreased by 1 degree per cycle for the first 10 cycles. For the final 25 cycles, the annealing temperature was held constant at 52° and the annealing time was 30 sec. For all cycles, denaturation was for 1 min at 94° and extension was for 1 min at 72°. PCR products were resolved on DNA sequencing gels, capillary-blotted onto Hybond N + ™ membranes and detected by probing with 5'[32P] labeled (GATA)7 oligomer. Acknowledgements: This work was supported by NIH Grant HG00022. We thank H. Lehrach for providing cosmid ICRFcl02B06134. References: 1) Lutty.J.A., Guo.Z., Willard.H.F., Ledbetter.D.H., Ledbetter.S. and Litt.M. (1990) Am. J. Hum. Genet. 46, 776-783. 2) Don.R.H., Cox,P.T., Wainwright.B.J., Baker.K. and Mattick.J.S. (1991) Nucl Acids Res. 19, 4008.
Downloaded from http://hmg.oxfordjournals.org/ at University of Saskatchewan on June 18, 2015
Source/Description: The GABRB3 gene has previously been mapped to proximal 15q in the AS/PWS region (1). Using primers generated from the sequence of the rat GABRB3 cDNA (2), a DNA probe was generated by PCR of human genomic DNA and used to screen a total human YAC libary (3). Clone B25E9 (150 kb) was identified and confirmed to map to proximal 15q by fluorescence in situ hybridization. Alu-PCR products from this YAC were screened for CA repeats by modification of a PCR method using a combination of Alu and CA or GT primers (4). Positive fragments were subcloned into pBS SK and sequenced; sequences flanking a ACAC(CA)17 repeat element were used to design PCR primers. (EMBL accession no. X63670.)
Tetranucleotide repeat polymorphism at the D21S11 locus
© 7992 Oxford University Press
Dinucleotide repeat polymorphism at the GABAA receptor 03 (GABRB3) locus in the Angelman/Prader - Willi region (AS/PWS) of chromosome 15 Apiwat Mutirangura, Susan A.Ledbetter, Akira Kuwano, A.Craig Chinault and David H.Ledbetter Institute for Molecular Genetics, Baylor College of Medicine, Houston, TX 77030, USA
Primer Sequences: CA strand: 5'-CTCTTGTTCCTGTTGCTTTCAATACAC-3' GT strand: 5'-CACTGTGCTAGTAGATTCAGCTC-3' Length of amplified fragment: 181 to 201 bp. Polymorphism: Estimated from 80 chromosomes of 40 unrelated individuals. Allele Basepairs Frequency Allele Basepairs Frequency Al 201 0.050 A7 189 0.013 A2 199 0.050 A8 187 0.013 A3 197 0.138 A9 185 0.088 A4 195 0.113 A10 183 0.088 A5 193 0.088 All 181 0.325 A6 191 0.038 % Heterozygosity = 82.5% PIC = 0.83. Chromosomal Localization: Assigned to the AS/PWS region at 15ql 1 —13. The localization of the YAC was confirmed by fluorescence in situ hybridization. The dinucleotide repeat was confirmed to be on chromosome 15 by PCR of somatic cell hybrids. PCR Conditions: PCR was performed in a total volume of 20 y\ containing 25 ng genomic DNA, 0.5 /iM of each primer, 50 mM KCL, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 0.01% (w/v) gelatin, 250 yM each dNTP, and 0.5 unit Taq polymerase (Perkin-Elmer/Cetus). Initial denaturation was at 95 °C for 4 min, followed by 26 cycles of 94°C/1 min, 55°C/2 min, 72°C/2 min, and a final extension of 7 min at 72°C. Acknowledgements: Supported by grants from the Beneficia Foundation and NIH (HD20619 and HG00210). References: 1) Wagstaff,J. etal. (1991) Am. J. Hum. Genet. 49, 330-337. 2) Ymer.S. etal. (1989) EMBO J. 8, 1665-1670. 3) Green.E.D. and Olson.M.V. (1990) Proc. Natl. Acad. Sci USA 87, 1213-1217. 4) Feener.C.A. etal. (1991) Am. J. Hum. Genet. 48, 621-627.
V.Sharma and M.Litt Departments of Biochemistry and Molecular Biology and Molecular and Medical Genetics, Oregon Health Sciences University, Portland, OR 97201, USA
Source and Description: Cosmid ICRFcl02BO6134 was from a flow sorted chromosome 21 cosmid library. DNA sequences flanking a (TCTA)4 (TCTG)6 (TCTA)3TA(TCTA)3TC(ATCT)io repeat within an Alu I subclone (VS17TB2) of this cosmid (GenBank accession no. M84567) were used to design PCR primers. PCR Primers: VS17T #3: 5'-GTGAGTCAATTCCCCAAG-3' VS17T #4: 5'-GTTGTATTAGTCAATGTTCTCC-3' Polymorphism: Allelic fragments were resolved on 4.5% denaturing acrylamide gels. Allele El E2 E3 E4 E5 E6
Length(nt)
Frequency
264
.006
248 240
.013 .117
236 232
.026 .065
228
.097
Allele E7 E8 E9 E10 Ell E12
Length(nt) 224 220 212 204
1% 172
Frequency .032 .247 .221 .156 .013 .006
Alleles found in four CEPH parents were: 133101 == A3, A9; 133102 = A4, A5; 137701 = A7, A8; 137702 = A9, A l l . Allele frequencies were measured in 72 unrelated European Caucasians. The heterozygosity was 90% and the PIC was 0.82. Chromosomal Localization and Mendelian Inheritance: Cosmid ICRFcl02B06134 was identified by hybridization with the D21S11 probe pPW236B. Linkage analysis with the D2IS 11 EcoRI RFLP in 5 CEPH families (50 informative meioses) gave a maximum LOD score of 14.1 at d = 0. Mendelian inheritance was observed in all cases. PCR Conditions: PCR was performed in a total volume of 12.5 jtl using standard buffer and dNTP concentrations according to (1) except that 0.25 mM spermidine was included and the products were not radioactively labelled. 'Touchdown' amplification (2) in a Perkin-Elmer/Cetus System 9600™ thermocycler was performed, using an initial annealing temperature of 62° for 1 min which was decreased by 1 degree per cycle for the first 10 cycles. For the final 25 cycles, the annealing temperature was held constant at 52° and the annealing time was 30 sec. For all cycles, denaturation was for 1 min at 94° and extension was for 1 min at 72°. PCR products were resolved on DNA sequencing gels, capillary-blotted onto Hybond N + ™ membranes and detected by probing with 5'[32P] labeled (GATA)7 oligomer. Acknowledgements: This work was supported by NIH Grant HG00022. We thank H. Lehrach for providing cosmid ICRFcl02B06134. References: 1) Lutty.J.A., Guo.Z., Willard.H.F., Ledbetter.D.H., Ledbetter.S. and Litt.M. (1990) Am. J. Hum. Genet. 46, 776-783. 2) Don.R.H., Cox,P.T., Wainwright.B.J., Baker.K. and Mattick.J.S. (1991) Nucl Acids Res. 19, 4008.
Downloaded from http://hmg.oxfordjournals.org/ at University of Saskatchewan on June 18, 2015
Source/Description: The GABRB3 gene has previously been mapped to proximal 15q in the AS/PWS region (1). Using primers generated from the sequence of the rat GABRB3 cDNA (2), a DNA probe was generated by PCR of human genomic DNA and used to screen a total human YAC libary (3). Clone B25E9 (150 kb) was identified and confirmed to map to proximal 15q by fluorescence in situ hybridization. Alu-PCR products from this YAC were screened for CA repeats by modification of a PCR method using a combination of Alu and CA or GT primers (4). Positive fragments were subcloned into pBS SK and sequenced; sequences flanking a ACAC(CA)17 repeat element were used to design PCR primers. (EMBL accession no. X63670.)
Tetranucleotide repeat polymorphism at the D21S11 locus
