BRIEF REPORTS 2O
Regional Localization of the Highly Polymorphic Locus D11S533 on the Linkage Map of Human Chromosome 1 lq
5.5:1
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I I
I 6.9 x 1011:1
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I,,,
13 8.1 x 1 0 : 1
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FIG. 1, Maximum likelihood order of marker loci. Maximum likelihood pairwise recombination fractions and odds against inversion of adjacent loci are indicated.
Michael Litt, *'1 James H. Eubanks, t Glen A. Evans, t and Thasanavadee Phromchotikul* *Departments of Biochemistry and Medical Genetics, Oregon Health Sciences University, Portland, Oregon 97201; and tMolecular Genetics Laboratory, The 5alk Institute for Biological Studies, La Jolla, California 92037
ReceivedFebruary3, 1992
Recently, two of us (J.H.E. and G.A.E.) described a highly informative polymorphic locus, D l l S 5 3 3 , on h u m a n chromosome l l q 1 3 (3). This locus is a V N T R based upon a degenerate repetitive hexanucleotide sequence [ T ( P u ) T ( P u ) T ( P u ) ] found in cosmid 4F7. I n preliminary studies, analysis of 15 unrelated individuals by means of P C R demonstrated at least 10 alleles ranging in size from 300 to 900 bp. Based on our typing of 40 C E P H families for the D l l S 5 3 3 VNTR, we have now localized this marker on the genetic linkage map of chromosome 11. We have also modified the P C R conditions from those described in our earlier report to improve significantly the reliability of typing at this marker locus. Because of its high heterozygosity a n d ease of typing, D 11 $533 should be a very useful addition to the index map of chromosome 11. P C R reactions were carried out in a total volume of 30 #1 according to Luty et al. (5), except that 0.25 m M spermidine was present in the reaction mixture. Primers 4F7A/f (CTCTGC C T A G T C C C T G G G T G ) a n d 4F7A/r ( T G G G G G T C T G G G AACATG) were each present at a concentration of 0.2 #M. P C R was performed with 38 cycles of denaturation at 94°C for 45 s, annealing at 64°C for 1 min, a n d extension at 72°C for 3 min. W i t h the shorter extension times (0.5-2 min) used in preliminary studies, the larger allele in heterozygotes often amplified very weakly compared to the smaller allele and was sometimes invisible. Lengthening the extension time to 3 m i n usually improved the yield of the larger allelic fragments. A few families were typed using a "touchdown" P C R protocol (2; A. E. Bale, personal communication). Use of this protocol significantly reduced the background often seen with the standard protocol. P C R products were resolved a n d visualized on ethidium bromide-stained 2% agarose gels. Using this method, 55 of 73 unrelated C E P H parents (75%) were found to be heterozygous. P a r e n t s that appeared homozygous by agarose gel analysis were reanalyzed by resolving 32P-labeled products on DNA sequencing gels. Using this method, 9 of the 18 individuals (56%) who appeared homozygous on agarose gels were shown to be heterozygous. Hence, we estimate the heterozygosity as 64/73 or 88%. Data on other l l q markers were obtained from the C E P H database, version 5. Pairwise a n d multipoint linkage analyses were performed using the L I N K A G E computer program pack1To whom correspondence should be addressed. GENOMICS 14, 820 (1992) 0888-7543/92 $5.00 Copyright © 1992by AcademicPress, Inc. All rights of reproductionin any form reserved.
6x 10:1
820
age (version 5.1) on a S u n Sparcstation 2. Results of pairwise linkage analysis (data not shown), taken together with the linkage map reported by Julier et al. (4), suggested t h a t D l l S 5 3 3 lies within the group of markers cen-D11S288PGA-PYGM-DllS97-INT2-DllS389-(DllS388,DllS85)qter. Using 3-point analysis, we excluded D l l S 5 3 3 from the intervals D l l S 2 8 8 - P G A and P G A - P Y G M , with odds >104:1 favoring these exclusions. To locate D l l S 5 3 3 within the region not excluded by these 3-point analyses, we next performed a 6-point analysis using data for D l l S 5 3 3 plus the five markers D l l S 9 7 , INT2, D l l S 3 8 9 , D l l S 3 8 8 , and D l l S 8 5 . The m a x i m u m likelihood order of markers derived from this analysis is shown in Fig. 1, together with the pairwise recombination fractions and the odds against inversion of adjacent markers. All other orders not shown in Fig. 1 were less likely t h a n the m a x i m u m likelihood order by odds of at least 104:1. Our localization of D l l S 5 3 3 proximal to D l l S 3 8 8 is consist e n t with in situ hybridization data placing D l l S 5 3 3 within 11q13 (3), proximal to the cytogenetically mapped position of D l l S 3 8 8 at 11q14.3 (1). Also, our map is consistent with the map reported by Julier et al. (4), with the exception that they obtained much stronger support for the order cen-D11S97I N T 2 - q t e r t h a n we did. This may perhaps be attributed to their use of data from 59 C E P H families, whereas we analyzed data from only 40 families. ACKNOWLEDGMENTS This project was supported by NIH Grant HG00022 to M.L. We thank Patricia Kramer and Bill Becker for assistance with linkage analysis. REFERENCES 1. Cherif, D., Julier, C., Delattre, O., Derr~, J., Lathrop, G. M., and Berger, R. (1990). Simultaneous localization of eosmids and chromosome R-banding by fluorescence microscopy: Application to regional mapping of human chromosome 11. Proc. Natl. Acad. Sci. USA 87: 6639-6643. 2. Don, R. H., Cox, P. T., Wainwright, B. J., Baker, K., and Mattick, J. S. (1991). Touchdown PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 19: 4008. 3. Eubanks, J. H., Selleri, L., Hart, R., Rosette, C., and Evans, G. A. (1991). Isolation, localization, and physical mapping of a highly polymorphie locus on human chromosome 11q13. Genornics 11: 720-729. 4. Julier, C., Nakamura, Y., Lathrop, M., O'Connell, P., Leppert, M., Litt, M., Mohandas, T., Lalouel, J.-M., and White, R. (1990). Detailed map of the long arm of chromosome 11. Genomics 7: 335-345. 5. Luty, J. A., Guo, Z., Willard, H. F., Ledbetter, D. H., Ledbetter, S., and Litt, M. (1990). Five polymorphic microsatellite VNTRs on the human X chromosome. Am. J. Hum. Genet. 46: 776-783.
Regional Localization of the Highly Polymorphic Locus D11S533 on the Linkage Map of Human Chromosome 1 lq
5.5:1
[
I I
I 6.9 x 1011:1
1.25:1
I,,,
13 8.1 x 1 0 : 1
I
FIG. 1, Maximum likelihood order of marker loci. Maximum likelihood pairwise recombination fractions and odds against inversion of adjacent loci are indicated.
Michael Litt, *'1 James H. Eubanks, t Glen A. Evans, t and Thasanavadee Phromchotikul* *Departments of Biochemistry and Medical Genetics, Oregon Health Sciences University, Portland, Oregon 97201; and tMolecular Genetics Laboratory, The 5alk Institute for Biological Studies, La Jolla, California 92037
ReceivedFebruary3, 1992
Recently, two of us (J.H.E. and G.A.E.) described a highly informative polymorphic locus, D l l S 5 3 3 , on h u m a n chromosome l l q 1 3 (3). This locus is a V N T R based upon a degenerate repetitive hexanucleotide sequence [ T ( P u ) T ( P u ) T ( P u ) ] found in cosmid 4F7. I n preliminary studies, analysis of 15 unrelated individuals by means of P C R demonstrated at least 10 alleles ranging in size from 300 to 900 bp. Based on our typing of 40 C E P H families for the D l l S 5 3 3 VNTR, we have now localized this marker on the genetic linkage map of chromosome 11. We have also modified the P C R conditions from those described in our earlier report to improve significantly the reliability of typing at this marker locus. Because of its high heterozygosity a n d ease of typing, D 11 $533 should be a very useful addition to the index map of chromosome 11. P C R reactions were carried out in a total volume of 30 #1 according to Luty et al. (5), except that 0.25 m M spermidine was present in the reaction mixture. Primers 4F7A/f (CTCTGC C T A G T C C C T G G G T G ) a n d 4F7A/r ( T G G G G G T C T G G G AACATG) were each present at a concentration of 0.2 #M. P C R was performed with 38 cycles of denaturation at 94°C for 45 s, annealing at 64°C for 1 min, a n d extension at 72°C for 3 min. W i t h the shorter extension times (0.5-2 min) used in preliminary studies, the larger allele in heterozygotes often amplified very weakly compared to the smaller allele and was sometimes invisible. Lengthening the extension time to 3 m i n usually improved the yield of the larger allelic fragments. A few families were typed using a "touchdown" P C R protocol (2; A. E. Bale, personal communication). Use of this protocol significantly reduced the background often seen with the standard protocol. P C R products were resolved a n d visualized on ethidium bromide-stained 2% agarose gels. Using this method, 55 of 73 unrelated C E P H parents (75%) were found to be heterozygous. P a r e n t s that appeared homozygous by agarose gel analysis were reanalyzed by resolving 32P-labeled products on DNA sequencing gels. Using this method, 9 of the 18 individuals (56%) who appeared homozygous on agarose gels were shown to be heterozygous. Hence, we estimate the heterozygosity as 64/73 or 88%. Data on other l l q markers were obtained from the C E P H database, version 5. Pairwise a n d multipoint linkage analyses were performed using the L I N K A G E computer program pack1To whom correspondence should be addressed. GENOMICS 14, 820 (1992) 0888-7543/92 $5.00 Copyright © 1992by AcademicPress, Inc. All rights of reproductionin any form reserved.
6x 10:1
820
age (version 5.1) on a S u n Sparcstation 2. Results of pairwise linkage analysis (data not shown), taken together with the linkage map reported by Julier et al. (4), suggested t h a t D l l S 5 3 3 lies within the group of markers cen-D11S288PGA-PYGM-DllS97-INT2-DllS389-(DllS388,DllS85)qter. Using 3-point analysis, we excluded D l l S 5 3 3 from the intervals D l l S 2 8 8 - P G A and P G A - P Y G M , with odds >104:1 favoring these exclusions. To locate D l l S 5 3 3 within the region not excluded by these 3-point analyses, we next performed a 6-point analysis using data for D l l S 5 3 3 plus the five markers D l l S 9 7 , INT2, D l l S 3 8 9 , D l l S 3 8 8 , and D l l S 8 5 . The m a x i m u m likelihood order of markers derived from this analysis is shown in Fig. 1, together with the pairwise recombination fractions and the odds against inversion of adjacent markers. All other orders not shown in Fig. 1 were less likely t h a n the m a x i m u m likelihood order by odds of at least 104:1. Our localization of D l l S 5 3 3 proximal to D l l S 3 8 8 is consist e n t with in situ hybridization data placing D l l S 5 3 3 within 11q13 (3), proximal to the cytogenetically mapped position of D l l S 3 8 8 at 11q14.3 (1). Also, our map is consistent with the map reported by Julier et al. (4), with the exception that they obtained much stronger support for the order cen-D11S97I N T 2 - q t e r t h a n we did. This may perhaps be attributed to their use of data from 59 C E P H families, whereas we analyzed data from only 40 families. ACKNOWLEDGMENTS This project was supported by NIH Grant HG00022 to M.L. We thank Patricia Kramer and Bill Becker for assistance with linkage analysis. REFERENCES 1. Cherif, D., Julier, C., Delattre, O., Derr~, J., Lathrop, G. M., and Berger, R. (1990). Simultaneous localization of eosmids and chromosome R-banding by fluorescence microscopy: Application to regional mapping of human chromosome 11. Proc. Natl. Acad. Sci. USA 87: 6639-6643. 2. Don, R. H., Cox, P. T., Wainwright, B. J., Baker, K., and Mattick, J. S. (1991). Touchdown PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 19: 4008. 3. Eubanks, J. H., Selleri, L., Hart, R., Rosette, C., and Evans, G. A. (1991). Isolation, localization, and physical mapping of a highly polymorphie locus on human chromosome 11q13. Genornics 11: 720-729. 4. Julier, C., Nakamura, Y., Lathrop, M., O'Connell, P., Leppert, M., Litt, M., Mohandas, T., Lalouel, J.-M., and White, R. (1990). Detailed map of the long arm of chromosome 11. Genomics 7: 335-345. 5. Luty, J. A., Guo, Z., Willard, H. F., Ledbetter, D. H., Ledbetter, S., and Litt, M. (1990). Five polymorphic microsatellite VNTRs on the human X chromosome. Am. J. Hum. Genet. 46: 776-783.
