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. 1989 Sep;86(17):6686–6690. doi: 10.1073/pnas.86.17.6686

Alu polymerase chain reaction: a method for rapid isolation of human-specific sequences from complex DNA sources.

D L Nelson 1, S A Ledbetter 1, L Corbo 1, M F Victoria 1, R Ramírez-Solis 1, T D Webster 1, D H Ledbetter 1, C T Caskey 1
PMCID: PMC297910  PMID: 2771952

Abstract

Current efforts to map the human genome are focused on individual chromosomes or smaller regions and frequently rely on the use of somatic cell hybrids. We report the application of the polymerase chain reaction to direct amplification of human DNA from hybrid cells containing regions of the human genome in rodent cell backgrounds using primers directed to the human Alu repeat element. We demonstrate Alu-directed amplification of a fragment of the human HPRT gene from both hybrid cell and cloned DNA and identify through sequence analysis the Alu repeats involved in this amplification. We also demonstrate the application of this technique to identify the chromosomal locations of large fragments of the human X chromosome cloned in a yeast artificial chromosome and the general applicability of the method to the preparation of DNA probes from cloned human sequences. The technique allows rapid gene mapping and provides a simple method for the isolation and analysis of specific chromosomal regions.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bender W., Spierer P., Hogness D. S. Chromosomal walking and jumping to isolate DNA from the Ace and rosy loci and the bithorax complex in Drosophila melanogaster. J Mol Biol. 1983 Jul 25;168(1):17–33. doi: 10.1016/s0022-2836(83)80320-9. [DOI] [PubMed] [Google Scholar]
  2. Bosma P. J., van den Berg E. A., Kooistra T., Siemieniak D. R., Slightom J. L. Human plasminogen activator inhibitor-1 gene. Promoter and structural gene nucleotide sequences. J Biol Chem. 1988 Jul 5;263(19):9129–9141. [PubMed] [Google Scholar]
  3. Britten R. J., Baron W. F., Stout D. B., Davidson E. H. Sources and evolution of human Alu repeated sequences. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4770–4774. doi: 10.1073/pnas.85.13.4770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burke D. T., Carle G. F., Olson M. V. Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science. 1987 May 15;236(4803):806–812. doi: 10.1126/science.3033825. [DOI] [PubMed] [Google Scholar]
  5. Chang S. M., Wager-Smith K., Tsao T. Y., Henkel-Tigges J., Vaishnav S., Caskey C. T. Construction of a defective retrovirus containing the human hypoxanthine phosphoribosyltransferase cDNA and its expression in cultured cells and mouse bone marrow. Mol Cell Biol. 1987 Feb;7(2):854–863. doi: 10.1128/mcb.7.2.854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Coulson A., Waterston R., Kiff J., Sulston J., Kohara Y. Genome linking with yeast artificial chromosomes. Nature. 1988 Sep 8;335(6186):184–186. doi: 10.1038/335184a0. [DOI] [PubMed] [Google Scholar]
  7. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  8. Gibbs R. A., Nguyen P. N., Caskey C. T. Detection of single DNA base differences by competitive oligonucleotide priming. Nucleic Acids Res. 1989 Apr 11;17(7):2437–2448. doi: 10.1093/nar/17.7.2437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gitschier J., Lawn R. M., Rotblat F., Goldman E., Tuddenham E. G. Antenatal diagnosis and carrier detection of haemophilia A using factor VIII gene probe. Lancet. 1985 May 11;1(8437):1093–1094. doi: 10.1016/s0140-6736(85)92388-8. [DOI] [PubMed] [Google Scholar]
  10. Gusella J. F., Keys C., VarsanyiBreiner A., Kao F. T., Jones C., Puck T. T., Housman D. Isolation and localization of DNA segments from specific human chromosomes. Proc Natl Acad Sci U S A. 1980 May;77(5):2829–2833. doi: 10.1073/pnas.77.5.2829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jelinek W. R., Schmid C. W. Repetitive sequences in eukaryotic DNA and their expression. Annu Rev Biochem. 1982;51:813–844. doi: 10.1146/annurev.bi.51.070182.004121. [DOI] [PubMed] [Google Scholar]
  12. Kariya Y., Kato K., Hayashizaki Y., Himeno S., Tarui S., Matsubara K. Revision of consensus sequence of human Alu repeats--a review. Gene. 1987;53(1):1–10. doi: 10.1016/0378-1119(87)90087-4. [DOI] [PubMed] [Google Scholar]
  13. Kim S. H., Moores J. C., David D., Respess J. G., Jolly D. J., Friedmann T. The organization of the human HPRT gene. Nucleic Acids Res. 1986 Apr 11;14(7):3103–3118. doi: 10.1093/nar/14.7.3103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Korenberg J. R., Rykowski M. C. Human genome organization: Alu, lines, and the molecular structure of metaphase chromosome bands. Cell. 1988 May 6;53(3):391–400. doi: 10.1016/0092-8674(88)90159-6. [DOI] [PubMed] [Google Scholar]
  15. Lee C. C., Wu X. W., Gibbs R. A., Cook R. G., Muzny D. M., Caskey C. T. Generation of cDNA probes directed by amino acid sequence: cloning of urate oxidase. Science. 1988 Mar 11;239(4845):1288–1291. doi: 10.1126/science.3344434. [DOI] [PubMed] [Google Scholar]
  16. Moyzis R. K., Torney D. C., Meyne J., Buckingham J. M., Wu J. R., Burks C., Sirotkin K. M., Goad W. B. The distribution of interspersed repetitive DNA sequences in the human genome. Genomics. 1989 Apr;4(3):273–289. doi: 10.1016/0888-7543(89)90331-5. [DOI] [PubMed] [Google Scholar]
  17. Nathans J., Thomas D., Hogness D. S. Molecular genetics of human color vision: the genes encoding blue, green, and red pigments. Science. 1986 Apr 11;232(4747):193–202. doi: 10.1126/science.2937147. [DOI] [PubMed] [Google Scholar]
  18. Nussbaum R. L., Airhart S. D., Ledbetter D. H. A rodent-human hybrid containing Xq24-qter translocated to a hamster chromosome expresses the Xq27 folate-sensitive fragile site. Am J Med Genet. 1986 Jan-Feb;23(1-2):457–466. doi: 10.1002/ajmg.1320230137. [DOI] [PubMed] [Google Scholar]
  19. Nussbaum R. L., Airhart S. D., Ledbetter D. H. Expression of the fragile (X) chromosome in an interspecific somatic cell hybrid. Hum Genet. 1983;64(2):148–150. doi: 10.1007/BF00327113. [DOI] [PubMed] [Google Scholar]
  20. Patel P. I., Framson P. E., Caskey C. T., Chinault A. C. Fine structure of the human hypoxanthine phosphoribosyltransferase gene. Mol Cell Biol. 1986 Feb;6(2):393–403. doi: 10.1128/mcb.6.2.393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Patterson M., Schwartz C., Bell M., Sauer S., Hofker M., Trask B., van den Engh G., Davies K. E. Physical mapping studies on the human X chromosome in the region Xq27-Xqter. Genomics. 1987 Dec;1(4):297–306. doi: 10.1016/0888-7543(87)90028-0. [DOI] [PubMed] [Google Scholar]
  22. Persico M. G., Viglietto G., Martini G., Toniolo D., Paonessa G., Moscatelli C., Dono R., Vulliamy T., Luzzatto L., D'Urso M. Isolation of human glucose-6-phosphate dehydrogenase (G6PD) cDNA clones: primary structure of the protein and unusual 5' non-coding region. Nucleic Acids Res. 1986 Mar 25;14(6):2511–2522. doi: 10.1093/nar/14.6.2511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Reilly D. S., Lewis R. A., Ledbetter D. H., Nussbaum R. L. Tightly linked flanking markers for the Lowe oculocerebrorenal syndrome, with application to carrier assessment. Am J Hum Genet. 1988 May;42(5):748–755. [PMC free article] [PubMed] [Google Scholar]
  24. Ruddle F. H. A new era in mammalian gene mapping: somatic cell genetics and recombinant DNA methodologies. Nature. 1981 Nov 12;294(5837):115–120. doi: 10.1038/294115a0. [DOI] [PubMed] [Google Scholar]
  25. Scott A. F., Phillips J. A., 3rd, Migeon B. R. DNA restriction endonuclease analysis for localization of human beta- and delta-globin genes on chromosome 11. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4563–4565. doi: 10.1073/pnas.76.9.4563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Singer M. F. SINEs and LINEs: highly repeated short and long interspersed sequences in mammalian genomes. Cell. 1982 Mar;28(3):433–434. doi: 10.1016/0092-8674(82)90194-5. [DOI] [PubMed] [Google Scholar]
  27. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  28. Triglia T., Peterson M. G., Kemp D. J. A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences. Nucleic Acids Res. 1988 Aug 25;16(16):8186–8186. doi: 10.1093/nar/16.16.8186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wahl G. M., Lewis K. A., Ruiz J. C., Rothenberg B., Zhao J., Evans G. A. Cosmid vectors for rapid genomic walking, restriction mapping, and gene transfer. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2160–2164. doi: 10.1073/pnas.84.8.2160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Warren S. T., Zhang F., Licameli G. R., Peters J. F. The fragile X site in somatic cell hybrids: an approach for molecular cloning of fragile sites. Science. 1987 Jul 24;237(4813):420–423. doi: 10.1126/science.3603029. [DOI] [PubMed] [Google Scholar]
  31. van Tuinen P., Rich D. C., Summers K. M., Ledbetter D. H. Regional mapping panel for human chromosome 17: application to neurofibromatosis type 1. Genomics. 1987 Dec;1(4):374–381. doi: 10.1016/0888-7543(87)90042-5. [DOI] [PubMed] [Google Scholar]

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