Summary
We have developed a novel technique that is useful for the rapid detection of previously characterized point mutations in the human genome. The method relies on the differential termination of primer extension being performed simultaneously on normal and mutant template molecules in the presence of a selectively limited complement of deoxynucleotide triphosphates. We have used this technique to determine the carrier status of two potential carriers of a hemophilia B mutation at codon 145 in the factor IX gene. In addition, the technique has been used to quantify the levels of mutant sequence in several tissues of a hemophilia B patient who is a somatic mosaic for a point mutation at codon 350.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bottema CDK, Koeberl DD, Sommer SS (1989) Direct carrier testing in 14 families with haemophilia B. Lancet II:526–529
Forrest S, Cotton RGH (1990) Methods of detection of single base substitutions in clinical genetic practice. Mol Biol Med 7:451–459
Giannelli F, Green PM, High KA, Sommer S, Lillicrap DP, Ludwig M, Olek K, Reitsma PH, Goossens M, Yoshioka A, Brownlee GG (1991) Haemophilia B: database of point mutations and short additions and deletions, 2nd edn. Nucleic Acids Res 19 [Suppl]:2193–2219
Green PM, Bentley DR, Mibashan RS, Nilsson I-M, Giannelli F (1989) Molecular pathology of haemophilia B. EMBO J 8:1067–1072
Grompe M, Gibbs RA, Chamberlain JS, Caskey CT (1989) Detection of new mutation in man and mouse. Mol Biol Med 6:511–521
Gyllensten UB, Erlich HA (1988) Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc Natl Acad Sci USA 85:7652–7656
Koeberl DD, Bottema CDK, Ketterling RP, Bridge PJ, Lillicrap DP, Sommer SS (1990a) Mutations causing hemophilia B: direct estimate of the underlying rates of spontaneous germline transitions, transversions, and deletions in a human gene. Am J Hum Genet 47:202–217
Koeberl DD, Bottema CDK, Sommer SS (1990b) Comparison of direct and indirect methods of carrier detection in an X-linked disease. Am J Med Genet 35:600–608
Kuppuswamy MN, Hoffmann JW, Kasper CK, Spitzer SG, Groce SL, Bajaj SP (1991) Single nucleotide primer extension to detect genetic diseases: experimental application to hemophilia B (factor IX) and cystic fibrosis genes Proc Natl Acad Sci USA 88:1143–1147
Lillicrap DP, Liddell MB, Matthews RJ, Peake IR, Bloom AL (1986) Comparison of phenotypic assessment and the use of two restriction fragment length polymorphisms in the diagnosis of the carrier state in haemophilia B. Br J Haematol 62:557–565
Sokolov BP (1990) Primer extension technique for the detection of single nucleotide in genomic DNA. Nucleic Acids Res 18:3671
Sylvanen A-C, Aalto-Setala K, Harju L, Kontula K, Soderlund H (1990) A primer guided nucleotide incorporation assay in the genotyping of apolipoprotein E. Genomics 8:684–692
Taylor SAM, Deugau KV, Lillicrap DP (1991) Somatic mosaicism and female to female transmission in a kindred with hemophilia B (factor IX deficiency). Proc Natl Acad Sci USA 88:39–42
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Picketts, D.J., Cameron, C., Taylor, S.A.M. et al. Differential termination of primer extension: a novel, quantifiable method for detection of point mutations. Hum Genet 89, 155–157 (1992). https://doi.org/10.1007/BF00217115
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00217115