Single nucleotide primer extension to detect genetic diseases: Experimental application to hemophilia B (factor IX) and cystic fibrosis genes

Journal Article · Fri Feb 15 04:00:00 EST 1991 · Proceedings of the National Academy of Sciences of the United States of America; (United States)

DOI:https://doi.org/10.1073/pnas.88.4.1143· OSTI ID:6107772

Kuppuswamy, M N; Hoffmann, J W; Spitzer, S G; Groce, S L; Bajaj, S P ^[1]; Kasper, C K ^[2]

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1. St. Louis Univ. School of Medicine, MO (United States)
2. Univ. of Southern California, Los Angeles (United States)

In this report, the authors describe an approach to detect the presence of abnormal alleles in those genetic diseases in which frequency of occurrence of the same mutation is high (e.g., hemophilia B). Initially, from each subject, the DNA fragment containing the putative mutation site is amplified by the polymerase chain reaction. For each fragment two reaction mixtures are then prepared. Each contains the amplified fragment, a primer (18-mer or longer) whose sequence is identical to the coding sequence of the normal gene immediately flanking the 5{prime} end of the mutation site, and either an {alpha}-{sup 32}P-labeled nucleotide corresponding to the normal coding sequence at the mutation site or an {alpha}-{sup 32}P-labeled nucleotide corresponding to the mutant sequence. An essential feature of the present methodology is that the base immediately 3{prime} to the template-bound primer is one of those altered in the mutant, since in this way an extension of the primer by a single base will give an extended molecule characteristic of either the mutant or the wild type. The method is rapid and should be useful in carrier detection and prenatal diagnosis of every genetic disease with a known sequence variation.

• OSTI ID: 6107772
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (United States), Journal Name: Proceedings of the National Academy of Sciences of the United States of America; (United States) Vol. 88:4; ISSN PNASA; ISSN 0027-8424
• Country of Publication: United States
• Language: English

Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
62 RADIOLOGY AND NUCLEAR MEDICINE
AUTORADIOGRAPHY
BLOOD COAGULATION FACTORS
COAGULANTS
DIAGNOSIS
DISEASES
DNA POLYMERASES
DRUGS
ELECTROPHORESIS
ENZYMES
FIBROSIS
GENE AMPLIFICATION
GENE MUTATIONS
HEMATOLOGIC AGENTS
HEMIC DISEASES
HEMOPHILIA
HEREDITARY DISEASES
MUTATIONS
NUCLEOTIDYLTRANSFERASES
ORGANIC COMPOUNDS
PATHOLOGICAL CHANGES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
PROTEINS
RFLPS
TRANSFERASES