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Trapped‐oligonucleotide nucleotide incorporation (TONI) assay, a simple method for screening point mutations
Author(s) -
Prezant Toni R.,
FischelGhodsian Nathan
Publication year - 1992
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.1380010212
Subject(s) - oligonucleotide , microbiology and biotechnology , biology , taq polymerase , biotinylation , point mutation , primer (cosmetics) , restriction enzyme , streptavidin , dna , mutant , primer extension , nucleotide , sequencing by ligation , genetics , dna polymerase , chemistry , genomic library , biotin , gene , base sequence , thermus aquaticus , organic chemistry
We present a simple screening method for detecting a known point mutation, using only one 5′‐biotinylated oligonucleotide primer, with its 3′ end adjacent to the mutation site. In parallel reactions, an amplified DNA template encompassing the biotinylated oligonucleotide and mutation site undergoes 40 step‐cycles of single nucleotide incorporation using Taq thermostable DNA polymerase and only one radioactive [α‐ 32 P]dNTP, specified by either the normal or mutant sequence. The oligonucleotides, now radioactively labelled at the 3′ end according to the template sequence, are then trapped by streptavidin‐coated magnetic beads, and the percent of radiolabel incorporated is determined directly by the Cerenkov method in a scintillation counter. The trapped‐oligonucleotide nucleotide incorporation (TONI) assay has been used for the screening of a mitochondrial polymorphism, and has also been shown to distinguish the genotypes of hemoglobin A/C, A/A, A/S, and S/S. It is reproducible over at least a 100‐fold range of radioisotope and a 10‐fold range of oligonucleotide primer. This method is particularly useful for diagnosing mutations which do not produce alterations detectable by restriction enzyme analysis, since optimization of conditions is rarely necessary. In addition, it requires only a single oligonucleotide, and no electrophoretic separation of the allele‐specific products. It thus represents an improved and simplified modification of the existing allele‐specific primer extension methods (Kuppuswamy et al., Proc Natl Acad Sci USA 88:1143–1147, 1991; Sokolov, Nucl Acids Res 18:3671, 1989; Syvanen et al., Genomics 8:684–692, 1990). © 1992 Wiley‐Liss, Inc.