A method for point mutation analysis that links SSCP and dye primer fluorescent sequencing.
A method is presented for mutation detection directly from single-strand conformational polymorphism (SSCP) variants. This approach is based on: (i) amplification of the exons to be analysed by SSCP using the forward primer with an eight-base tail to form a universal SSCP cassette; (ii) excision from the gel of the shifted silver-stained bands; (iii) reamplification of the eluted DNAs using, as the forward primer, a 26-base universal adaptor primer corresponding to the 18-base-21M13 sequence plus the eight nucleotides of the universal SSCP cassette; and (iv) direct sequencing of the purified products using the standard-21M13 fluorescent primer. This procedure presents several advantages including the avoidance of a cloning step which leads to significant time reduction, while maintaining comparable accuracy at relatively low costs. In conclusion, the presence of the universal SSCP cassette and subsequent reamplification with the same adaptor primer for direct sequencing makes the method universal for scanning and identification of gene alterations.
Dipartimento di Fisiopatologia Clinica, Unità di Genetica Umana, Firenze, Italy.
I
I
Chiarelli
B
B
Porfirio
P L
PL
Mattiuz
M
M
Seri
F
F
Caroli
I
I
Celli
G
G
Romeo
S
S
Volorio
M
M
Zollo
D001483:Base Sequence; D001706:Biopsy; D002294:Carcinoma, Squamous Cell / Q000235:genetics* / Q000473:pathology; D017931:DNA Primers; D005091:Exons; D005456:Fluorescent Dyes; D016158:Genes, p53; D014644:Genetic Variation; D006258:Head and Neck Neoplasms / Q000235:genetics* / Q000473:pathology; D006801:Humans; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational
D017931:DNA Primers; D005456:Fluorescent Dyes