METHOD OF PARTIAL DNA SEQUENCING FOR DETECTION OF MUTATIONS IN SHORT FRAGMENTS OF SINGLE-STRANDED DNA USING MICROCHIP

摘要

FIELD: molecular biology, medicine. SUBSTANCE: mutations are detected by partial sequencing and using oligonucleotide microchip containing cells with immobilized taken sequences of synthetic oligonucleotides of definite length with hairpin structure. Method involves selection (theoretically) of oligonucleotides with length N (N-oligonucleotides) comprising self-closing hairpin structure. N-oligonucleotides are immobilized on micromatrix and nonself-complementary oligonucleotides with length n (n-oligonucleotides) are immobilized for hybridization butt. Melting point (T_m) of three variants of duplexes, namely: (1) DNA-immobilized N-oligonucleotide; (2) self-closing hairpin; (3) n- oligonucleotide immobilized N-oligonucleotide corresponds to inequality: T_m(1)T_m(2)T_m3. Test microchip is prepared with theoretically selected immobilized N-oligonucleotides and their hybridization is carried out with mixture of fluorescently labeled model DNA and each of theoretically selected n-oligonucleotides by selection of conditions for equilibration of energetics of AT- and GC-enriched oligonucleotides. Results are recorded by plotting melting curves using method of fluorescent analysis. Based on obtained data N-oligonucleotides are taken for preparing analytical microchip, n-oligonucleotides for hybridization butt and conditions for hybridization. EFFECT: simplified analysis, reduced time of analysis. 14 cl, 4 dwg, 2 ex