Discrimination of single nucleotide polymorphisms by strand exchange assay using partially double-stranded probes.
Previously we designed the partially double-stranded (PDS) probes that have protruding single-stranded portion for a single-base mismatch analysis. The single-stranded portion is engineered to sense existences of mismatches in the counterparts and to transduce it in strand exchange rates. Here we report the influence of probe length and operating conditions, such as temperature and buffer conditions, on the mismatch resolution using the PDS probes. Reliable detection of single-base mismatches was achieved even with a 45mer-long probe. By lowering operating temperature, the higher and faster discrimination of the mismatches was demonstrated. Addition of cationic comb-type copolymers (CCCs) in the buffer increased the reaction rate 3-4 orders without disordering the resolving power.
Hirata
Kazuya
K
Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
Ishii
Daisuke
D
Kano
Arihiro
A
Yamayoshi
Asako
A
Akaike
Toshihiro
T
Maruyama
Atsushi
A
D020137:Base Pair Mismatch; D002021:Buffers; D015342:DNA Probes / Q000737:chemistry*; D003911:Dextrans / Q000737:chemistry; D009693:Nucleic Acid Hybridization / Q000379:methods*; D011107:Polylysine / Q000737:chemistry; D020641:Polymorphism, Single Nucleotide; D013696:Temperature
D002021:Buffers; D015342:DNA Probes; D003911:Dextrans; D011107:Polylysine