Amplicons were sequenced by using the same primers applied for the RT-PCR cycling and the ABI PRISM Dye Terminator Kit (Applied Biosystems, Foster City, CA, USA) by using the
dideoxyribonucleotide chain terminator method (11).
The PCR products are then used as templates for extension of SBE primers with dideoxyribonucleotide triphosphates (ddNTPs) attached to a biotin moiety.
[4] Nonstandard abbreviations: SNP, single-nucleotide polymorphism; SPC, solid-phase capture; SBE, single-base extension; CYP450, cytochrome P450; NCBI, National Center for Biotechnology Information; ddNTP, dideoxyribonucleotide triphosphate.
Amplicons were direct sequenced in an automated sequencer (AB1377, Applied Biosystems, Foster City, CA, USA) using the Kit ABI PRISM Dye Terminator (Applied Biosystems) by the
dideoxyribonucleotide chain terminator method (11).
In minisequencing, a primer anneals to a DNA template immediately upstream of a mutation site to be analyzed and is extended by a single base as a result of incorporating a
dideoxyribonucleotide triphosphate (ddNTP) (3).
In minisequencing, a primer is hybridized to DNA next to a variant nucleotide site and extended with DNA polymerase by a single appropriate dideoxyribonucleotide triphosphate (ddNTP) that matches the nucleotide at the target site.
[6] Nonstandard abbreviations: ARMS, amplification refractory mutation system; ddNTP, dideoxyribonucleotide triphosphate; and SAP, shrimp alkaline phosphatase