Annealing control primer system for improving specificity of PCR amplification.
A novel primer designed to improve the specificity of PCR amplification, called the annealing control primer (ACP), comprises a tripartite structure with a polydeoxyinosine [poly(dI)] linker between the 3' end target core sequence and the 5' end nontarget universal sequence. We show that this ACP linker prevents annealing of the 5' end nontarget sequence to the template and facilitates primer hybridization at the 3' end to the target sequence at specific temperatures, resulting in a dramatic improvement of annealing specificity. The effect of this linker is demonstrated by the incorporation of ACP sequences as primers during the amplification of target nucleotide sequence and as hybridization probes in the genotyping of single nucleotide polymorphisms. This is the first report to show that a poly(dI) linker between two different sequences of ACP forms a bubble-like structure and disrupts or destabilizes DNA duplex formation at certain annealing temperatures.
Hwang
In-Taek
IT
Seegene Life Science Laboratory, Seoul, South Korea.
Kim
Yun-Jee
YJ
Kim
Seung-Hyun
SH
Kwak
Chae-Il
CI
Gu
Young-Yun
YY
Chun
Jong-Yoon
JY
D000818:Animals; D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics*; D006358:Hot Temperature; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D017931:DNA Primers