Pyrosequencing on templates generated by asymmetric nucleic acid sequence-based amplification (asymmetric-NASBA).
Pyrosequencing is an ideal tool for verifying the sequence of amplicons. To enable pyrosequencing on amplicons from nucleic acid sequence-based amplification (NASBA), asymmetric NASBA with unequal concentrations of T7 promoter primer and reverse transcription primer was proposed. By optimizing the ratio of two primers and the concentration of dNTPs and NTPs, the amount of single-stranded cDNA in the amplicons from asymmetric NASBA was found increased 12 times more than the conventional NASBA through the real-time detection of a molecular beacon specific to cDNA of interest. More than 20 bases have been successfully detected by pyrosequencing on amplicons from asymmetric NASBA using Human parainfluenza virus (HPIV) as an amplification template. The primary results indicate that the combination of NASBA with a pyrosequencing system is practical, and should open a new field in clinical diagnosis.
Department of Analytical Chemistry, China Pharmaceutical University, Nanjing, China. jiahuning@sina.com
Huning
H
Jia
Zhiyao
Z
Chen
Haiping
H
Wu
Hui
H
Ye
Zhengyu
Z
Yan
Guohua
G
Zhou
D004247:DNA / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D010222:Parainfluenza Virus 1, Human / Q000235:genetics; D021161:Self-Sustained Sequence Replication; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D004247:DNA