Journal
ANALYTICAL CHEMISTRY
Volume 82, Issue 5, Pages 2074-2081Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ac902825r
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Funding
- National Natural Science Foundation of China [20975113]
- Natural Science Foundation of Jiangsu Province of China [BK2008067]
- Genetically Modified Organisms, Breeding Major Projects, Ministry of Environmental Protection of China [2008ZX08012-005]
- 973 Program [2009CB941602]
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Although the pyrosequencing method is simple and fast, the step of ssDNA preparation increases the cost, labor, and cross-contamination risk. In this paper, we proposed a method enabling pyrosequencing directly on dsDNA digested by nicking endonucleases (NEases). Recognition sequence of NEases was introduced using artificially mismatched bases in a PCR primer (in the case of genotyping) or a reverse-transcription primer (in the case of gene expression analysis). PCR products were treated to remove excess amounts of primers, nucleotides, and pyrophosphate (PPi) prior to sequencing. After the nicking reaction, pyrosequencing starts at the nicked 3' end, and extension reaction occurs when the added dNTP is complementary to the non-nicked strand. Although the activity of strand displacement by Klenow is limited, similar to 10 bases are accurately sequenced; this length is long enough for genotyping and SRPP-based differential gene expression analysis. It was observed that the signals of two allele-specific bases in a pyrogram from nicked dsDNA are highly quantitative, enabling quantitative determination of allele-specific templates; thus, Down's Syndrome diagnosis as well as differential gene expression analysis was successfully executed. ne results indicate that pyrosequencing using nicked dsDNA as templates is a simple, inexpensive, and reliable way in either quantitative genotyping or gene expression analysis.
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