期刊
NUCLEIC ACIDS RESEARCH
卷 37, 期 2, 页码 -出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkn956
关键词
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资金
- Ministry of Education, Culture, Sports, Science and Technology [19201046, 18710197]
- Targeted Proteins Research Program
- RIKEN Structural Genomics/Proteomics Initiative
- National Project on Protein Structural and Functional Analyses
- Grants-in-Aid for Scientific Research [18710197, 19201046] Funding Source: KAKEN
Toward the expansion of the genetic alphabet, we present an unnatural base pair system for efficient PCR amplification, enabling the site-specific incorporation of extra functional components into DNA. This system can be applied to conventional PCR protocols employing DNA templates containing unnatural bases, natural and unnatural base triphosphates, and a 35 exonuclease-proficient DNA polymerase. For highly faithful and efficient PCR amplification involving the unnatural base pairing, we identified the natural-base sequences surrounding the unnatural bases in DNA templates by an in vitro selection technique, using a DNA library containing the unnatural base. The system facilitates the site-specific incorporation of a variety of modified unnatural bases, linked with functional groups of interest, into amplified DNA. DNA fragments (0.15 amol) containing the unnatural base pair can be amplified 10(7)-fold by 30 cycles of PCR, with 1 total mutation rate of the unnatural base pair site. Using the system, we demonstrated efficient PCR amplification and functionalization of DNA fragments for the extremely sensitive detection of zeptomol-scale target DNA molecules from mixtures with excess amounts (pmol scale) of foreign DNA species. This unnatural base pair system will be applicable to a wide range of DNA/RNA-based technologies.
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