A method for the preparation of normalized cDNA libraries enriched with full-length sequences
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We developed a new method for the preparation of normalized cDNA libraries enriched with full-length sequences. It is based on the properties of the recently characterized duplex-specific nuclease from the hepatopancreas of the Kamchatka crab. The duplex-specific nuclease is thermostable, effectively cleaves double-stranded DNA, and is inactive toward single-stranded DNA (Shagin et al., Genome Res., 2002, vol. 12, pp. 1935–1942). Our method enables the normalization of cDNA samples enriched with full-length sequences without use of laborious and ineffective stages of physical separation. The efficiency of the method was demonstrated in model experiments using cDNA samples from several human tissues.
Key wordscDNA normalization duplex-specific nuclease normalized cDNA libraries
- ds cDNA
inverted terminal repeat
polymerase chain reaction
switching mechanism at the 5′-end of the RNA transcript
- ss cDNA
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- 1.Young, B.D. and Anderson, M., in Nucleic Acids Hybridization: a Practical Approach, Hames, B.D.. and Higgins, S.J., Eds., Oxford-Washington, DC: IRL, 1985, pp. 47–71.Google Scholar
- 17.Chenchik, A., Zhu, Y.Y., Diatchenko, L., Li, R., Hill, J., and Siebert, P.D., in BioTechniques Books, Siebert, P. and Larrick, J., Eds., Natick, MA: np, 1998, pp. 305–319.Google Scholar
- 21.Moreno-Palanques, R.F and Fuldner, R.A., in Automated DNA Sequencing and Analysis, Adams, M.D., Fields, C., and Venter, J.C., Eds., London: Academic, 1994, pp. 102–109.Google Scholar
- 23.Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989.Google Scholar