NDLI: Global amplification of cDNA from limiting amounts of tissue : An improved method for gene cloning and analysis

Content Provider	Springer Nature Link
Author	Reddy, M. K. Nair, Suresh Sopory, S. K.
Copyright Year	2002
Abstract	In this study we present an improved polymerase chain reaction (PCR)-based methodology to generate large amounts of high-quality complementary DNA (cDNA) from small amounts of initial total RNA. Global amplification of cDNA makes it possible to simultaneously clone many cDNAs and to construct directional cDNA libraries from a sequence-abundance-normalized cDNA population, and also permits rapid amplification of cDNA ends (RACE), from a limited amount of starting material. The priming of cDNAs with an adapter oligo-deoxythymidine (oligo-dT) primer and the ligation of a modified oligonucleotide to the 3′ end of single-stranded cDNAs, through the use of T4 RNA ligase, generates known sequences on either end of the cDNA population. This helps in the global amplification of cDNAs and in the sequence-abundance normalization of the cDNA population through the use of PCR. Utilization of a long-range PCR enzyme mix to amplify the cDNA population helps to reduce bias toward the preferential amplification of shorter molecules. Incorporation of restriction sites in the PCR primers allows the amplified cDNAs to be directionally cloned into appropriate cloning vectors to generate cDNA libraries. RACE-PCR done with biotinylated primers and streptavidin-coated para-magnetic particles are used for the efficient isolation of either full-length coding or noncoding strands.
Starting Page	223
Ending Page	230
Page Count	8
File Format	PDF
ISSN	10736085
Journal	Molecular Biotechnology
Volume Number	22
Issue Number	3
e-ISSN	15590305
Language	English
Publisher	Humana Press
Publisher Date	2002-01-01
Publisher Place	Totowa
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Neurosciences Neurology
Content Type	Text
Resource Type	Article
Subject	Molecular Biology Biochemistry Bioengineering Applied Microbiology and Biotechnology Biotechnology

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