Construction of cDNA Libraries for Highly Efficient DNA Sequencing from the 3′ End of Expressed Genes

Abstract

The majority of expressed sequence tag (EST) sequences available today have been derived from the 5′ ends of cDNA clones. Obtaining high-quality DNA sequences from the 3′ ends of oligo(dT)-primed cDNA on a large scale has been difficult because of slippage of the DNA polymerase enzyme used in direct PCR and cycle sequencing. With the completion of whole genome sequencing for more and more organisms, mRNA 3′-UTR sequences can be particularly useful for clustering large numbers of ESTs for the effective discrimination of individual genes and gene families. We have identified a flaw in the widely used oligo(dT) primers for cDNA synthesis, and here we describe an improved priming approach to effectively synthesize cDNA devoid of homopolymeric nucleotide stretches from mRNA poly(A) tails to enable highly efficient and reliable DNA sequence determination from 3′ mRNA ends. Using this method, we produced a rat lung cDNA library and successfully sequenced the 3′ ends of 98% of all attempted clones.