Method for generating five prime biased tandem tag libraries of cDNAs

#1. Field of the Invention

#The sequences of whole genomes from several organisms have now been elucidated and are available as searchable databases. This enables rapid identification of full-length messenger RNAs (mRNAs) expressed in a biological sample once a partial sequence is known. The method described here allows generation of such partial sequences consisting of a minimal length of expressed cDNA sequences of at least 20 bases from biological samples to rapidly identify novel expressed transcripts.

#2. Description of the Related Art

#FIGS. 1A, 1B and 1C show a flow chart of an embodiment of the present method for generating five primed biased tandem tag libraries of cDNAs.

#A. Brief Description of the Method

#1. The first and second strand cDNA synthesis is carried out according the standard procedure. In a preferred embodiment, the first strand synthesis is carried out with olido-dT 3′ primer covalently linked to magnetic beads according to the manufacturer's protocol (Dynal Inc.).

#2. The 5′ ends of the ds-cDNAs are flushed using T4 DNA polymerase in the presence of dNTP, followed by the ligation of a double stranded adaptor. The adaptor can be of any sequence but contains the recognition sequence for a type IIS restriction enzyme that cleaves double stranded DNA substrates at some length downstream of the recognition site. In a preferred embodiment, the recognition sequence for a type IIS enzyme, Bpm I (also known as Gsu I) was placed at the 3′ end of the adaptor so that the nucleotide sequence immediately following the Bpm I site is from cDNAs. In addition, optionally, the recognition site for a rare six cutter such as the Mlu I enzyme can also be incorporated into the adaptor at just upstream of the Bpm I site to be utilized at a later step. The remaining adaptor sequence serves as the forward primer site for a subsequent PCR amplification step.