Screening Poly [dA/dT(−)] cDNA for Gene Identification
The goal for developing the SPGI (screening poly [da/dT(−)] cDNA for gene identification technique was to generate an efficient tool for maximal identification of the expressed genes from eukaryotic genomes. The impetus for developing the SPGI method was triggered by the observation that a large number of human novel transcripts have not been identified in spite of intensive efforts in the past decades using the expressed sequence tag (EST) approach (1, 2, 3, 4, 5, 6, 7, 8, 9). Based on our analysis, we believe that the use of complementary DNA (cDNA) libraries generated by regular oligo-(dT) primers contributes significantly to this problem (10,11). The cDNAs generated by oligo-(dT) priming contain various lengths of poly(dA/dT) tail sequences at the 3′ end. Most of the cDNA libraries are processed through normalization/subtraction before being used for sequencing analysis (7). During the process of normalization/subtraction, poly(dA)–poly(dT) hybrids will form randomly between unrelated cDNA templates. The removal of these hybrids causes the loss of cDNA templates. This phenomenon affects especially the low-copy cDNA templates, which represent most of the genes. To overcome this problem, we developed the SPGI method. In this method, a set of anchored oligo-(dT) primers is used for mRNA priming to prevent the inclusion of the poly(dA/dT) sequences from the 3′ ends of cDNAs. Using the poly [dA/dT(−)] cDNA for subtraction/normalization prevents the formation of poly(dA)–poly(dT) hybrids, therefore preventing the nonspecific loss of cDNAs because of the poly(dA)–poly(dT) hybrids.
KeywordsMagnetic Particle Concentrator M280 Streptavidin Nonspecific Loss NlaIII Restriction Site Dynabeads M280 Streptavidin