3' RACE LaNe: a simple and rapid fully nested PCR method to determine 3'-terminal cDNA sequence.

Abstract

3′ Rapid amplification of cDNA ends (RACE) has been an invaluable tool for molecular biologists since its inception (1). This method and the subsequent method of 3′ RACE walking (2) enable the simple and effective amplification of 3′ flanking cDNA sequence in a ligation-independent manner (3). However, despite their capacity for the suppression of nonspecific PCR products (4), they remain essentially one-sided reactions. As such, they can be compromised in their selection of specific products in difficult scenarios, such as long coding or 3′ untranslated regions and regions including high GC content, secondary structure, or homopolymeric tracts (5,6). Here the best features of 3′ RACE and universal fast walking (7) are combined to produce 3′ RACE lariat-dependent nested PCR (3′ RACE LaNe), a method as quick and simple to perform as 3′ RACE (Figure 1). The method enables gene-specific fully nested PCR series (8) without the requirement for random priming or efficient exonuclease I processing. I demonstrate that the method outperforms a conventional heminested anchored PCR 3′ RACE series (9) when it is applied to the long and complex tammar wallaby ATRX gene (10) and the mouse PGK1 and GAPD genes (Figure 2). Total RNA was extracted from adult tammar wallaby (Macropus eugenii) testis tissue (for ATRX experiments) and the mouse mammary epithelial cell line, HC11 (for PGK1 and GAPD experiments), using the GenEluteTM Mammalian Total RNA Kit (Sigma, Sydney, NSW, Australia). Tissue was collected during an earlier study (10) using protocols approved by the Institutional Animal Ethics Committees (Melbourne University, Melbourne, Australia). All gene-specific primers sequences are listed in Table 1. The ATRX primer design was based on cDNA sequence characterized previously by a reverse transcription PCR (RT-PCR) approach (unpublished data). The PGK1 and GAPD primer designs were based on mouse cDNA clone database submissions, BC_ 023819 and NM_008084, respectively. Reverse transcription of 200 or 20 ng total RNA (for the ATRX or PGK1 and GAPD experiments, respectively) was 3′ RACE LaNe: a simple and rapid fully nested PCR method to determine 3′-terminal cDNA sequence