Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer.

Abstract

We have devised a simple and efficient cDNA cloning strategy that overcomes many of the difficulties encountered in obtaining full-length cDNA clones of lowabundance mRNAs. In essence, cDNAs are generated by using the DNA polymerase chain reaction technique to amplify copies of the region between a single point in the transcript and the 3' or 5' end. The minimum information required for this amplification is a single short stretch of sequence within the mRNA to be cloned. Since the cDNAs can be produced in one day, examined by Southern blotting the next, and readily cloned, large numbers of full-length cDNA clones of rare transcripts can be rapidly produced. Moreover, separation of amplified cDNAs by gel electrophoresis allows precise selection by size prior to cloning and thus facilitates the isolation of cDNAs representing variant mRNAs, such as those produced by alternative splicing or by the use of alternative promoters. The efficacy of this method was demonstrated by isolating cDNA clones of mRNA from int-2, a mouse gene that expresses four different transcripts at low abundance, the longest of which is =2.9 kilobases. After <0.05% of the cDNAs produced had been screened, 29 independent int-2 clones were isolated. Sequence analysis demonstrated that the 3' and 5' ends of all four int-2 mRNAs were accurately represented by these clones.Despite the development of numerous cDNA cloning strategies (1-5), obtaining full-length cDNA copies of lowabundance mRNAs remains a formidable task. We describe here a simple, rapid, and efficient cDNA cloning strategy that is based on the DNA polymerase chain reaction (PCR) technique developed by Saiki et al. (6). PCR employs two oligonucleotide primers, one complementary to a sequence on the (+) strand and the other to a downstream sequence on the (-) strand. Reiterative cycles of denaturation, annealing, and extension are used to generate multiple copies of the DNA that lies between the two primers. PCR has been used for a variety of purposes (7-13), including the detection of allelic polymorphisms and of DNA sequences unique to rare cells in a population, as well as the cross-species isolation of homologous genes. The feature common to all applications of PCR to date has been the use of primers designed to match two known or presumed genomic or cDNA sequences. In contrast, we have devised an application of this technique that achieves amplification and cloning of the region between a single short sequence in a cDNA molecule and its unknown 3' or 5' end. We demonstrate here the utility of this strategy, termed "rapid amplification of cDNA ends" (RACE), by using it to obtain clones representing transcripts of a gene, int-2, expressed at low abundance in the early mouse embryo. METHODS 3'-End Amplification of cDNAs. See Fig. 1 and the substitution of 20 pmol of 5RT primer for (dT)17-adaptor. Excess 5RT was removed as follows: the 20-Al cDNA pool was applied to a Bio-Gel A-5m (Bio-Rad) column (in a 2-ml serological pipette plugged with silane-treated glass wool) equi...

The galanin receptor type 2 initiates multiple signaling pathways in small cell lung cancer cells by coupling to Gq, Gi and G12 proteins

“…The cDNA coding for the human type 2 galanin receptor (GALR2) was isolated from H69 cells employing the rapid ampli®cation of cDNA ends technique (RACE), exactly following the procedure described by Frohman et al (1988). Brie¯y, 2 mg of total RNA were reverse transcribed using the Q T primer.…”

cDNA cloning of a human 100 kDa de‐ubiquitinating enzyme: the 100 kDa human de‐ubiquitinase belongs to the ubiquitin C‐terminal hydrolase family 2 (UCH2)

“…Clone RST4 was sequenced on both strands and fused to HIBAA66 clone, thus yielding a 3 kb cDNA clone encoding 803 amino acids. The 5' end of the eDNA was obtained through the 5' anchored PCR technique [8][9][10]; the full length cDNA sequence comprises 3158 bp and a putative AUG codon, which suggest that the cDNA clone encodes a protein of 858 amino acids (calculated Mr = 95,743 Da) with a pI of 4.90 (Fig. 1).…”

“…The full length cDNA clone was obtained by PCR using selected primers and the 5' anchored PCR amplification technique [9][10][11].…”

Stromelysin-1 expression is activated in vivo by Ets-1 through palindromic head-to-head Ets binding sites present in the promoter

“…We first determined the sequences of the 5 0 -and 3 0 -terminal regions of rabbit ets-1 cDNA. The rapid amplification of cDNA ends (RACE) method (Frohman et al, 1988) was performed onto cDNA fragments, obtained from HIG-82 cells by RT-PCR, using the following primers 5 0 -CTGCAGGATCTCTAGATGCTC-3 0 and 5 0 -GGCTGGGAATTCAAACTTTCT-3 0 for 5 0 -and 3 0 -RACE, respectively. The DNA fragments were purified, cloned into the pCR s II-TOPO s vector (Invitrogen, Carlsbad, CA, USA) and sequenced allowing us to design primers flanking the open-reading frame of rabbit ets-1 cDNA.…”