Ligation-anchored PCR: a simple amplification technique with single-sided specificity.

Abstract

A simple, efficient, and sensitive technique has been developed for amplification of cDNAs encoding molecules with 5' regions of unknown sequence. In this ligationanchored PCR, T4 RNA ligase is used to covalently link an "anchor" oligonucleotide to first-strand cDNAs. These anchored cDNAs are then amplified by using one PCR primer specific for the anchor and another specific for a sequence within the molecule of interest. The anchor oligonucleotide has been especially designed to facilitate subsequent analysis and cloning of the resultant PCR products. This three-stage procedure does not require purification of product between steps and avoids many of the technical difficulties associated with established anchored PCR protocols. The efficacy of ligationanchored PCR was demonstrated by amplification of a specific IgG1 cDNA; total RNA equivalent to as few as 100 cells yielded the expected PCR product.The anchored or single-sided PCR allows specific amplification ofDNA where the 5' sequence ofthe molecule of interest is unknown (1). This approach is based on homopolymer tailing of cDNA, subsequent amplification using one primer specific for the molecule of interest and a second primer containing a defined "anchor" sequence attached to a homopolymer sequence complementary to the tail, and, finally, reamplification using one primer specific for the cDNA of interest and one specific for the anchor. This technique has been very useful, but the established protocols have a number of disadvantages that have restricted their application. These protocols have often proved technically difficult, require multiple purification steps, often need relatively large amounts of starting RNA, and have the potential to generate nonspecific products due to use of homopolymer-containing primers in the PCR. We have developed an alternative approach in which an anchor of defined sequence is directly ligated to first-strand cDNA, and the resultant product is amplified by using primers specific for both the cDNA of interest and the anchor. We have used this technique to amplify a specific immunoglobulin cDNA from as little as 1 ng of total RNA. The simplicity and sensitivity of this ligation-anchored PCR (LA-PCR) suggest that it could have widespread utility.

E6AP gene suppression and characterization with in vitro selected hammerhead ribozymes

“…Most importantly, introduction of LA-PCR in the detection step will certainly increase the sensitivity of the method. 19,30,31 Finally, sequence alignment was used, thereby eliminating the need for cloning and sequencing of individual LA-PCR products. The sum of these modifications resulted in the ability to identify favorable ribozyme target sites more quickly and efficiently.…”

“…Using a synthetic combinatorial library of ribozymes and ligation-anchored PCR (LA-PCR), 19 several ribozyme-cleavable sites within the E6AP transcript were identified. Characterization of ribozymes designed to the selected sites enabled the identification of active agents to gain further insight into the role of E6AP within cells.…”

FGFR-1 signaling is involved in spermiogenesis and sperm capacitation

“…The missing 3Ј end was cloned using 3Ј rapid amplification of cDNA ends (RACE), utilizing the FirstChoice TM RLM-RACE kit (Ambion, Texas) as previously described (O'Bryan et al, 2000b;Troutt et al, 1992), using the primers F1701A 5Ј-AGC-CCAATGCACCTATGAG-3Ј, F1701B 5Ј-CTTCCTCCTGTGTGGGAGAG-3Ј and the supplied adaptor primer, AP3Ј. The sites of cellular expression of Snt-2 within the developing testis was determined by Northern blotting using a 1.2 kb (5Ј fragment) Snt-2 specific probe (O'Bryan et al, 1998).…”

Molecular cloning and functional expression of a novel rat heart P2X purinoceptor

“…The forward primer was: 5'-TG(G/T)CIG(C/G)ITGGTGCCC(G/T)GT(G/A)GAG-3'; the reverse primer was: 5'-TAC(C/T)TGGCAAA(T/C)CTGAA(A/G)TTG(A/ T)AGC-3'. To isolate the unknown 5' and 3' sequences we used the Marathon cDNA amplification kit (Clontech) based on a modification of the ligation-anchored PCR amplification technique [18]. Specific oligonucleotides targeted to the P2X5 sequence were extended by nested PCR (Expand Long Template PCR system, Boehringer-Mannheim) towards the 5' and 3' mRNA ends, using a library of adaptorligated cDNA (constructed from rat brain polyA + mRNA) and following the manufacturer's recommendations.…”