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Flow chart of the experiments for Figs 1 and 2. From the chromosomal DNA of E. coli (λ), a lambda DNA fragment was amplified by IPCR under a variety of conditions.
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fig01: Flow chart of the experiments for Figs 1 and 2. From the chromosomal DNA of E. coli (λ), a lambda DNA fragment was amplified by IPCR under a variety of conditions.

Mentions: In the first series of experiments (Fig. 1), IPCR of a lambda-phage sequence was carried out without RCA. DNA (0.5 μg) extracted from E. coli cells was digested with 7.5 U of HindIII (TaKaRa-Bio) at 37°C for 5 h. The digested DNA was purified by using Microcon YM-100 (Millipore Corporation, Bedford, MA), and c. 200 ng of the DNA was self-circularized in 80 μl of the ligation buffer at 16°C overnight by using the Mighty Mix DNA ligation kit (TaKaRa-Bio). DNA in the reaction buffer was purified by using YM-100, and amounts equivalent to 1.0 × 106, 1.0 × 105, 1.0 × 104, 1.0 × 103, 1.0 × 102 and 1.0 × 101 copies of the purified DNA were used as templates for IPCR. Each of the templates was mixed with 0.2 mM dNTP, 0.2 μM each of primers Ecol-iFw and Ecol-iRv (Table 1), and 2.5 U of LA Taq DNA polymerase (TaKaRa-Bio) in 50 μl of GC Buffer I (TaKaRa-Bio), and IPCR was performed under the following conditions: 1 min of denaturation at 94°C, 50 cycles of 30 s at 94°C, 30 s at 60°C and 3 min at 72°C followed by 7 min of final extension at 72°C. Inverse PCR products were analysed by electrophoresis using 1.0% (w/v) of SeaKem GTG Agarose Gel (TaKaRa-Bio).

Retrieval of entire genes from environmental DNA by inverse PCR with pre-amplification of target genes using primers containing locked nucleic acids

Yamada K, Terahara T, Kurata S, Yokomaku T, Tsuneda S, Harayama S - Environ. Microbiol. (2007)

Bottom Line: To enrich the target DNA sequences prior to IPCR, a rolling-circle amplification was used with a site-specific primer containing locked nucleic acids (LNAs).We then applied the PAI-PCR method to isolate glycosyl hydrolase genes from DNAs extracted from vermiform appendixes of horses and termite guts.The flanking sequences of the target genes were amplified and cloned successfully using PAI-PCR, whereas standard IPCR resulted in no amplification.

Affiliation: Technological Research Laboratory, Nippon Steel Kankyo Engineering Co., Ltd, 2-1-38 Shiohama, Kisarazu-shi, Chiba 292-0838, Japan. kyamada@nbrc.nite.go.jp

ABSTRACT
We had been unsuccessful to amplify desired nucleotide sequences from various environmental DNA samples by using the inverse polymerase chain reaction (IPCR) technique, most probably because the copy numbers of target DNA sequences had been quite low. To enrich the target DNA sequences prior to IPCR, a rolling-circle amplification was used with a site-specific primer containing locked nucleic acids (LNAs). This pre-amplified IPCR (PAI-PCR) method increased the sensitivity of PCR almost 10,000 times compared with the standard IPCR in model experiments using Escherichia coli. We then applied the PAI-PCR method to isolate glycosyl hydrolase genes from DNAs extracted from vermiform appendixes of horses and termite guts. The flanking sequences of the target genes were amplified and cloned successfully using PAI-PCR, whereas standard IPCR resulted in no amplification.

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