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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: 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.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.

View Article: PubMed Central - PubMed

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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Related in: MedlinePlus

Capillary electrophoreses of IPCR (odd-numbered lanes) and PAI-PCR (even-numbered lanes) products amplified from DNA extracted from horse vermiform appendixes. The electrophoreses followed by the analysis of the electrophoregrams were automatically performed by using an Agilent 2100 Bioanalyser with a DNA 7500 LabChip® kit (TaKaRa-Bio). The results are shown as a gel-like image. L is the ladder obtained with DNA fragment size markers (bp). The green and pink peaks are the lowest 50 bp and highest 10 380 bp markers respectively. The substrates used for IPCR or PAI-PCR were BamHI-digested DNA (lanes 1 and 2), EcoRI-digested DNA (lanes 3 and 4), HindIII-digested DNA (lanes 5 and 6), PstI-digested DNA (lanes 7 and 8) and XbaI-digested DNA (lanes 9 and 10).
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fig04: Capillary electrophoreses of IPCR (odd-numbered lanes) and PAI-PCR (even-numbered lanes) products amplified from DNA extracted from horse vermiform appendixes. The electrophoreses followed by the analysis of the electrophoregrams were automatically performed by using an Agilent 2100 Bioanalyser with a DNA 7500 LabChip® kit (TaKaRa-Bio). The results are shown as a gel-like image. L is the ladder obtained with DNA fragment size markers (bp). The green and pink peaks are the lowest 50 bp and highest 10 380 bp markers respectively. The substrates used for IPCR or PAI-PCR were BamHI-digested DNA (lanes 1 and 2), EcoRI-digested DNA (lanes 3 and 4), HindIII-digested DNA (lanes 5 and 6), PstI-digested DNA (lanes 7 and 8) and XbaI-digested DNA (lanes 9 and 10).

Mentions: Based on the cloned sequences, IPCR primer sets for the amplification of the flanking regions of the targets were designed by using OLIGO software (TaKaRa-Bio). DNA extracted from the horse vermiform appendix was digested with several restriction enzymes and self-circularized. Then, PAI-PCR was performed, IPCR without RCA pre-amplification being carried out as a control. The results with Xyn-RC(LNA-Even) as the RCA primer, and Xyn-0101LF plus Xyn-0101LR as the IPCR primers are shown in Fig. 4 (even-numbered lanes). A single amplification product was observed from all the five samples that had been digested by five different restriction enzymes. Pre-amplified inverse PCR amplification products were also recovered using other IPCR primer sets such as Xyn-0022LF and Xyn-0022LR (data no shown). However no amplification product was observed in any IPCR without RCA reaction (Fig. 4, odd-numbered lanes).


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)

Capillary electrophoreses of IPCR (odd-numbered lanes) and PAI-PCR (even-numbered lanes) products amplified from DNA extracted from horse vermiform appendixes. The electrophoreses followed by the analysis of the electrophoregrams were automatically performed by using an Agilent 2100 Bioanalyser with a DNA 7500 LabChip® kit (TaKaRa-Bio). The results are shown as a gel-like image. L is the ladder obtained with DNA fragment size markers (bp). The green and pink peaks are the lowest 50 bp and highest 10 380 bp markers respectively. The substrates used for IPCR or PAI-PCR were BamHI-digested DNA (lanes 1 and 2), EcoRI-digested DNA (lanes 3 and 4), HindIII-digested DNA (lanes 5 and 6), PstI-digested DNA (lanes 7 and 8) and XbaI-digested DNA (lanes 9 and 10).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2327201&req=5

fig04: Capillary electrophoreses of IPCR (odd-numbered lanes) and PAI-PCR (even-numbered lanes) products amplified from DNA extracted from horse vermiform appendixes. The electrophoreses followed by the analysis of the electrophoregrams were automatically performed by using an Agilent 2100 Bioanalyser with a DNA 7500 LabChip® kit (TaKaRa-Bio). The results are shown as a gel-like image. L is the ladder obtained with DNA fragment size markers (bp). The green and pink peaks are the lowest 50 bp and highest 10 380 bp markers respectively. The substrates used for IPCR or PAI-PCR were BamHI-digested DNA (lanes 1 and 2), EcoRI-digested DNA (lanes 3 and 4), HindIII-digested DNA (lanes 5 and 6), PstI-digested DNA (lanes 7 and 8) and XbaI-digested DNA (lanes 9 and 10).
Mentions: Based on the cloned sequences, IPCR primer sets for the amplification of the flanking regions of the targets were designed by using OLIGO software (TaKaRa-Bio). DNA extracted from the horse vermiform appendix was digested with several restriction enzymes and self-circularized. Then, PAI-PCR was performed, IPCR without RCA pre-amplification being carried out as a control. The results with Xyn-RC(LNA-Even) as the RCA primer, and Xyn-0101LF plus Xyn-0101LR as the IPCR primers are shown in Fig. 4 (even-numbered lanes). A single amplification product was observed from all the five samples that had been digested by five different restriction enzymes. Pre-amplified inverse PCR amplification products were also recovered using other IPCR primer sets such as Xyn-0022LF and Xyn-0022LR (data no shown). However no amplification product was observed in any IPCR without RCA reaction (Fig. 4, odd-numbered lanes).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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.

Show MeSH
Related in: MedlinePlus