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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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Agarose gel electrophoresis of PAI-PCR products using (A) an E. coli (λ) DNA and (B) an E. coli (λ) DNA mixed with 104-fold excess of competing DNA. M indicates the 1.0 kb ladder lanes, the intensity of the 3.0 kb band being higher than other bands.
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fig03: Agarose gel electrophoresis of PAI-PCR products using (A) an E. coli (λ) DNA and (B) an E. coli (λ) DNA mixed with 104-fold excess of competing DNA. M indicates the 1.0 kb ladder lanes, the intensity of the 3.0 kb band being higher than other bands.

Mentions: To improve the sensitivity of IPCR, we tried to enrich the target DNA prior to IPCR by RCA using several target-specific primers (Table 1). After the RCA reaction, DNA was purified and used as a template for IPCR. The pre-amplification was effective to improve the sensitivity of IPCR. The effectiveness was different with different RCA primers: the sensitivity increased almost 100 times with the DNA primer, Ecol-RC(DNA), almost 1000 times with the LNA-containing primers, Ecol-RC(LNA-5′) and Ecol-RC(LNA-3′), and more than 105 times with Ecol-RC(LNA-Even) (Fig. 3A). These results demonstrated that the RCA pre-amplification effectively increased the sensitivity of IPCR especially with LNA-containing primers. Next, we examined the sensitivity of PAI-PCR in the presence of excess competing DNA. The results with 104-fold excess of the competing DNA are shown in Fig. 3B. Under the conditions, the sensitivity decreased approximately 10-fold compared with that in the absence of the competing DNA. Even so, the amplification was possible using the primer Ecol-RC(LNA-even) when more than 102 copies of the target DNA existed as templates. These results indicated that target gene sequences from a gene of a bacterium constituting a small percentage (e.g. 0.01%) of the total microbial population could be recovered by PAI-PCR.


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)

Agarose gel electrophoresis of PAI-PCR products using (A) an E. coli (λ) DNA and (B) an E. coli (λ) DNA mixed with 104-fold excess of competing DNA. M indicates the 1.0 kb ladder lanes, the intensity of the 3.0 kb band being higher than other bands.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2327201&req=5

fig03: Agarose gel electrophoresis of PAI-PCR products using (A) an E. coli (λ) DNA and (B) an E. coli (λ) DNA mixed with 104-fold excess of competing DNA. M indicates the 1.0 kb ladder lanes, the intensity of the 3.0 kb band being higher than other bands.
Mentions: To improve the sensitivity of IPCR, we tried to enrich the target DNA prior to IPCR by RCA using several target-specific primers (Table 1). After the RCA reaction, DNA was purified and used as a template for IPCR. The pre-amplification was effective to improve the sensitivity of IPCR. The effectiveness was different with different RCA primers: the sensitivity increased almost 100 times with the DNA primer, Ecol-RC(DNA), almost 1000 times with the LNA-containing primers, Ecol-RC(LNA-5′) and Ecol-RC(LNA-3′), and more than 105 times with Ecol-RC(LNA-Even) (Fig. 3A). These results demonstrated that the RCA pre-amplification effectively increased the sensitivity of IPCR especially with LNA-containing primers. Next, we examined the sensitivity of PAI-PCR in the presence of excess competing DNA. The results with 104-fold excess of the competing DNA are shown in Fig. 3B. Under the conditions, the sensitivity decreased approximately 10-fold compared with that in the absence of the competing DNA. Even so, the amplification was possible using the primer Ecol-RC(LNA-even) when more than 102 copies of the target DNA existed as templates. These results indicated that target gene sequences from a gene of a bacterium constituting a small percentage (e.g. 0.01%) of the total microbial population could be recovered by PAI-PCR.

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