Limits...
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.

Show MeSH

Related in: MedlinePlus

Capillary electrophoreses of PAI-PCR products amplified from DNA extracted from 12 different termite gut samples. All DNA samples were digested with EcoRI, and an RCA primer [(A) Xyn-RC(LNA-Even); or (B) Xyn-RC(LNA-5′)] was used in the RCA reaction. Products were analysed with an Agilent 2100 Bioanalyser with a DNA 7500 LabChip® kit (TaKaRa-Bio).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2327201&req=5

fig05: Capillary electrophoreses of PAI-PCR products amplified from DNA extracted from 12 different termite gut samples. All DNA samples were digested with EcoRI, and an RCA primer [(A) Xyn-RC(LNA-Even); or (B) Xyn-RC(LNA-5′)] was used in the RCA reaction. Products were analysed with an Agilent 2100 Bioanalyser with a DNA 7500 LabChip® kit (TaKaRa-Bio).

Mentions: Similar experiments were carried out with 12 DNA samples extracted from termite guts (Table 1). Polymerase chain reaction fragments (c. 160 bp) were successfully amplified from all the 12 samples, then cloned and sequenced. When clones with sequence similarity higher than 90% were grouped, they were divided into 10 different groups. Between these 10 groups, the inter-group similarities ranged between 41% and 79%. A blast analysis demonstrated that the amino acid sequences translated from the DNA sequences revealed 40–74% amino acid sequence similarities to known xylanases categorized as GHF10. An IPCR primer set was designed for each of the 12 clones and PAI-PCR reactions were carried out. Pre-amplified inverse PCR amplification products were obtained from all the 12 DNA samples when an appropriate IPCR primer set was used together with either Xyn-RC(LNA-Even) or Xyn-RC(LNA-5′) as an RCA primer. When the RCA primer was Xyn-RC(LNA-Even), the sizes of the major products were generally larger than 2 kb suggesting successful PAI-PCR (Fig. 5A). To the contrary, the PAI-PCR products obtained with Xyn-RC(LNA-5′) contained both of large and small (< 0.5 kb) fragments, the smaller ones most probably being non-specific amplification products (Fig. 5B).


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 PAI-PCR products amplified from DNA extracted from 12 different termite gut samples. All DNA samples were digested with EcoRI, and an RCA primer [(A) Xyn-RC(LNA-Even); or (B) Xyn-RC(LNA-5′)] was used in the RCA reaction. Products were analysed with an Agilent 2100 Bioanalyser with a DNA 7500 LabChip® kit (TaKaRa-Bio).
© Copyright Policy
Related In: Results  -  Collection

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

fig05: Capillary electrophoreses of PAI-PCR products amplified from DNA extracted from 12 different termite gut samples. All DNA samples were digested with EcoRI, and an RCA primer [(A) Xyn-RC(LNA-Even); or (B) Xyn-RC(LNA-5′)] was used in the RCA reaction. Products were analysed with an Agilent 2100 Bioanalyser with a DNA 7500 LabChip® kit (TaKaRa-Bio).
Mentions: Similar experiments were carried out with 12 DNA samples extracted from termite guts (Table 1). Polymerase chain reaction fragments (c. 160 bp) were successfully amplified from all the 12 samples, then cloned and sequenced. When clones with sequence similarity higher than 90% were grouped, they were divided into 10 different groups. Between these 10 groups, the inter-group similarities ranged between 41% and 79%. A blast analysis demonstrated that the amino acid sequences translated from the DNA sequences revealed 40–74% amino acid sequence similarities to known xylanases categorized as GHF10. An IPCR primer set was designed for each of the 12 clones and PAI-PCR reactions were carried out. Pre-amplified inverse PCR amplification products were obtained from all the 12 DNA samples when an appropriate IPCR primer set was used together with either Xyn-RC(LNA-Even) or Xyn-RC(LNA-5′) as an RCA primer. When the RCA primer was Xyn-RC(LNA-Even), the sizes of the major products were generally larger than 2 kb suggesting successful PAI-PCR (Fig. 5A). To the contrary, the PAI-PCR products obtained with Xyn-RC(LNA-5′) contained both of large and small (< 0.5 kb) fragments, the smaller ones most probably being non-specific amplification products (Fig. 5B).

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