Preparation of Phi29 DNA polymerase free of amplifiable DNA using ethidium monoazide, an ultraviolet-free light-emitting diode lamp and trehalose

Hirokazu Takahashi, Hiroyuki Yamazaki, Satoshi Akanuma, Hiroko Kanahara, Toshiyuki Saito, Tomoyuki Chimuro, Takayoshi Kobayashi, Toshio Ohtani, Kimiko Yamamoto, Shigeru Sugiyama, Toshiro Kobori

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

We previously reported that multiply-primed rolling circle amplification (MRPCA) using modified random RNA primers can amplify tiny amounts of circular DNA without producing any byproducts. However, contaminating DNA in recombinant Phi29 DNA polymerase adversely affects the outcome of MPRCA, especially for negative controls such as non-template controls. The amplified DNA in negative control casts doubt on the result of DNA amplification. Since Phi29 DNA polymerase has high affinity for both single-strand and double-stranded DNA, some amount of host DNA will always remain in the recombinant polymerase. Here we describe a procedure for preparing Phi29 DNA polymerase which is essentially free of amplifiable DNA. This procedure is realized by a combination of host DNA removal using appropriate salt concentrations, inactivation of amplifiable DNA using ethidium monoazide, and irradiation with visible light from a light-emitting diode lamp. Any remaining DNA, which likely exists as oligonucleotides captured by the Phi29 DNA polymerase, is degraded by the 3′-5′ exonuclease activity of the polymerase itself in the presence of trehalose, used as an anti-aggregation reagent. Phi29 DNA polymerase purified by this procedure has little amplifiable DNA, resulting in reproducible amplification of at least ten copies of plasmid DNA without any byproducts and reducing reaction volume. This procedure could aid the amplification of tiny amounts DNA, thereby providing clear evidence of contamination from laboratory environments, tools and reagents.

Original languageEnglish
Article numbere82624
JournalPLoS One
Volume9
Issue number2
DOIs
Publication statusPublished - 2014 Feb 5
Externally publishedYes

Fingerprint

Trehalose
DNA-directed DNA polymerase
trehalose
DNA-Directed DNA Polymerase
Ultraviolet Rays
Electric lamps
Light emitting diodes
DNA
Amplification
byproducts
Byproducts
8-azidoethidium
ethidium-DNA complex
ethidium
circular DNA
recombinant DNA
Light
Circular DNA
Exonucleases
Recombinant DNA

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Preparation of Phi29 DNA polymerase free of amplifiable DNA using ethidium monoazide, an ultraviolet-free light-emitting diode lamp and trehalose. / Takahashi, Hirokazu; Yamazaki, Hiroyuki; Akanuma, Satoshi; Kanahara, Hiroko; Saito, Toshiyuki; Chimuro, Tomoyuki; Kobayashi, Takayoshi; Ohtani, Toshio; Yamamoto, Kimiko; Sugiyama, Shigeru; Kobori, Toshiro.

In: PLoS One, Vol. 9, No. 2, e82624, 05.02.2014.

Research output: Contribution to journalArticle

Takahashi, H, Yamazaki, H, Akanuma, S, Kanahara, H, Saito, T, Chimuro, T, Kobayashi, T, Ohtani, T, Yamamoto, K, Sugiyama, S & Kobori, T 2014, 'Preparation of Phi29 DNA polymerase free of amplifiable DNA using ethidium monoazide, an ultraviolet-free light-emitting diode lamp and trehalose', PLoS One, vol. 9, no. 2, e82624. https://doi.org/10.1371/journal.pone.0082624
Takahashi, Hirokazu ; Yamazaki, Hiroyuki ; Akanuma, Satoshi ; Kanahara, Hiroko ; Saito, Toshiyuki ; Chimuro, Tomoyuki ; Kobayashi, Takayoshi ; Ohtani, Toshio ; Yamamoto, Kimiko ; Sugiyama, Shigeru ; Kobori, Toshiro. / Preparation of Phi29 DNA polymerase free of amplifiable DNA using ethidium monoazide, an ultraviolet-free light-emitting diode lamp and trehalose. In: PLoS One. 2014 ; Vol. 9, No. 2.
@article{055a8d1f144e4eb8a24c7b4fbb41c5c3,
title = "Preparation of Phi29 DNA polymerase free of amplifiable DNA using ethidium monoazide, an ultraviolet-free light-emitting diode lamp and trehalose",
abstract = "We previously reported that multiply-primed rolling circle amplification (MRPCA) using modified random RNA primers can amplify tiny amounts of circular DNA without producing any byproducts. However, contaminating DNA in recombinant Phi29 DNA polymerase adversely affects the outcome of MPRCA, especially for negative controls such as non-template controls. The amplified DNA in negative control casts doubt on the result of DNA amplification. Since Phi29 DNA polymerase has high affinity for both single-strand and double-stranded DNA, some amount of host DNA will always remain in the recombinant polymerase. Here we describe a procedure for preparing Phi29 DNA polymerase which is essentially free of amplifiable DNA. This procedure is realized by a combination of host DNA removal using appropriate salt concentrations, inactivation of amplifiable DNA using ethidium monoazide, and irradiation with visible light from a light-emitting diode lamp. Any remaining DNA, which likely exists as oligonucleotides captured by the Phi29 DNA polymerase, is degraded by the 3′-5′ exonuclease activity of the polymerase itself in the presence of trehalose, used as an anti-aggregation reagent. Phi29 DNA polymerase purified by this procedure has little amplifiable DNA, resulting in reproducible amplification of at least ten copies of plasmid DNA without any byproducts and reducing reaction volume. This procedure could aid the amplification of tiny amounts DNA, thereby providing clear evidence of contamination from laboratory environments, tools and reagents.",
author = "Hirokazu Takahashi and Hiroyuki Yamazaki and Satoshi Akanuma and Hiroko Kanahara and Toshiyuki Saito and Tomoyuki Chimuro and Takayoshi Kobayashi and Toshio Ohtani and Kimiko Yamamoto and Shigeru Sugiyama and Toshiro Kobori",
year = "2014",
month = "2",
day = "5",
doi = "10.1371/journal.pone.0082624",
language = "English",
volume = "9",
journal = "PLoS One",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "2",

}

TY - JOUR

T1 - Preparation of Phi29 DNA polymerase free of amplifiable DNA using ethidium monoazide, an ultraviolet-free light-emitting diode lamp and trehalose

AU - Takahashi, Hirokazu

AU - Yamazaki, Hiroyuki

AU - Akanuma, Satoshi

AU - Kanahara, Hiroko

AU - Saito, Toshiyuki

AU - Chimuro, Tomoyuki

AU - Kobayashi, Takayoshi

AU - Ohtani, Toshio

AU - Yamamoto, Kimiko

AU - Sugiyama, Shigeru

AU - Kobori, Toshiro

PY - 2014/2/5

Y1 - 2014/2/5

N2 - We previously reported that multiply-primed rolling circle amplification (MRPCA) using modified random RNA primers can amplify tiny amounts of circular DNA without producing any byproducts. However, contaminating DNA in recombinant Phi29 DNA polymerase adversely affects the outcome of MPRCA, especially for negative controls such as non-template controls. The amplified DNA in negative control casts doubt on the result of DNA amplification. Since Phi29 DNA polymerase has high affinity for both single-strand and double-stranded DNA, some amount of host DNA will always remain in the recombinant polymerase. Here we describe a procedure for preparing Phi29 DNA polymerase which is essentially free of amplifiable DNA. This procedure is realized by a combination of host DNA removal using appropriate salt concentrations, inactivation of amplifiable DNA using ethidium monoazide, and irradiation with visible light from a light-emitting diode lamp. Any remaining DNA, which likely exists as oligonucleotides captured by the Phi29 DNA polymerase, is degraded by the 3′-5′ exonuclease activity of the polymerase itself in the presence of trehalose, used as an anti-aggregation reagent. Phi29 DNA polymerase purified by this procedure has little amplifiable DNA, resulting in reproducible amplification of at least ten copies of plasmid DNA without any byproducts and reducing reaction volume. This procedure could aid the amplification of tiny amounts DNA, thereby providing clear evidence of contamination from laboratory environments, tools and reagents.

AB - We previously reported that multiply-primed rolling circle amplification (MRPCA) using modified random RNA primers can amplify tiny amounts of circular DNA without producing any byproducts. However, contaminating DNA in recombinant Phi29 DNA polymerase adversely affects the outcome of MPRCA, especially for negative controls such as non-template controls. The amplified DNA in negative control casts doubt on the result of DNA amplification. Since Phi29 DNA polymerase has high affinity for both single-strand and double-stranded DNA, some amount of host DNA will always remain in the recombinant polymerase. Here we describe a procedure for preparing Phi29 DNA polymerase which is essentially free of amplifiable DNA. This procedure is realized by a combination of host DNA removal using appropriate salt concentrations, inactivation of amplifiable DNA using ethidium monoazide, and irradiation with visible light from a light-emitting diode lamp. Any remaining DNA, which likely exists as oligonucleotides captured by the Phi29 DNA polymerase, is degraded by the 3′-5′ exonuclease activity of the polymerase itself in the presence of trehalose, used as an anti-aggregation reagent. Phi29 DNA polymerase purified by this procedure has little amplifiable DNA, resulting in reproducible amplification of at least ten copies of plasmid DNA without any byproducts and reducing reaction volume. This procedure could aid the amplification of tiny amounts DNA, thereby providing clear evidence of contamination from laboratory environments, tools and reagents.

UR - http://www.scopus.com/inward/record.url?scp=84895510855&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84895510855&partnerID=8YFLogxK

U2 - 10.1371/journal.pone.0082624

DO - 10.1371/journal.pone.0082624

M3 - Article

C2 - 24505243

AN - SCOPUS:84895510855

VL - 9

JO - PLoS One

JF - PLoS One

SN - 1932-6203

IS - 2

M1 - e82624

ER -