Jpn. J. Infect. Dis., 52, 1999

Laboratory and Epidemiology Communications

Non‑radioactive Screening of DNA Polymerases by Using Oligo r(A‑U) as●Primer/Template for Reverse Transcnptase Activity
Ken‑ichi Hanaki* , Hiroshi Nakatake, Takashi Odawaral and Kenji Yamamoto2
The Chemo‑Sero Therapeutic Research Institute,

Kyokushi, Kikuchi‑gun, Kumamoto 869‑1298,
1AIDS Resewch Center, National Institute of Infectious Diseases,

Gakuen 4‑7‑1 , Musashimurayama, Tokyo 208‑0011 and2LaborqtoTy Of Clinical Microbiology and lmmunology, Bun Tin Hospital of UniversityofTokyo,
Mejirodai 3‑28‑6, Bunkyo‑ku, Tokyo 112‑0015‑

Communicated by Hiroshi Yoshikura(Accepted October 26, 1 999)

Reverse transcription‑polymerase chain reaction provides

is generally used fわr converting RNA to cDNA･ However,(lane c) which lacks RT activity did not. Probably, oligo d(A‑T) was first reverse‑transcribed from oligo r(A‑U)10, and thenthe oligo d(A‑T) was elongated to the huge DNA molecule･
Other DNA polymerases were also tested by incubation at
650C for only 2 h･ Bacillus cardotenu‑derived BcaBESTTM

these mesophilic viral RTs inemciently transcribe highly

DNA polymerase (lane d), Pyr10COCCuS SP.‑derived KOD DNA

folded RNA into cDNA･ The problem can be solved by tran‑
scrlption at a high temperature. So far, thermostable Rr
activity of Thermus thermophilus DNA polymerase (3) andthat of Bacillus calldoteyuu DNA polymerase have been used,
but further screenlng for such activity in DNA polymerases

polymerase (lane e), and Thermococcus litoralis‑derived
Vent@ DNA polymerase (lane f) synthesized poly d(A‑T),
while Pyrococcus sp･‑derived PyrobestTM DNA polymerase

a way of detecting rare messages such as those of HIV andHCV in sera (1 , 2).･ Reverse transcriptase (RT) derived fromavian myeloblastosis virus or Moloney murine leukemia virus(lane g) failed to synthesize poly d(A‑T). ThoughBcaBESTTM●DNA polymerase was known to have RT activity (including
Takara's RT‑PCR kit), the RT activities of KOD and Vent@

is needed.

RI‑labelled dUTP on poly rA template‑Oligo dT pnmer have

DNA polymerases have not been yet reported. It should be
mentioned that some thermostable DNA polymerases, suchbeen used thus far (4,5). In this report, we showthat polyas Taq or Tth, have the characteristic of "pnmer/template‑

For such screening, polymerization of PH】dTTP or non‑●●d(A‑T) synthesis primed by oligo r(A‑U) can be used as a

independent DNA synthesisM (6,8,9). The presence of such

simpler alternative method. The idea was derived from theactivity should be checked in parallel. All the DNA poly一observation that oligo d(A‑T) was elongated by a Stoffel frag‑ment of Taq DNA polymerase to a huge poly d(A‑T) DNA

as no DNA bands were obseⅣed in the controls (lanes (‑) inby self‑priming and elongation (6).

Fig.).

merases examined in this report were devoid of this activity,

The amount of poly d(A‑T) synthesized on oligo r(A‑U)

01igo r(A‑U)10, Which would seⅣe as both template and●●●pnmer/template is detemined by at least three factors: efficiency

prlmer for DNA polymerases, was transcribed by uslng T7‑

MEGA shortscriptTM (Ambion lnc., Austin, Tex.) from aof imitial reverse transcription, that of elongation which follows,

double stranded DNA template whose sense strand sequence
was 5 '‑GGATCTAATACGACTCACTATAGGGATATATATIATATATATATAT‑ 3 '(Sawady Technology, Tokyo). The transcrip‑and digestion by 5'to 3'or 3'to 5'exonuclease activities oftion mixture in a volume of 20 Lil contained a 5 ELM template,
7･5 mM each of NTP and 2LLI T7 MEGAshortscrlPt Enzymeof the DNA in the top of the gel by both Pol I's exonucleaseactivities. The intensity of the high molecular weight DNA

Mix･ It was incubated at 37oC for 3 h fわr transcription andband does not necessarily reflect RT activity itself. Withthis

the DNA polymerase. The diffuse band in the Pot I‑mediatedreaction (Fig., lane a (+)) probably represents the digestion

then the template DNA was digested away by incubation at37oC for 2 h with 2 U RNase‑free DNase I. After treatment
twice with phenol/chloroform/isoamyl‑alcohol (25:24: 1 ), theoligo r(A‑U)10 Was precipitated with ammonium acetate‑reservation in mind, our method, poly d(A‑T) synthesis on
oligo r(A‑U) primer/template, Could be used for screening
the RT activity of DNA polymerases. It requlreS nO labelled●precursor nucleotides and is very simple toperform･ethanol. The material obtained from the reaction mixture with‑out T7 MEGAshortscript Enzyme Mix was used as a nega‑

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Jpn. J. Infect. Dis., 52, 1999abc

M (‑) (+) (‑) (+) (‑) (+)

dfgh(‑) (+) (‑) (+) (‑) (+) (‑) (+)

Figure･ Poly d(A‑T) synthesis of DNA polymerases byusing oligo r(A‑U)I. aS Primer/template･
Five umits of E･ coli DNA polymerase Croyobo, Osaka) (lane a), Klenow fragment (Toyobo) (lane b), T4 DNA polymerase(Takara, Shiga) (lane c), BcaBESTTM DNA polymerase (Takara) (lane d), KOD DNA polymerase CToyobd) (lane e), Vent@

DNA polymerase (New England BioLabs, Beverly, Mass.) (lane f), or PyrobestTM DNA polymerase (Takara) (lane g) weredissolved in a 50 LLl reaction mixture optimized for each DNA polymerase with 600 ng of oligo r(A‑U)I., and 200 LLM eachof dATP and dTrP (GeneAmp@ dNTPs, PE Applied Biosystems Japan, Chiba). The incubation conditions were 37oC for 16 hfor Pot I, Klenow fragment, and T4 DNA polymerase, and 65oC for 2 h for BcaBESTTM, KOD, Vent@, and PyrobestTM DNA

polymerases･ The reaction mixture for Pot I was 50 mM TriS‑HCl, pH 7･8, 10 mM MgCl2, 100 mM DTr, l･25 jig BSA, thatfor Klenow fragment was 10 mM TriS‑HCl, pH 7.5, 7 mM MgCl2, 100 mM DTr, that for T4 DNA polymerase was 50 mM
lⅥS‑HCl, pH 7･5, 7 mM MgC12, 100 mM DTr, that for βCαBESTTM DNApolymerase was 20 mM Tris‑HCl, pH 8･5, 10 mM

MgCl2, that for KOD DNApolymerase was 120 mMTriS‑HCl, pH 8.0, 2･5 mM MgC12, 10 mM KCI, 6 mM (NH.)2S0., 0･1%
Triton@ X‑loo and O･001% BSA,that for Vent@ DNA polymerase was 20 mM TriS‑HCI, pH 8･8, 2･5 mM MgCl2, 10 mM KCl,and that for PyrobestTM DNA polymerase was 20 mM TriS‑HCl, pH 8.3, 2 mM MgCl2, 10 mM KCI, 6 mM (NH.)2S0., 0･1%
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Oriental, Inc･, Tokyo)･ The materials run in (+) lanes were those obtained with oligo r(A‑U)1. aS Primer/template;those run ln(‑) lanes were those obtainedusing the mock transcripts obtained by omitting T7 RNA polymerase.
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