Plant Molecular Biology Reporter pages 127 to 130 Volume 9(2) 1991

Protocol

Oligolabeling DNA Probes to High Specific Activity with Sequenase

Partha Sen and Norimoto Murai

Department of Plant Pathology and Crop Physiology, College of Agriculture, Louisiana State University, Baton Rouge, LA 70803-1720 USA

Key Words: Oligolabeling, Sequenase

Abstract: In this protocol we present a reproducible method of preparing DNA probes of high specific activity using Sequenase. The probes produced by this method had a specific activity of 2.8Xl(Ycpm/~ag with 69% of the total radioac- tivity incorporated into the TCA-precipitable materials. Probes with 5 to 1()-fold lower specific activity were obtained using commercially available kits or using currently employed methods.

R adiolabeled DNA probes are routinely prepared by nick transla- tion (Kelly et al., 1970, Maniatis et al., 1975) or oligolabeling with random primers (Feinberg and Vogelstein, 1984; Hodgson and

Fisk, 1987). Both reactions use the Klenow fragment of DNA polymerase I. Here we report a protocol in which Sequenase is used instead of Klenow fragment to obtain radiolabeled probes of high specific activity (2.8X109cpm/~tg) with a maximum of 69% incorporation of the total radioactivity into the TCA precipitable materials. In a parallel experi- ment probes produced by the method of Hodgson and Fisk (1987) had a 5-fold lower specific activity. A commercially available kit (Pharmacia) consistently yielded a specific activity of less than 3x10 s cpm/~tg.

Materials and Methods All reagents used were of AR grade.

10 X Sequenase Buffer: 75 mM Tris-HC1 pH 7.5, 55 mM dithiothreitol (DTT), 50 mM MgC12

Template DNA Plasmid DNA was dissolved in water to a final concentration of 100 ~g/mL.

127

128 Sen and Mural

Random Primer was purchased from New England Biolabs (NEB) and was dissolved in water to a final concentration of 50/.tg/mL.

Deoxynucleotide Triphosphates (dATP, dGTP, TTP) were pur- chased as 10 mM solutions from Bethesda Research Laboratories (BRL) and mixed together to a final concentration of 0.6 mM each.

Sequenase was purchased from US Biochemicals. cz-P2P]-dCTP (10.0 mCi/mL) was purchased from ICN Biomedicals,

Inc.

Protoco l

�9 50 ng of Template DNA (0.5 ~L) and 50 ng of random primer (1.0 pL) were mixed with 8.5 BL of water and heated together in a boiling water bath for 5 min. and then quick chilled in ice water.

�9 2 ~L of 10 X Sequenase buffer was added. �9 2 ~L of the dNTP mix (0.6 raM) was added. �9 5 ~L of (z-[32p]-dCTP (50 ~Ci) was added. �9 1 ktL of Sequenase (13 units) was added. �9 The solutions were mixed thoroughly and incubated overnight at

room temperature (25 ~ �9 The probe was separated from the unincorporated nucleotides by

passing through a Sephadex G-50 Column (Pharmacia). �9 The void volume fractions containing the probe were pooled and

the probe was concentrated by precipitating with cold ethanol in the presence of 25/.tg/mL of tRNA.

Note We have found that random primer from NEB gives better results.

R e s u l t s a n d D i s c u s s i o n

We have optimized the template to primer ratio, enzyme concentra- tion, amount of radioisotope and the duration of the reaction. In our hands a reaction with 50 ng each of template and primer, 0.06 mM each of dATP, dGTP and TTP, 50 juCi of r and 13 units of Se- quenase when incubated overnight at 25 ~ has produced the best results. Varying the primer concentration from 5 to 500 ng neither changed significantly the specific activity of the probe (1.8X109cpm/~g) nor the percent incorporation (69%). Using higher amounts of enzyme (26 units) or of ct-[32p]-dCTP (150 BCi) did not change the specific activity (2.0X109cpm/lzg) notably. The time course reaction indicated that 4 hr of

Oligolabeling DNA Probes 129

100.

- - 80" =

6O.

c 40 .

c

20- o

a .

0 �9 i " , i �9 �9 i , , i ,

200 400 600 800

T ime in minutes

25 O0

2 0 0 0

1500

1000

5 O0

. , . 0 O0 iooo

Fig. 1. Time course of oligolabeling DNA reaction using Sequenase. The reaction was carried out with 50 ng template DNA, 50 ng primer, 13 units Sequenase and 50 p.Ci [o~32P] - dCTP at room temperature. Four hundredths of the reaction mixture was taken at the time specified and the reaction product was pre- cipitated with TCA and quantified in a liquid scintillation counter.

1107 -

7 3 8 -

4 9 2 -

3 6 9 -

2 4 6 -

1 2 3 -

6 5

1 2 3

i Fig. 2. Size distribution of labeled D N A products obtained with the three reaction protocols. Products of three labeling reactions purified on a G-50 Sephadex column were fractiona ted on a 3% NuSieve agarose gel. Seq, products obtained with Sequenase; H+F, products obtained with the method of Hodgson and Fisk (1987); Pharm., products obtained with Pharmacia kit. The numbers on the left represent size markers in base pairs

incubat ion was sufficient to reach the m a x i m u m percent incorpora t ion (Fig. 1 ). The labeled probes were found to be of 50 to 250 base pairs in size when ana lyzed on a 3% NuSieve agarose gel (Fig. 2). Reducing the p r imer concent ra t ion to 5 ng p roduced little detectable effect on the probe size. This is con t ra ry to the f inding of H o d g s o n and Fisk (1987) using Klenow f ragmen t and appears to be characterist ic of the Sequenase reaction. DNA probes p repa red by this me thod have been used successfully in Sou them, Nor the rn and dot blot hybr id iza t ion analysis in ou r laboratory.

Acknowledgments : We thank Dr. M. D. Burow for critical reading ot the manuscript. This work was supported partly by the LSU College of Agriculture and by grants from Louisiana Education Quality Support Fund.

130 Sen and Murai

References Kelly, R.G., N. Cozzarelli, M.P. Deutscher, IR. Lehman and A. Kornberg. 1970. Enzymatic

synthesis of deoxyribonudeic acid. J. Biol. Chem. 245, 39-45. Maniatis, T., T.A. Jeffery and D.G. Kleid. 1975. Nucleotide sequence of the rightward

operator of phage k. Proc. Natl. Acad. Sci. USA. 72, 1184-1188. Feinberg, A.P. and B. Vogelstein. 1984. A technique for radiolabeling DNA restriction

fragments to high specific activity. Anal. Biochem. 137, 266-267. Hodgson, C.P. and R.Z. Fisk. 1987. Hybridization probe size control: optimal 'Oligolabeling'.

Nucl. Acids Res. 15, 6295.