[9] NICK TRANSLATION 91

[9] N i c k T r a n s l a t i o n
By JUDY MEINKOTH and GEOFFREY M. WAHL

Nick translation, or more precisely nick translocation, is a specific

procedure for incorporating radioactive nucleotides into double-stranded
DNA. The method takes advantage of the ability of Escherichia coli DNA
polymerase I to combine the sequential addition of nucleotide residues to
the 3'-hydroxyl terminus of a nick [generated by pancreatic deoxyribonu-
clease (DNase) I] with the elimination of nucleotides from the adjacent 5'-
phosphoryl terminus. Linear, supercoiled, nicked, or gapped circular
double-stranded molecules can be labeled to specific activities > 108 cpm/
/zg with deoxynucleotide 5'-[a-32P]triphosphates by this technique. 1,2
Since the nicks are introduced at random sites in the duplex, the method
generates a population of radioactive fragments which partially overlap
each other. At saturating levels of nucleotide triphosphates the size of the
fragments is determined by the DNase concentration. Fragments approxi-
mately 500-1500 nucleotides long produce optimal signal-to-noise ratios
when hybridized to immobilized DNA or RNA (see this volume [45, 61]),
presumably due to their ability to hybridize with each other in overlapping
complementary regions to form "networks" or "hyperpolymers."2 While
experiments consistent with hyperpolymer formation of nick-translated
probes have been reported, 3,4 the reproducibility and extent of hyperpoly-
mer formation seem to be difficult to obtain, probably because of the
critical dependence on probe size. 2,4 Longer probes have been correlated
with higher backgrounds (G. Wahl, unpublished observations).

Nick Translation Reaction

1. Dilute stock DNase in 50 mM Tris-HCl at pH 7.4, 10 mM MgC12, 1
mg/ml DNase-free bovine serum albumin just before use. A DNase con-
centration of about 40-80 pg//zl is usually adequate.
2. Mix 2 ~1 10x NT (nick translation) buffer [10x = 50 mM MgCI2,
200-250/zM each unlabeled deoxyribonucleoside triphosphate (dNTP),
omitting those corresponding to the labeled nucleotide(s), 50 mM Tris-

I p. W. J. Rigby, M. Dieckmann, C. Rhodes, and P. Berg, J, Mol. Biol. 113, 237 (1977).
2 j. Meinkoth and G. Wahl, Anal. Biochem. 138, 267 (1984).
3 G. M. Wahl, M. Stern, and G. R. Stark, Proc. Natl. Acad. Sci. U.S.A. 76, 3683 (1979).
4 R. H. Singer, J. B. Lawrence, and C. Villnave, BioTechniques 4, 230 (1986).

Copyright © 1987by AcademicPress, Inc.

METHODS IN ENZYMOLOGY,VOL. 152 All fightsof reproductionin any form reserved.
92 ENZYMATICTECHNIQUES [9]
HCI at pH 7.4] with 100-400 ng DNA, and 50-300/zCi of deoxyribonucle-
oside 5'-[a-32p]triphosphate (3000 Ci/mmol, 10/zCi//.d). Add 1/zl diluted
DNase and water to 19/xl. Usually three of the four triphosphates (e.g.,
dATP, dGTP, and TTP) are unlabeled whereas the fourth (dCTP in this
example) is supplied as a radioisotope. Any combination of labeled and
unlabeled deoxyribonucleoside triphosphates will suffice, however, pro-
vided the concentration of each dNTP is greater than 1-3 /.tM, a value
which is probably not saturating for the enzyme. Incubate 2 min at room
temperature.
3. Add 1 txl E. coli DNA polymerase I (Pol I, 0.8 units//zl) and incubate
at 15° for 30 min to 2.5 hr. The progress of the reaction may be monitored
by measuring incorporation into trichloroacetic acid-insoluble material
using the techniques detailed in this volume [6]. Purify the nick-translated
DNA as detailed in steps 4-6.
4. Add 0.1 volume of 2% SDS, 250 mM EDTA, or an equal volume of
phenol-saturated H20 to stop the reaction. Phenol-saturated H20 is made
by vortexing equal volumes of redistilled phenol (often containing 0.1%
hydroxyquinoline as an antioxidant) and H20 for 30 sec. After centrifuga-
tion for 30 sec in an Eppendorf Microfuge, or the equivalent, the upper
phase consisting of phenol-saturated H20 is recovered.
5. Remove unincorporated nucleotides by passing the reaction
through a P-60 (100-200 mesh, Bio-Rad) column poured in a Pasteur pipet
or through a spun Sephadex G-50 fine column. Such a column is made in a
1-ml syringe plugged with glass wool by adding aliquots of swelled Sepha-
dex G-50 fine resin (Pharmacia) and centrifuging at -1000 g for 5 min until
a packed volume of - 0 . 9 - 1 ml has been attained. Centrifugation is ac-
complished by placing the column inside a 15-ml conical disposable tube.
The lip of the syringe conveniently hangs on the edge of the tube; thus,
the column does not come in contact with the effluent. Add 100 ~1 H20 to
the column. Centrifuge at 1000 g for 5 min, transfer the column to a fresh
disposable tube, and add the sample brought to 100/xl with H20. Centri-
fuge at I000 g for 5 min. The eluate in the conical tube is virtually free of
unincorporated deoxyribonucleoside triphosphates and may be used
without further purification. Alternatively, transfer the DNA to a micro-
fuge tube and precipitate it with 5-25 /xg tRNA, 0.5 volume of 7.5 M
ammonium acetate, and 2.5 volumes of ethanol by incubating 10 min in
dry ice. Recover the DNA by centrifuging in a microfuge (12,000 g) for 15
min. The pellet contains the sample. Determine the radioactivity incorpo-
rated by any of the methods described in this volume [6]. Dissolve the
material in -100/xl H20, denature by boiling for 5 min or by heating at 37 °
in 0.1-0.3 M N a O H for at least 5 min, quench in ice for 2 min, and use for
hybridization.
[9] NICKTRANSLATION 93

Optimizing Nick-Translated Probe Size

1. Store DNase I (Worthington DPFF) at a concentration of 1 mg/ml
in 50% glycerol, 50 mM Tris-HC1 (pH 7.4), 10 mM MgCI2, 1 mM
dithiothreitol at - 7 0 ° in small aliquots.
2. Titrate new stocks of DNase to determine the concentration which
produces probes 500-1500 bp long as follows. Set up a nick translation
reaction as above using a fixed concentration of DNase and perform a
time course experiment with the concentrations of dNTPs chosen to
mimic experimental conditions. Determine when optimal incorporation
occurs. Repeat at different DNase concentrations.

Sizing Nick-Translated Probes

1. Remove approximately 2 x 105 cpm of probe from each reaction.
2. Add NaOH to 0.I M. Incubate at 37° for 10-15 min.
3. Add 0.2 volume 50% glycerol, 0.5% bromphenol blue.
4. Load samples onto a 2% agarose gel with appropriate denatured size
markers (in range of 100-2000 bp). Run at approximately 50 V until the
dye has migrated 8-10 cm. Stain, photograph, and then expose gel to film.
(See this volume [8].) A good probe migrates as a smear with most of the
counts in the range 250-1500 bp. Probes with material which smears from
the gel origin have been correlated with high backgrounds.

Notes
I. Nick translation kits are now available from Bethesda Research
Laboratories, New England Nuclear, and Amersham. In general they
provide instructions, a stock mixture of Pol I and DNase I, and a series of
buffers lacking one or more unlabeled deoxyribonucleoside triphos-
phate(s). The radioactive triphosphates are the sole source of the missing
nucleotides.
2. Failure to nick translate a fragment is almost invariably a function of
the quality of the DNA. Once the enzymes have been eliminated as the
cause by successfully nick translating a known control DNA and once the
quality of the radioisotope has been established (by successfully using it
to label control DNA), one should focus attention on the sample DNA.
The problem can sometimes be alleviated by phenol extracting the DNA
(this volume [4]) or by purifying it on a column such as Elutip (Schleicher
and Schuell) or NACS (Bethesda Research Laboratories). If neither pro-
cedure solves the problem, the concentration of DNase and the incuba-
94 ENZYMATIC TECHNIQUES [10]

tion time before Pol I is added may have to be increased. If one is using a
kit with premixed enzymes, better results can sometimes be obtained by
simply nick translating in a larger volume. Buffer and enzymes are scaled
up accordingly, but sample DNA and sometimes radioisotopes are not
increased.

[10] E n z y m e s f o r M o d i f y i n g a n d L a b e l i n g D N A a n d R N A
By FABIO COBIANCHI a n d SAMUEL H . WILSON

In this chapter, we outline practical applications for 12 of the nucleic

acid enzymes now in routine use in the molecular biology laboratory.
Properties of these enzymes have been reviewed elsewhere, 1-4 and vari-
ous methods for their use also have appeared. Here we describe a set of
simple procedures we have used along with methods for isolating and
monitoring reaction products of interest.

K l e n o w F r a g m e n t o f Escherichia coli D N A P o l y m e r a s e I

The Klenow fragment enzyme is a single polypeptide chain obtained

by proteolytic digestion of DNA polymerase I or by molecular cloning of
the appropriate portion of the gene. The enzyme carries the polymerase
and 3' --> 5' exonuclease activities of intact DNA polymerase I, but lacks
the 5' ---->3' exonuclease activity of the intact enzyme.
The Klenow fragment is used primarily for filling in and labeling re-
cessed 3' ends of double-stranded DNA produced by digestion with re-
striction enzymes and for repairing the ends of DNA duplexes. Nucleases
such as $1 and Ba131 and some restriction enzymes often create staggered
ends consisting of one or more unpaired bases. Since these unpaired
bases prevent subsequent blunt-end ligation (as in the ligation of linkers),
Klenow fragment is used to fill in the missing nucleotides. The Klenow
fragment is the enzyme of choice in DNA sequencing protocols by the
chain termination method (see this volume [58]), although reverse
transcriptase is also useful because of its superior ability to read through

t This series, Vols. 65, 68, 100.

2 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Man-
ual." Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1982.
3 j. G. Chirikjian and T. S. Papas, eds., "Gene Amplification and Analysis" Vol. 2. Else-
vier/North-Holland, New York, 1981.
4 p. D. Boyer, ed., "The Enzymes," 3rd ed., Vol. 15. Academic Press, New York, 1982.

Copyright © 1987 by Academic Press, Inc.

METHODS IN ENZYMOLOGY, VOL. 152 All rights of reproduction in any form reserved.