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.
[10] MODIFYING AND LABELING D N A AND R N A 95

secondary structures. A recent application of the enzyme has been the

production of single-stranded probes by primer extension in the single-
stranded phage MI3 system. 5,6 The probes generated by these methods
can be used for in situ hybridization and SI nuclease analysis.

Filling-in Reaction
Mix, in order, to a total volume of 20 ~1 using water to adjust the
volume,
DNA with 3'-recessed ends 0.1 to 1/xg
10× fill-in buffer [500 mM Tris-HCl at pH 7.5, 2 txl
100 m M MgC12, 10 m M dithiothreitol (DTT),
500/xg/ml bovine serum albumin]
2 mM each of three deoxyribonucleoside triphos- 3 t~l
phates (dNTPs)
[~-32p]dNTP at 400 Ci/mmol (I-2/zCi total) 1-2/zl
(Choose the nucleotide not represented among
nonradioactive dNTPs)
Klenow fragment 1 unit
Incubate at 23° for 15 min and add
2 m M dNTP, unlabeled dNTP corresponding to la- 2/zl
beled nucleotide above
Incubate 10 min at 23 °.
Stop the reaction by adding 1 ~1 0.5 M EDTA (pH 8.0), add 100/A of
TE at pH 8.0 (10 mM Tris-HC1 at pH 8.0, 1 mM EDTA), extract the
solution with phenol-chloroform 2 times, and precipitate with ethanol
and ammonium acetate as detailed in this volume [5]. Wash the pellet with
70% ethanol. Alternatively, unincorporated triphosphates may be re-
moved using a spun Sephadex G-50 column (this volume [9]) or by drop
dialysis (this volume [5]).
Monitoring the Reaction. Incorporation of radioactivity into trichlo-
roacetic acid (TCA)-insoluble material is measured by spotting a tiny
aliquot onto GF/C glass filters as detailed in this volume [6].

Production of Single-Stranded Probes

Procedure 1. In this first protocol, the M13 phage recombinant DNA
(plus strand) is primed with a commercially available primer upstream of
the insert and the primer extended away from the insert in the presence of

5 j. Meinkoth and G. Wahl, Anal. Biochem. 138, 267 (1984).

6 A. Seiler-Tuyns, J. D. Eldridge, and B. M. Paterson, Proc. Natl. Acad. Sci. U,S.A. 81,
2980 (1984).
96 ENZYMATIC TECHNIQUES [10]

radioactive nucleotides. The resultant radioactive probe consists of a par-

tially duplex molecule in which the specificity is dictated by the insert
which is in single-stranded configuration.
In a siliconized Eppendorf tube, mix
500 ng single-stranded M13 recombinant DNA 5/zl
8 ng M13 primer (Bethesda Research Laborato- 4/zl
ries catalog no. 8238 SA)
TM buffer (100 m M Tris-HCl at pH 7.6, 600 m M I/zl
NaCI, 66 m M MgC12)
Heat at 90 ° for 5 min, incubate at 23 ° for 45 min, and add
solution containing 660/xM each dATP, dGTP, 3/.d
TTP and 50/xCi [a-32p]dCTP at 3000 Ci/mmol
40 m M dithiothreitol (DTT) 1/A
1 to 2 units Klenow fragment 1-2/xl
Incubate at 23 ° for 30 min.
Stop the reaction by adding 1 /xl 0.4 M EDTA, 2% sodium dodecyl
sulfate (SDS). Remove the unincorporated label by ethanol precipitation
as described above. The probe is ready for immediate use and is not to be
denaturated before hybridization.
Procedure 2. In the second procedure, uniformly labeled single-
stranded DNA probes are prepared in which only the cloned insert is
specifically labeled. In this protocol, the M13 phage DNA-containing in-
sert is primed with one of the standard sequencing primers (downstream
of the insert), and the primer is extended to traverse the insert in the
presence of radioactive nucleotides. The partially duplex product is di-
gested with a restriction enzyme that will cut at the end of the insert distal
to the primer. The strands are dissociated, and the single-stranded labeled
probe is resolved and recovered by acrylamide gel electrophoresis in the
presence of urea.
In a siliconized Eppendorf tube, mix
500 ng single-stranded MI3 recombinant DNA 5/zl
3 ng M I3 primer (New England BioLabs catalog 1 /zl
no. 1211)
TM buffer (see above) 2/zl
Water 2/zl
Incubate at 60 ° for 1 hr and add
solution containing 1 m M each dCTP, dGTP, 10/xl
TTP
50/xCi [a-32p]dATP at 600 Ci/mmol 5/xl
50 mM DTT 1 /xl
Water 3/~1
5 units Klenow fragment I p.l
Incubate 30 min at 23 ° and add
[10] MODIFYINGAND LABELINGDNA AND RNA 97

chase solution (2 mM each dATP, dCTP, dGTP, 1 /zl

TTP)
Incubate at 23 ° for 20 min.
Inactivate the Klenow fragment by incubating at 70° for 5 min. Add 4
/xl of the appropriate 10× restriction buffer, 1 ~1 restriction enzyme, and
water to 40/zl. Incubate at 37° for 1 hr. Purify the single-stranded labeled
DNA probe by gel electrophoresis under denaturing conditions as de-
scribed in this volume [8].
Procedure 3. This technique is aimed at synthesizing highly radioac-
tive oligomers for use in hybridization experiments. It is basically a varia-
tion of procedures 1 and 2 using an oligomer as template and a smaller
oligomer complementary to the 3' end of the template as the primer. 7
Since the primer and template are often identical in size after the reaction,
a method has been developed to facilitate electrophoretic separation of
the two fragments in gels. The blocking group at the 5' end of the primer,
a dimethoxytrityl group, is not removed following organic synthesis of the
primer. Since this group is very large, it decreases mobility of the labeled
primer. The extended primer may be excised from a denaturing polyacryl-
amide gel without fear of contamination by unlabeled template. The
blocking group does not interfere with subsequent hybridization. 7 As an
alternative, an internal primer that does not form a flush 3' end with the
template may be used.
Mix in a total of 10/.d, using water to adjust the volume,
extension buffer (600 mM Tris-HCl at pH 7.5, 600 1 /zl
mM NaCI, 60 m M MgC12, 20 mM DTT)
primer oligomer with blocked 5' end (4-fold molar 100 ng
excess over template)
template oligomer 60 ng
Heat at 55 ° for 10 rain; cool slowly to room temperature.
Transfer the entire mixture to a fresh tube containing
300/zCi [a-32p]dNTP at 3000 Ci/mmol (all four dry
dNTPs may be labeled; unlabeled dNTPs each at
I0/zM)
Place in ice 5 min and add
Klenow fragment (Boehringer-Mannheim) 2.5 units
Incubate 2-3 hr in ice.
Purify labeled strand in a 20% polyacrylamide-urea gel using the
methods in this volume [8].
Monitoring Reactions in Procedures 1 through 3. Use methods de-
scribed under Filling-in Reaction.

7 A. B. Studencki and R. B. Wallace, D N A 3, 7 (1984).

98 ENZYMATIC TECHNIQUES [10]

Notes. The filling-in reaction described is intended as a "repairing

reaction" for generating blunt ends in DNA molecules. In order to moni-
tor the activity of the enzyme the labeled deoxyribonucleoside triphos-
phate is added at low specific activity, 400-600 Ci/mmol, as a tracer.
Since the probe prepared by procedure 2 is to be used in Sl analysis
experiments, the specific activity of the labeled triphosphate is chosen
low in order to minimize the formation of breakdown products. The spe-
cific activity of probes obtained with procedures 1 and 2 should be in the
order of 109 and 108 cpm//.~g of DNA, respectively. Procedure 3 provides
the highest specific activities possible, > 109 cpm//xg.

Second-Strand DNA Synthesis with Random Oligodeoxynucleotides

as Primers
It is well known that DNA polymerases can use oligonucleotides as
primers for second-strand synthesis from a single-stranded DNA tem-
plate. 8,9 A technique was developed by Feinberg and Vogelstein l° in
which the Klenow fragment is used to copy single-stranded DNA tem-
plates primed with random oligodeoxynucleotides prepared from calf thy-
mus DNA or random hexamers obtained commercially.
Using this technique a stable high specific activity DNA can be ob-
tained using very small amounts of DNA template. Probes obtained by
this procedure are suitable for filter hybridization techniques such as
Southern and Northern analysis, colony screening, plaque hybridization,
and in situ hybridization.
In an Eppendorf centrifuge tube in ice mix
l0 mg/ml bovine serum albumin (nucleic acid grade) 1/zl
LS solution (440 m M H E P E S - N a O H at pH 7.6, 44 11.5/xl
/zM each dATP, dGTP, TTP, 110 mM Tris-HCl at
pH 7.6, 11 m M MgCI2, 22 mM 2-mercaptoethanol,
oligodeoxyribonucleotide hexamers at 300/zg/ml,
commercially available, or prepared by DNase I
digestion of calf thymus DNA)
Water 12.5/xl
The purified DNA restriction fragment (1-5/.d, 20-100 ng in water) is
sealed in a capillary tube and heated at 100° for 2 min, immediately cooled
to 0°, and added to the reaction mixture. To the primers-template mixture
in ice add
50/xCi [a-32p]dCTP at 3000 Ci/mmol 5/xl
(2.5 units) Klenow fragment 1 /xl
Incubate at 23° for 3 hr.

g M. Goulian, Cold Spring Harbor Symp. Quant. Biol. 33, 11 (1984).

9 j. M. Taylor, R. Illmensee, and J. Sumers, Biochirn. Biophys. Acta 442, 324 (1976).
~0A. P. Feinberg and B. Vogelstein, Anal. Biochern. 132, 6 (1983).
[10] MODIFYINGAND LABELINGDNA AND RNA 99

Stop the reaction by adding 1/xl 0.5 M EDTA and process the sample
as described for the Filling-in Reaction.
Monitoring the Reaction. Incorporation of radioactivity into acid-in-
soluble material is monitored by TCA precipitation on filters as described
in this volume [6]. The reaction product is analyzed by polyacryamide gel
electrophoresis under denaturing conditions (this volume [8]). From a 1.2-
kb restriction fragment, the average size of single-stranded probe was 0.8
kb. Over 70% of the precursor triphosphate is incorporated into DNA and
a specific activity of 109 cpm//xg of DNA can be obtained.

Removing 5'-Terminal Phosphates from DNA

Removal of Y-phosphates from DNA or RNA is performed prior to
labeling 5' ends with 3zp or to prevent undesired intramolecular ligation of
either vector DNA or RNA. Bacterial alkaline phosphatase (BAPF) and
calf intestine alkaline phosphatase (CIP) are commercially available and
are used for this purpose. The amount of enzyme necessary may vary
depending upon the substrate. Therefore, it is best to determine the
amount of enzyme required empirically. Generally 1 unit of enzyme is
needed for the complete removal of terminal phosphates from 100 pmol of
5' ends of DNA. Both enzymes also cleave Y-phosphates.

Bacterial Alkaline Phosphatase Reaction

Mix in a total of 50/zl, using water to adjust the volume
DNA with 5'-terminal phosphates 10 pmol of ends
500 mM Tris-HCl at pH 8.0 5/zl
Bacterial alkaline phosphatase 0.1 unit
Incubate at 37° (5'-protruding or flush ends) or at 60° (recessed 5' ends)
for 30 min.

Calf lntestine Alkaline Phosphatase Reaction

Mix in a total of 50 tzl, using water to adjust the volume
DNA with Y-terminal phosphates, 10 pmol of ends
10x CIP buffer (500 mM Tris-HC1 at pH 5/zl
9.0, 10 mM MgCI2, 1 mM ZnClz, 10 mM
spermidine)
Calf intestine phosphatase (CIP) 0.1 unit
Incubate at 37° (5'-protruding ends) for 30 rain, add another 0.1 unit of
CIP, and continue incubation for an additional 30 min. For blunt ends or
recessed 5' ends use 56° .
Stop the reaction by adding 1/zl 0.5 M EDTA at pH 8.0. Add 100/xl
TE at pH 8.0. Extract twice with an equal volume of phenol-chloroform
(1 : 1) saturated with TE at pH 8.0, and twice with chloroform. Extract
100 ENZYMATICTECHNIQUES [10]

with abundant ether; add sodium acetate at pH 5.3 to a final concentration

of 0.3 M and precipitate the DNA with 2.5 volumes of ethanol. Centrifuge
and resuspend the pellet in a minimal volume of TE at pH 8.0. The DNA
can be further purified by chromatography in Sephadex G-50 and is now
ready for subsequent ligation or end labeling.
Monitoring the Reaction. If some of the DNA has already been end
labeled and is available as tracer, the disappearance of radioactivity from
trichloroacetic acid-precipitable material, hence the extent of the reac-
tion, can be measured by means of a filter assay (this volume [6]). Alterna-
tively the extent of dephosphorylation of vector DNA can be followed by
self-ligation (of the vector DNA) and subsequent bacterial transforma-
tion. The efficiency of transformation with dephosphorylated DNA prod-
uct above should be less than the efficiency observed with vector not
dephosphorylated. See elsewhere in this chapter for ligation methods.

T4 Polynucleotide Kinase: Uses for 5'-End Labeling with a2p

Bacteriophage T4-induced polynucleotide kinase catalyzes transfer of
the y-phosphate of ATP to the 5'-hydroxyl terminus of either DNA or
RNA. ~1 Because of the requirement for a 5'-hydroxyl terminus, the sub-
strate often must be dephosphorylated with alkaline phosphatase prior to
the [32p]phosphotransfer reaction. Alternatively, a 5'-phosphoryl termi-
nus can be labeled by phosphate exchange, taking advantage of the re-
versibility of the T4 polynucleotide kinase-catalyzed reaction.~2
Labeling Y-Hydroxyl Termini (Forward Reaction)
For protruding 5' ends, mix in an Eppendorf tube in ice, adjusting the
volume to 50/xl with water
Dephosphorylated DNA 1-50 pmol of 5' ends
10x kinase buffer I (500 mM Tris-HCl 5/zl
at pH 7.6, I00 m M MgCI2, 50 m M
DTT, I m M spermidine hydrochlo-
ride, 1 mM EDTA)
150 ~Ci [y-32p]ATP at 3000 Ci/mmol 15/zl
T4 polynucleotide kinase 20 units
Incubate at 37 ° for 30 min.
For flush or recessed 5' ends, mix in an Eppendorf tube in ice, adjust-
ing the volume to 40/zl with water
Dephosphorylated DNA 1-50 pmol of 5' ends
10× TSE buffer (200 m M Tris-HC1 at 4/zl
pH 9.5, 10 m M spermidine hydrochlo-
ride, 1 mM EDTA)
II C. C. Richardson, Proc. Natl. Acad. Sci. U.S.A. 54, 158 (/965).
~2j. H. Van de Sande, K. Kleppe, and H. G. Khorana, Biochemistry 12, 5050 (1973).
[10] MODIFYINGAND LABELINGDNA AND RNA 101

Incubate at 90° for 2 min, chill by placing in ice water, and add
10x kinase buffer II (500 mM Tris-HCI 5/zl
at pH 9.5, 100 mM MgCI2, 50 mM DTT,
50% glycerol (v/v)
150 p.Ci [y-32p]ATP at 3000 Ci/mmol dry
T4 polynucleotide kinase 20 units
Water to 50/zl
Incubate at 37° for 15 min.

Exchange Reaction
Mix in a total of 50/xl, using water to adjust the volume
DNA 5'-PO4 1-50 pmol of 5' ends
10x kinase buffer III (500 mM imida- 5/xl
zole-HC1 at pH 6.6, 100 mM MgC12,
50 mM DTT, 1 mM spermidine hy-
drochloride, 1 mM EDTA)
5 mM ADP 3/xl
100/zCi [y-3Zp]ATP at 3000 Ci/mmol 10/zl
T4 polynucleotide kinase 20 units
Incubate at 37° for 30 min.

Coupled Phosphatase-Kinase Reaction

This method is particularly useful for end labeling of restriction frag-
ments since the restriction digestion, dephosphorylation, and terminal
labeling can be performed sequentially in one reaction tube. In this proce-
dure, the dephosphorylation of the substrate is carried out as usual with
calf intestine alkaline phosphatase; subsequently, the labeling reaction is
performed in the presence of inorganic phosphate under conditions that
will inhibit alkaline phosphatase but not the phosphorylation reaction.~3
In an Eppendorf tube in ice mix
50 pmol 5' ends of restricted DNA in pre-
vious restriction buffer 10/xl
Calf intestine alkaline phosphatase 0.1 unit
Incubate at 45 ° for 45 min. Add
10x kinase buffer IV (500 mM glycine-NaOH 5/xl
at pH 9.2, 100 mM DTT, 50 mM MgCI2)
Sodium phosphate at pH 9.2 see note below
[y-32p]ATP at 1000 Ci/mmoi see note below
T4 polynucleotide kinase 4 units
Water to a final volume of 50/xl
Incubate at 37° for 15 min.

13G. Chaconas and J. H. Van de Sande, this series, Vol. 65, p. 75.
102 ENZYMATICTECHNIQUES [10]

Stop the reaction with 1 /~l of 0.5 M EDTA and purify the DNA by
phenol extraction and ethanol precipitation as described for Klenow frag-
ment filling-in reaction.
N o t e s . For restriction fragments with flush or 3' protruding ends, the
ATP concentration in the reaction should be 10-30/zM with an ATP to 5'
ends ratio of at least l0 : l and a sodium phosphate concentration of 1
raM. For restriction fragments with Y-protruding ends, an ATP concen-
tration in the reaction of l0/zM is recommended with an ATP to 5' ends
ratio of 10 : 1 and sodium phosphate concentration of 5 mM.
Monitoring the Reactions. Incorporation of 32p can be monitored by
TCA precipitation as described in this volume [6]. For oligonucleotides of
10 residues or less, use DE-81 filters and wash with concentrated phos-
phate as described in this volume [6]. Under conditions described above,
the forward reaction yields 2 x l 0 6 cpm/pmol end, the exchange reaction
yields 6 × 105 cpm/pmol end, and the coupled reaction yields 105 cpm/
pmol end.

The 3'-Phosphatase Activity of T4 Polynucleotide Kinase

T4 polynucleotide kinase possesses a 3'-phosphatase activity, j4 in ad-
dition to the activity that transfers the y-phosphate of ATP to the 5'-OH
terminus of DNA. The kinase and phosphatase activities of the enzyme
may be used independently by choosing appropriate reaction conditions.
By incubating the substrate at low pH in the absence of ATP or ADP, 3'-
phosphates will be removed from DNA or RNA molecules without alter-
ing the structure of the 5' terminus. Note that a commercially available
cloned polynucleotide kinase lacks the 3'-phosphatase activity of the na-
tive enzyme and cannot be used for this reaction.

Reaction Conditions 15
Mix in a total volume of 50/xl, using water to adjust the volume
DNA with 3'-phosphate ends 1-50 pmol of ends
10x reaction buffer (1 M Tris-HCl at pH 5/~1
6.5, 1 M magnesium acetate, 50 mM 2-
mercaptoethanol)
T4 polynucleotide kinase 6 units
Incubate at 37° for 12 hr.
Stop the reaction by adding 1/A 0.5 M EDTA and recover the DNA by
phenol extraction and ethanol precipitation as described above.
Monitoring the Reaction. If 3'-end-labeled DNA is available as tracer,

14V. Cameronand O. C. Uhlenbeck,Biochemistry 16, 5120 (1977).

15B. Royer-Pokorna, L. K. Gordon, and W. A. Haseltine, Nucleic Acids Res. 9, 4595
(1981).
[10] MODIFYING AND LABELINGDNA AND RNA 103

the extent of the reaction can be followed using either loss of trichlo-
roacetic acid-precipitable material or loss of DE-81 binding material (this
volume [6]).

3'-Terminal Labeling of RNA with T 4 RNA Ligase j6

Bacteriophage T4 R N A ligase catalyzes the ligation of a 5'-phosphoryl
donor to a 3'-hydroxyl acceptor. Although the minimal acceptor must be a
trinucleoside diphosphate, mononucleoside 3',5'-bisphosphates (pNps)
are effective donors. ~7,~8 One consequence of this observation is that a
convenient method for labeling the 3' end of RNA molecules in vitro is
available. By using a [5'-32p]pNp as a donor and RNA as acceptor, the
product of the reaction is an R N A molecule extended by one nucleotide
and containing a 3'-terminal phosphate and a [32p]phosphate in the last
internucleotide linkage.
5' 3' [5'-32p]pCp 5' 3'
.... pC-pG-pA-OH ~ .... pC-pG-pA-[32p]pCp
ATP

In an E p p e n d o r f tube in ice mix

RNA 1 tzg
0.15 m M ATP 1 /zl
0.75 M H E P E S - N a O H at pH 8.3 2/zl
0.3 M MgCI2 1 /zl
0.1 M DTT 1 /zl
Dimethyl sulfoxide 3/zl
300/xg/ml BSA 1 pA
100 p,Ci [5'-32P]pCp at 3000 Ci/mmol I0/zl
T4 R N A ligase at 3000 units/ml 4/zl
Water to final volume of 30 izl
Incubate at 4 ° for 24 hr.
M o n i t o r i n g the R e a c t i o n . The reaction is monitored by measuring
incorporation of radioactivity into trichloroacetic acid-precipitable mate-
rial. The reaction products can be analyzed by polyacrylamide gel electro-
phoresis under denaturing conditions; the labeled R N A can be purified by
drop dialysis (this volume [5]). The yield may vary, depending on the
sequence and the secondary structure at the 3' end of the RNA; between
l 0 4 and 105 cpm//xg of RNA is expected. In general, poly(A) + RNA and
tRNA are better substrates than rRNA.

16T. E. England, A. G. Bruce, and O. C. Uhlenbeck, this series, Vol. 65, p. 65.
17T. E. England, R. I. Gumpert, and O. C. Uhlenbeck, Proc. Natl. Acad. Sci. U.S.A. 74,
4839 (1977).
ig y. Kikuchi, F. Hishinuma, and K. Sakaguchi, Proc. Natl. Acad. Sci. U.S.A. 75, 1270
(1978).
 104 ENZYMATICTECHNIQUES [10]
3'-End Labeling of DNA Using [a-a2P]ddATP
Recently, a method t9 for efficient 3'-end labeling of DNA was devel-
oped in which blunt or 3'-protruding ends are labeled with [a-32P]ddATP
using terminal transferase (TdT). In the past, the method of choice for
labeling 3'-protruding ends had been with cordycepin 5'-[a-32p]triphos-
phate ([a-32p]KTP). L o w incorporation of precursor was a problem, how-
ever, especially for subsequent sequencing (with the chemical method)
from the 3' end. Since the new ddATP method of labeling is more efficient
than the KTP method, the 3'-end labeling of DNA is now a valid alterna-
tive to 5'-end labeling before DNA sequencing.
In a total volume of 50/xl, using water to adjust the volume mix
DNA fragment 10 pmol of 3' ends
5× TdT buffer (700 mM sodium cacodyl- 10/xl
ate at pH 7.2, 5 m M COC12, 0.5 mM
DTT)
150/~Ci [a-32p]ddATP at 3000 Ci/mmol 15/A
TdT 10 units
Incubate at 37° for 1 hr.
Stop the reaction by adding 1/A 0.5 M EDTA, and purify the DNA by
phenol extraction and ethanol precipitation as described in this chapter or
this volume [4, 5].
Monitoring the Reaction. The reaction is monitored by measuring
incorporation of [a-32p]ddATP into acid-precipitable material as described
in this volume [6] and can be expected to yield 3 x 106 cpm/pmol of 3'
ends.

Exonucleases

Nuclease Bal 31
Bal 31 nuclease, purified from the culture medium of Alteromonas
espejiana, carries a double-stranded DNA exonuclease activity that pro-
gressively and bidirectionally removes mononucleotides from both
strands of linear DNA. 2° Since degradation occurs only at ends of the
duplex DNA molecule, the enzyme is useful for such applications as
restriction enzyme fragment mapping and controlled removal of se-
quences from the ends of molecules to b e cloned or expressed. The en-
zyme is highly sequence dependent, and the rate of shortening of DNA
molecules can vary considerably. The incubation time required to achieve

19 S. I. Yousaf, A. R. Carroll, and B. E. Clarke, Gene 27, 309 (1984).

2o H. B. Gray, Jr., D. A. Ostrander, J. L. Hodnett, R. J. Legersky, and D. L. Robberson,
Nucleic Acids Res. 2, 1459 (1975).
[10] MODIFYINGAND LABELINGDNA AND RNA 105

the desired resection of a DNA molecule can be estimated according to

the following equation2~:
Mt = Mo - 2 M n V m a x t / ( K m + So)

where M0 is the original MW of the DNA, Mt is the MW after t minutes of

incubation, M, is the average MW of a mononucleotide (330), Vmaxis the
maximum reaction velocity, Km is the Michaelis constant, and So is the
molar concentration of duplex termini. For the conditions given below, a
value of g m = 2 × 10-s M and V m a x = 3 × 10-6 mol/liter-min should be
used.
In an Eppendorf tube in ice mix
Linear DNA 1.5-15/zg
10× Bal31 buffer (200 mM Tris- 10 tA
HCI at pH 8.1, 125 mM M g S O 4 ,
125 mM MgCI2, 6 M NaC1, 10
mM EDTA)
Bal 31 1-4 units per/xg of DNA
Water to a final volume of 100 ~1
Incubate at 23° .
Aliquots of the reaction are removed at various times and the reac-
tions are stopped by adding EGTA to 20 mM final concentration. Measure
the extent of digestion by analyzing the products electrophoretically (this
volume [8]).

Exonuclease III
Exonuclease III of Escherichia coli catalyzes the release of mononu-
cleotides from the 3' end of a DNA strand. In addition, the enzyme is a
DNA 3'-phosphatase, an apurine endonuclease that can cleave phospho-
diester bonds at apurinic sites, and an RNase H preferentially degrading
RNA in a D N A - R N A hybrid. The enzyme will attack a nick, but will not
cleave either single-stranded DNA or double-stranded DNA with a 5'
recessed end. 22,23 Exo III followed by S~ nuclease can be used to shorten
double-stranded DNA in a controlled manner for production of deletions
in particular regions of interest. 24 Wu and colleagues 25 have developed a
method for the control of exonucleolytic degradation of duplex DNA with
Exo III in which the products are molecules with 3' ends shortened to
varying lengths. These 3' ends can then be extended or sequenced using
the chain termination procedure. In the following protocol, using a rela-

2i R. J. Legersky, J. L. Hodnett, and H. B. Gray, Jr., Nucleic Acids Res, 5, 1445 (1978).
22 C. Richardson, I. Lehman, and A. Kornberg, J. Biol. Chem. 239, 51 (1964).
23 S. Rogers and B. Weiss, this series, Vol. 65, p. 201.
24 T. Roberts, R. Kacicj, and M. Ptashne, Proc. Natl. Acad. Sci. U.S.A. 76, 760 (1979).
2~ G. Li-He and R. Wu, this series, Vol. 100, p. 60.
106 ENZYMATICTECHNIQUES [10]
TABLE I
DIGESTION OF DNA WITH EXONUCLEASEIII ~

Number of nucleotides
removed from each Incubation Exo III
end of DNA (min) (units/pmol DNA)

100-250 10-15 15-20

250-500 25-50 20-25
500-750 50-75 25-30
750-1000 75-100 30-35
1000-1500 100-150 35-45

a To remove approximately 10 nucleotides per minute from

each 3' end of DNA, the concentration of Escherichia coli
exonuclease III (Exo III) needed is given in the table. A unit
of exonuclease III is the amount of enzyme that liberates 1
nmol of mononucleotides from a sonicated DNA substrate
in 30 min at 37°. Table reproduced from Li-He and Wu. 2~

tively high ratio of enzyme to DIffA ends and a moderate concentration of

salt, the digestion is synchronous, removing approximately 10 nucleotides
per minute per 3' end at 230.25
In an Eppendorf tube in ice mix
DNA with 3' recessed or blunt ends 1 pmol
10x Exo III buffer (660 mM Tris-HCl at pH 8.0, 4/~1
770 mM NaCI, 50 mM MgCI2, 100 mM DTT)
Exonuclease III (see Table I)
Water to a final volume of 40/.d
Incubate at 23° for the required time (see Table I).
Stop the reaction by addition of 2/zl of 0.5 M EDTA.

h Exonuclease
Several different catalytic properties of h exonuclease make this en-
zyme a useful reagent for the analysis and modification of DNA structure.
The enzyme processively degrades duplex DNA from the 5' end (flush or
recessed) 100 times faster than single-stranded DNA, with preference for
a 5'-phosphoryl group over a 5'-hydroxyl group. To cleave each molecule
at a uniform rate, a molar excess of enzyme is required. The enzyme
cannot start digestion of duplex DNA at a nick or a gap. h exonuclease
has been used in a variety of different applications such as determination
of DNA structure at termini, restriction mapping, preparation of substrate
for terminal transferase, determination of direction of transcription, prep-
aration of substrate for DNA sequencing, and preparation of substrates
for other enzymes. See Little26 for a review.
26 j. W. Little, in "Gene Amplification and Analysis" (J. G. Chirikjian and T. S. Papas,
eds.), Vol. 2, p. 135. Elsevier/North-Holland, New York, 1981.
[10] MODIFYING AND LABELING D N A AND R N A 107

h Exonuclease Reaction for Preparation of Template for Terminal

Transferase. Jackson et al. 27 demonstrated that the low efficiency of 3'
labeling by terminal deoxynucleotidyl transferase in the presence of Co 2÷
can be enhanced by using h exonuclease to remove 5' unpaired terminal
nucleotides that are often generated when DNA fragments are prepared
by restriction enzyme digestion.
In an Eppendorf tube in ice mix
DNA fragment 10 pmol of 5' ends
10× exonuclease buffer (670 mM glycine- 10 ~1
K O H at pH 9.4, 30 mM MgCI2, 5 mM
DTT)
h exonuclease 50 units
Water to a final volume of 100/xl
Incubate at 14° for 4-5 min, and stop the reaction by adding 2/zl of
0.5 M EDTA.

T4 DNA Polymerase
In the absence of deoxyribonucleoside triphosphates, the T4 DNA
polymerase 3' exonuclease activity can be used to resect molecules of
linear duplex DNA. The product of the reaction is a partially single-
stranded template that can be used in resynthesis of double-stranded
DNA by adding deoxyribonucleoside triphosphates. The resulting DNA
molecule can be full-length, double-stranded, unnicked, and radioactive
with high specific activity 28 (see Fig. 1). In addition, in the presence of a
saturating concentration of the four canonical triphosphates, the enzyme
will create blunt-ended molecules starting with either 5'-protruding or 5'-
recessed ends.
In an Eppendorf tube in ice mix
DNA fragment 1-2/~g
10x T4 buffer (330 mM Tris-acetate at pH 7.9 5/~1
660 m M potassium acetate, 100 mM magnesium
acetate)
T4 DNA polymerase 5-10 units
Water to a final volume of 50 #1
Incubate at 37° for the appropriate time (20-120 rain) (resection reac-
tion). Add
Resynthesis mix (I x T4 buffer with 50 mM each 150/~1
dATP, dGTP, TTP and 5/~M [a-32P]dCTP at
400 Ci/mmol,and 14% (v/v) glycerol)
Incubate at 37° for the appropriate time (40-120 rain) (resynthesis
reaction).
27 D. A. Jackson, R. H. Symons, and P. Berg, Proc. Natl. Acad. Sci. U.S.A. 69, 2904 (1972).
2s K. C. Deen, J. A. Landers, and M. Berninger, Anal. Biochem. 135, 456 (1983).
108 ENZYMATICTECHNIQUES [10]

5' 3'

~ + T4 DNA polymerase
- dNTPs

5' 3'

resection

5' 3'

~ + dNTPs

5' --------------~---- 3' resynthesis

FIG. I. Replacement synthesis by T4 DNA polymerase. The time of incubation for the 3'-
exonuclease reaction depends on the length of the DNA fragment and must be determined
experimentally. In order to maximize label incorporation into a DNA fragment, the resec-
tion reaction is allowed to continue until just before the middle point of the DNA molecules.
This is done by removing aliquots from the resection reaction mixture (4 tzl) and incubating
with 11/~1 of resynthesis mix at 37° for a time that is at least twice as long as the resection
reaction. 29Aliquots (1/zl) of the resynthesis reaction mixture are precipitated with 10% TCA
to determine the extent of incorporation. Figure redrawn from Deen et al. 2s

Stop the reaction by adding EDTA to 20 mM final concentration. The

reaction products are purified by phenol extraction and ethanol precipita-
tion as described in this volume [4,5]. Under these conditions, specific
activity as high as 108 cpm//xg can be obtained.
Monitoring the Reaction. Exonuclease reactions are monitored by
characterizing the products electrophoretically in agarose or polyacryl-
amide gels (depending on the expected size of the products) and then,
more precisely, by subcloning and nucleotide sequence analysis.
[Editors' Note. Conditions for the use of exonuclease VII are found in
this volume [66]. The single-strand specific nucleases, $1 and mung bean
nuclease, are discussed in this volume [34] and [35], respectively.]

Ligation
From the many sets of reaction conditions described in the literature,
it is apparent that conditions for enzymatic activity of T4 DNA ligase
sometimes are suboptimal for use in cloning experiments. It is well
known, for example, that a high concentration of ATP in the ligation
reaction leads to accumulation of 5'-adenylated intermediates 29 with con-
sequent low yield of recombinant molecules. Lathe et al. 3° developed a
method for linker tailing DNA molecules without the need for restriction
enzyme digestion of the linkers and thus for the methylation of the tailed
29 V. Sgaramella and H. G. Khorana, J. Mol. Biol. 72, 427 (1972).
3o R. Lathe, M. P. Kieny, S. Skory, and J. P. Lecocq, D N A 3, 173 (1984).
[10] MODIFYING AND LABELING D N A AND R N A 109

molecules. In this study, low ionic strength (30 mM NaC1) and low ATP
concentration (0.25 mM) were found to be optimal. Recently, King et al. 31
undertook a study to determine optimal T4 DNA ligase reaction condi-
tions (for blunt end ligation) using the efficiency of bacterial transforma-
tion as assay.

Blunt E n d Ligation (Optimal); Vector~Insert Molar Ratio = 3

In a siliconized Eppendorf tube in ice mix
Dephosphorylated vector DNA (~4 kb) 160 ng
DNA fragment ( - 1 kb) 13 ng
10× ligase buffer I (250 mM Tris-HCl at pH 7.5, 50 4/zl
mM MgCI2, 25% (w/v) polyethylene glycol 8000, 5
mM DTT, 4 mM ATP) (no effect on the efficiency
of transformation was detected when the ATP final
concentration was varied from 10/zM to 1 mM)
T4 DNA ligase 1 unit
Water to a final volume of 20/zl
Incubate at 23° for 4 hours and stop the reaction by adding 1/zl of 0.5
M EDTA. Dilute 5-fold before adding the mixture to competent cells for
transformation.
Sticky E n d Ligation; Vector~Insert Ratio = 0.5
In a siliconized Eppendorf tube in ice mix:
Vector DNA ( - 4 kb) 160 ng
DNA fragment ( - 1 kb) 80 ng
10x ligase buffer II (250 mM Tris-HCl at pH 7.5, 2/zl
100 m M MgCI2, 100 mM DTT, 4 mM ATP)
T4 DNA ligase 0.01 unit
Water to a final volume of 20/zl
Incubate overnight at 8° .
Linker Tailing 31
In a siliconized Eppendorf tube in ice mix
Linear blunt-ended plasmid DNA 5/zg
10x ligation buffer III (300 m M Tris- 2/xl
HCI at pH 7.5,300 mM NaCI, 80
mM MgC1, 20 mM DTT, 2 mM
EDTA, 70 mM spermidine hydro-
chloride, 1 mg/ml bovine serum al-
bumin, and 2.5 mM ATP)
Unphosphorylated linker 500 ng if 10 or 12-mer
12.5/zg if 8-mer

31 p. V. King and R. W. Blakesley, Focus (Bethesda, Md.) 8, No. 1, 1 (1986).

110 ENZYMATIC TECHNIQUES [10]

T4 DNA ligase 2 units

Water to a final volume of 20/zl
Incubate overnight at 4 °.
Monitoring the Reaction. Ligation reactions are monitored by charac-
terizing the products by agarose gel electrophoresis and by bacterial
transformation.

DNase I

Pancreatic deoxyribonuclease I is an endonuclease catalyzing the hy-

drolysis of both double- and single-stranded DNA to produce 5'-
phosphoryloligodeoxynucleotides. In molecular biology, DNase I is used,
for example, to introduce random nicks in DNA substrates, as in the
preparation of substrate for the nick translation reaction, n The enzyme
has been used, in the presence of ethidium bromide, to introduce one nick
at random per circular DNA molecule, in order to obtain sensitive sites
for the bisulfite-mediated mutagenesis protocol. 33 Other applications have
been described such as production of random DNA fragments for shotgun
cloning and sequencing in the M13 system, 34 studies of chromatin struc-
ture, 35 and analysis of DNA-protein complexes. 36
Reaction Conditions for the Production of Random Fragments. 37 Re-
suspend the plasmid DNA to be cloned and sequenced in 6× SSC at a
concentration of 12 mg/ml. Incubate for l0 min in boiling water (relaxed
form production). Cool the solution slowly by holding it for 19 rain at 65 °
and then for 10 min at 23 ° (renaturation). Precipitate the DNA with etha-
nol and resuspend in 50 mM Tris-HCl at pH 7.6, to a final concentration
of I mg/ml.
In an Eppendorf tube in ice mix
Resuspended DNA from previous step 50/xl
10× DNase buffer (500 m M T r i s - H C l at pH 7.5, 10raM l0/xl
MgCl2, I mg/ml bovine serum albumin)
DNase I (electrophoretically pure) l0 ng
Water to a final volume of 100/xl
Incubate at 23 °. Remove aliquots of 25/xl every 5 min and stop the
reactions by addition of 1 /xl of 0.5 M EDTA.
Monitoring the Reaction. The digestion is checked by electrophoresis
of the DNA in a 6% polyacrylamide gel using a portion of each sample
(10 ptl).
32 p. W. Rigby, M. Dieckmann, C. Rhodes, and P. Berg, J. Mol. Biol. 113, 237 (1977).
33 L. Greenfield, L. Simpson, and D. Kaplan, Biochim. Biophys. Acta 407, 365 (1975).
34 S. Anderson, Nucleic Acids Res. 9, 3015 (1981).
J5 H. Weintranb and M. Groudine, Science 193, 848 0976).
36 A. Prunell, R. D. Kornherg, L. Lutter, A. Klug, M. Levitt, and F. H. C. Crick, Science
204, 855 (1979).
37 j. Messing, this series, Vol. 101, p. 20.