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Art 9
Art 9
USA
Vol. 74, No. 12, pp. 5463-5467, December 1977
Biochemistry
ABSTRACT A new method for determining nucleotide se- a stereoisomer of ribose in which the 3'-hydroxyl group is ori-
quences in DNA is described. It is similar to the "plus and ented in trans position with respect to the 2'-hydroxyl group.
minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. The arabinosyl (ara) nucleotides act as chain terminating in-
94,441-4481 but makes use of the 2',3'-dideoxy and arabinonu- hibitors of Escherichia coli DNA polymerase I in a manner
cleoside analogues of the normal deoxynucleoside triphosphates,
which act as specific chain-terminating inhibitors of DNA comparable to ddT (4), although synthesized chains ending in
polymerase. The technique has been applied to the DNA of 3' araC can be further extended by some mammalian DNA
bacteriophage 4bX174 and is more rapid and more accurate than polymerases (5). In order to obtain a suitable pattern of bands
either the plus or the minus method. from which an extensive sequence can be read it is necessary
to have a ratio of terminating triphosphate to normal triphos-
The "plus and minus" method (1) is a relatively rapid and phate such that only partial incorporation of the terminator
simple technique that has made possible the determination of occurs. For the dideoxy derivatives this ratio is about 100, and
the sequence of the genome of bacteriophage 4X174 (2). It for the arabinosyl derivatives about 5000.
depends on the use of DNA polymerase to transcribe specific
regions of the DNA under controlled conditions. Although the
method is considerably more rapid and simple than other METHODS
available techniques, neither the "plus" nor the "minus" Preparation of the Triphosphate Analogues. The prepa-
method is completely accurate, and in order to establish a se- ration of ddTTP has been described (6, 7), and the material is
quence both must be used together, and sometimes confirma- now commercially available. ddA has been prepared by
tory data are necessary. W. M. Barnes (J. Mol. Biol., in press) McCarthy et al. (8). We essentially followed their procedure
has recently developed a third method, involving ribo-substi- and used the methods of Tener (9) and of Hoard and Ott (10)
tution, which has certain advantages over the plus and minus to convert it to the triphosphate, which was then purified on
method, but this has not yet been extensively exploited. DEAE-Sephadex, using a 0.1-1.0 M gradient of triethylamine
Another rapid and simple method that depends on specific
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| :..< S: _
.. _ ..
0*..
AxaA
C,-t;ri.
4040 I IT
A GAC~ ~
40320 /t -'~ (-AG-(" QiT
1-Ta G-A-
t e
2:e.
_
P-t..A'AAAIT 35,10
IT AT A 3500
4Ozc20C1
401A0A 1 (A -1 A A33)40
AT c AtUAs4
OFO
4000 T(,1 Q ( 1 04-A-A-- -
jij~~~C.'G
A; A ( $48(
3999)(;001(oT I- 1 A 1 A- -
; jA 7T-3- by C
C
34980 0
39601AT -C A 3ox4810
A ~~~~~~~( AC 34 7
39T0 T
-t it1-to3430
A A A 3460
3950Q( ACs. iAA
oA A
_ Ad GAT 34150
3940 GrT
AC AC34 300
3930 T A A 440
-C3
4.m. A
C A A 3420
AA 3
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FIG. 1. Autoradiograph of the acrylamide gel from the sequence determination using restriction fragments A12d and A14 as primers on the
complementary strand of IX174 DNA. The inhibitors used were (left to right) ddGTP, ddATP, ddTTP, and araCTP. Electrophoresis was on
a 12%6 acrylamide gel at 40 mA for 14 hr. The top 10 cm of the gel is not shown. The DNA sequence is written from left to right and upwards beside
the corresponding bands on the radioautograph. The numbering is as given in ref. 2.
al. (4) have shown that the didehydrodideoxy-TTP is also active ddG: 0.1 mM dCTP, 0.1 mM dTTP, 0.005 mM dGTP, 0.5
as a terminator. However, we were unsuccessful in this. These mM ddGTP
compounds seem much less stable than the dideoxy deriva- ddC: 0.1 mM dGTP, 0.1 mM dTTP, 0.005 mM dCTP,
tives. approximately 0.25 mM ddCTP
araATP and araCTP were obtained from P-L Biochemicals (The concentration of the ddCTP was uncertain because there
Inc., Milwaukee, WI. was insufficient yield to determine it, but the required dilution
Sequencing Procedure. Restriction enzyme fragments were of the solution was determined experimentally.)
obtained from OX174 replicative form and separated by elec- araC: 0.1 mM dGTP, 0.1 mM dTTP, 0.005 mM dCTP, 12.5
trophoresis on acrylamide gels. The material obtained from 5 mM araCTP
,ug of OX174 replicative form in 5 ,Al of H20 was mixed with Incubation was at room temperature for 15 min. Then 1 Ml
1 Al of viral or complementary strand 4X174 DNA (0.6 jg) and of 0.5 mM dATP was added and incubation was continued for
1 Mi of H X 10 buffer (13) and sealed in a capillary tube, heated a further 15 min. If this step (chase) was omitted some termi-
to 1000 for 3 min, and then incubated at 670 for 30 min. The nation at A residues occurred in all samples due to the low
solution was diluted to 20 Al with H buffer and 2 Al samples concentration of the [a-32PldATP. With small primers, where
were taken for each incubation and mixed with 2 Ml of the ap- it was unnecessary to carry out a subsequent splitting (as in the
propriate "mix" and 1 il of DNA polymerase (according to experiment shown in Fig. 1), the various reaction mixtures were
Klenow, Boehringer, Mannheim) (0.2 units). Each mix con- denatured directly and applied to the acrylamide gel for elec-
tained 1.5 X H buffer, 1 MCi of [a -32P]dATP (specific activity trophoresis (1). If further splitting was necessary (see Fig. 2),
approximately 100 mCi/,Mnwol) and the following other tri- 1 Mil of the appropriate restriction enzyme was added shortly
phosphates. after the dATP "chase, and incubation was at 370.
ddT: 0.1 mM dGTP, 0.1 mM dCTP, 0.005 mM dTTP, 0.5 The single-site ribo-substitution procedure (N. L. Brown,
mM ddTTP unpublished) was carried out as follows. The annealing of
ddA: 0.1 mM dGTP, 0.1 mM dCTP, 0.1 mM dTTP, 0.5 mM template and primer was carried out as above but in "Mn
ddATP buffer" (66 mM TrisCl, pH 7.4/1.5 mM 2-mercaptoethanol/
M~A .-
Biochemistrv: Sanger et al. Proc. Nati. Acad. Sci. USA 74 (1977) 5465
G A T C T aC ddC G A
Si E ~I~A9TiTTC
CGTT -CA7 4370
-
4380
- -. _ L>TG 3480
ohsPAGACAGA 4360 T C 3490
CGAG 4330 T A 4350 _ ""Nw*~~~~~u -A T
TG T T4320-6-.
ACCA T 3500
- ------AC--A
-
A 4340 3510
= * - -G-GA-C GAAAA 4340 A EC 3520-
A
TI
^cGAAG 4330) I
q
l~~~~~
- ----- 3530
- - ACTA -
-- A
A - C4270 A
----A 3580
G0
TT
A
T 4260
primer and the newly synthesized DNA. This is the most simple of about 110 nucleotides starting 33 residues from the priming
and rapid procedure, requiring only a preliminary annealing site can be read off. In the provisional sequence (2) this region
of template and primer, incubation of the four separate samples was regarded as very tentative. Most of it is confirmed by this
with DNA polymerase and appropriate triphosphates, followed experiment, but there is a clear revision required at positions
by a chase with unlabeled dATP and application to the gel for 3524-3530. The sequence of the viral strand should read A-
electrophoresis. In these experiments the inhibitors used were T-C-A-A-C, replacing A-T-T-C- - -A-C given in the provisional
ddGTP, ddATP, ddTTP, and araCTP. The conditions used for sequence. There is difficulty in reading the sequence at
the "T" samples were not entirely optimal, resulting in the 3543-3550, where there is considerable variation in the distance
faster-moving bands being relatively weak. between bands, suggesting the presence of a looped structure.
The sequences can be read with reasonable accuracy starting Further work in which the electrophoresis was carried out at
at 88 nucleotides from the 5' end of the primer for about 80 a higher temperature indicates that the sequence here is actu-
nucleotides (apart from some difficulty at position 3459 with ally G-C-T-C-G-C-G (viral strand); i.e., an insertion of C be-
A12d). For the next 50 nucleotides there is some uncertainty tween positions 3547 and 3548 in the provisional sequence.
in the number of nucleotides in "runs" because bands are not
actually resolved. DISCUSSION
With longer restriction enzyme fragments as primers it is The method described here has a number of advantages over
necessary to split them off from the newly synthesized DNA the plus and minus methods. First, it is simpler to perform be-
chains before the electrophoresis. This is normally done by di- cause it requires no preliminary extension, thus avoiding one
gestion with a restriction enzyme. Fig. 2 shows such an exper- incubation and purification on a Sephadex column. It requires
iment in which fragment R4 was used as primer on the com- only the commercially available DNA polymerase I (Klenow
plementary strand of OX174 DNA. In this experiment only fragment). The results appear to be more clear-cut with fewer
dideoxynucleoside triphosphates were used as inhibitors because artefact bands, and can usually be read further than with the
the results with araC were much less satisfactory when a re- plus and minus methods. Intermediate nucleotides in "runs"
striction enzyme was used for the subsequent splitting. This may show up as bands, thus avoiding a source of error in the plus and
be due to the araC being removed by the 3'-exonuclease activity minus method-estimating the number of nucleotides in a run.
of the DNA polymerase during the incubation at 370 (which Theoretically one would expect the different bands in a run to
is necessary for the restriction enzyme splitting), resulting in be of the same strength, but this is not always the case. Fre-
a few C bands being either very faint or missing. Alternatively, quently, the first nucleotide is the strongest, but in the case of
the enzyme may be able to extend some chains beyond the araC ddCTP the second is the strongest (see Fig. 2). The reasons for
at the higher temperature while being unable to do so at lower these effects are not understood, but they do not usually cause
temperatures. araATP, which has been used only under these difficulties with deducing the sequences. For the longer se-
conditions, shows the same limitations as araCTP. These quences in which the separate bands in a run are not resolved,
problems do not arise when ddCTP is used in this reaction. experience has shown that it is frequently possible to estimate
With one exception (positions 4330-4343, see below), a se- the number of nucleotides from the strength and width of the
Downloaded from https://www.pnas.org by 82.154.68.114 on April 2, 2023 from IP address 82.154.68.114.
other technique or by priming on the opposite strand. This & Moffatt, J. G. (1969) Biochemistry 8, 4897-4904.
consideration probably applies to all other available methods 5. Hunter, T. & Francke, B. (1975) J. Virol. 15, 759-775.
also. The main disadvantage of the present method is the dif- 6. Russell, A. F. & Moffatt, J. G. (1969) Biochemistry 8, 4889-
ficulty in obtaining all the inhibitors-particularly ddGTP, 4896.
7. Geider, K. (1974) Eur. J. Biochem. 27,555-563.
which is not commercially available. 8. McCarthy, J. R., Robins, M. J., Townsend, L. B. & Robins, R. K.
We wish to thank Dr. K. Geider for a gift of ddTTP, Dr. F. E. Baralle (1966) J. Am. Chem. Soc. 88, 1549-1553.
for a gift of N-isobutyryl-5'-O-monomethoxytrityldeoxyguanosine, 9. Tener, G. M. (1961) J. Am. Chem. Soc. 83, 159-168.
and Dr. M. J. Gait for useful advice on the synthetic work. 10. Hoard, D. E. & Ott, D. G. (1965) J. Am. Chem. Soc. 87,1785-
1788.
1. Sanger, F. & Coulson, A. R. (1975) J. Mol. Blol. 94, 441-448. 11. Buchi, H. & Khorana, H. G. (1972) J. Mol. Biol. 72,251-288.
2. Sanger, F., Air, G. M., Barrell, B. G., Brown, N. L., Coulson, A. 12. Robins, M. J., McCarthy, J. R. & Robins, R. K. (1966) Biochem-
R., Fiddes, J. C., Hutchison, C. A., Slocombe, P. M. & Smith, M. istry 5,224-231.
(1977) Nature 265,687-695. 13. Air, G. M.,'Sanger, F. & Coulson, A. R. (1976) J. Mol. Biol. 108,
3. Maxam, A. M, & Gilbert, W. (1977) Proc. Nat!. Acad. Sci. USA 519-533.
74,560-564. 14. Slocombe, P. M. (1976) Ph.D. Dissertation, University of Cam-
4. Atkinson, M. R., Deutscher, M. P., Kornberg, A., Russell, A. F. bridge.
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