Vol. 45, No. 2, 1971
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A NEW METHOD FOR THE SEQUENCE ANALYSIS OF OLIGODEOXYNlJCIEOTIDES+ Ranajit Institut
Received
Roychoudhury, Dietrich Fischer and Hans Kijssel fiir Biologie III (Genetik und Molekularbiologie) Universitat Freiburg 78 Freiburg, W-Germany
August
11, 1971
Limited degradation of an oligodeoxynucleotide (I) with snake venom phosphodiesterase leads to a population of oligodeoxynucleotides of successively smaller chain lengths. Labelling of these oligonucleotides at the 3'-ends with [ 32 PI-riboadenylic acid residues followed by sepsxation according to their chain lengths and degradation with spleen phosphodiesterase allows the sequence determination of the original oligodeoxynucleotide (I) except for the two nucleotide residues towards the 5'-end. The enzyme terminal catalyzes
the
addition
end group
for
steps
communication the partial
(see
1. Limited
also
2. terminal
3. separation basis
of their
chain
3 '-ends
to describe
to the
thymus
residue(s)
gland
to a given
is
used
development
as sub-
of a new
of oligodeoxynucleotides the
application
of the sequence
(2).
of this
oligodeoxynucleotide consists
of the
follow-
oligodeoxynucleotide
(I)
oligodeoxynucleotide
with
population
snake
venom
of successively
(II), of one [32 PI-riboadenylic
of the of the
acid
calf
scheme I):
to yield
addition
of each species
led
reaction
from
triphosphate
determination
The whole
of the
lengths
the
we want
the
digestion
chain
for
sequence (I).
phosphodiesterase smaller
has recently
technique
(A-C-C-A-T-C-C-A)a ing
when riboadenosine
observation
labelling
In this method
primer
This
(1).
transferase
of one or two riboadenylic
oligodeoxynucleotide strate
deoxynucleotidyl
population riboadenylic
lengths
acid
residue
to the
3'-end
(III), acid
followed
terminated by complete
+
radioactive digestion
species of the
The abbreviations and symbols in this paper follow recommendations of IUPAC-IUB Commission on Biochemical Nomenclature, published in Eur. J. Biochem. l& 203 (1970), except for "p, which symbolizes [32P]-phosphate groups in contrast to the nonradioactive phosphate groups which are represented by hyphens.
430
on the
Vol. 45, No. 2, 1971
individual
BIOCHEMICAL
species
from the
with
[32P]pAr
neighbours
residues
(IV),
ginally
spleen
which
present
at the
phosphodiesterase.
thus
are
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
will
The radioactive
be transferred
identical
with
3'-terminus
the
of the
to their
nucleotide
respective
label
nearest
residues
ori-
oligodeoxynucleotide
species. Scheme I (A-C-C-A-T-C-C-A)%A (A-C-C-A-T-C-~)d snake venom
[OL:~P]ATP, terminal transferase
(A-C-C-A-T-~)d
I
>
d
r
. ..A$
(A-C-C-A-T-C-C)~&~
. ..I$
(A-C-C-A-T-C)~&,
. ..Cp"
(A-C-C-A-T)~$A~
. ..T$
(A-c-c-A)~$A~
. ..A$
(A-C-C)a$Ar
. ..Cp"
>
phosphodiesterase
(A-C-C-A-~)d
followed by alkali and phosphatase treatment
(A-C-C-A)~ (A-C-C)a
spleen II
III
>
IV
phosphodiesterase Materials Terminal
deoxynucleotidyl
Bollum
(3)
tested
with
had a specific (PT~)~
was obtained
tested
Boehringer.
(5)
in the
using
units
Center,
buffer
and calf
spleen
as substrate)
was prepared
by the
as described
(1.5
was prepared
as intermediates single
terminal
previously
[Oc-32P)rATP
with
unitslmg
a fragment
by organic (6).
were
was obtained
A-C-C-A-T-C-C-A,
fd (4),
as
phosphodiesterase
bis-p-nitrophenylphosphate
blocks
system.
and
Amersham.
venom phosphodiesterase
of the phage
to Yoneda
per mg protein
Snake
dinucleotide
(A-C-C-A-T-C)d
according
cacodylate
The deoxyoctanucleotide
strand
[14C]Ar
of 25.000
phosphatase
of Worthington. with
minus
as primer
alkaline
isolated
activity
from Radiochemical
E.coli products
transferase,
of [
[OL-~~P]ATP
of the DNA
chemical
A marker
addition
from
14
synthesis
of (A-C-C-A-T-C)dC]ATP to
(2).
Results It
is
evident
from figure
1
that
the 431
[32P]-radioactivity
of peak
5
Vol. 45, No. 2, 1971
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0 FRACTION
80
10
NUMBER
Figure 1. Separation of the oligonucleotide mixture resulting from partial digestion of (A-C-C-A-T-C-C-A)d with snake venom phosphodiesterase subsequent labelling of the resulting oligonucleotide species with 99 [ PIriboadenylic acid residues. 0.8 A -unit of the octanucleotide was incubated for 5 minutes at 37’ in 30 mM pg e %ssium cacodylate (pH 6.8), 6 mM MgCl , 0.3 mM dithiothreitol and 1 pg of venom phosphodiesterase in a final vofume of 170,ii. Phosphodiesterase was then inactivated by heating the incubation mixture in a boiling water bath for 3 minutes. The reaction mixture after cooling to 0' was adjusted to 40 m&I otassium cacodylate, 8 ml4 MgCl threitol and 1 mM [CG3%]rATP (20~~ per umole). Afte?'a~d?t?!n"~~$>g of terminal transferase (final volume 400 ,ul) incubation was carried out at 37' for 4 hours. Subsequent addition of 100 pl of 2 N NaOH was followed by incubation of the resulting mixture for 21 hours at 37O (2). Neutralization was then achieved by passing the whole incubation mixture through a 0.5 ml column of pyridinium Dowex 50 in the presence of 33 % pyridine. The radioactive material after evaporation of the solvent was dissolved in 100 mM Tris-HCl (pH 8.1) and incubated with 1.2 units of alkaline phosphatase in 400~11 for a period of 12 hours at 37'. The incubation mixture together with (A-C-C-A-T-C) -[14C]A as internal marker finally was diluted tenfold with buffer contain&g 0.05 fi triethylammonium bicarBonate (pH 8.0) and 7 M urea, loaded onto a DE!U+cellulose column (0.3 cm x 25 cm) preequilibrated with the same buffer and eluted with a linear gradient using 100 ml of 0.05 M triethylsmmonium bicarbonate, 7 M urea in the mixing vessel and 100 ml of 0.4 M triethylammonium bicarbonate, 7 M urea in the reservoir at room temperature. Twenty drop fractions (approx. 1.4 ml) were collected about every 12 minutes. Aliquots (100 ~1) from each fraction were placed on filter paper discs, air dried and counted for radioactivity. cochromatographes marker
carrying
with
the
6 negative
internal
oligonucleotide
charges.
Therefore, 432
(A-C-C-A-T-c),~['~c]A the
[32P]-radioactivity
r of
BIOCHEMICAL
Vol. 45, No. 2, 1971
the
peaks
negative the
2-7 must charges
undigested
excess
correspond
completely
with
octanucleotide
degraded
and nonradioactive
to the
respectively,
of [32 P]rATP
AND BIOPHYSICAL
As shown in figure
CATHODE
the
during
to inorganic adenosine
oligodeoxynucleotides last
peak
the
terminal
[ 32P]-phosphate
DISTANCE
FROM
has been added. addition 1
The
reaction of figure
is 1)
treatment. from the
ORIGIN
3-8
7) representing
(peak
by the phosphatase
2, the radioactivity
carrying
(peak
one [ 32 PIrAMP
to which
unutilized
RESEARCH COMMUNICATIONS
(CM)
individual
peaks
ANODE
Figure 2. Elect phoretic separation of the deoxynucleoside-3'-monophosphates obtained from [ 58 PI-riboadenylic acid terminated oligodeoxynucleotides spleen phosphodiesterase treatment.
by
For desalting, the peak fractions of figure 1 were pooled, diluted with 3 volumes of water and loaded separately onto small DEAF-cellulose columns (bed volume 1 ml). After washing the columns with 20 ml of water the materials were el.uted with 1 M triethylammonium bicarbonate buffer (6 ml). The material was freed from the buffer by evaporation in the presence of excess pyridine. Traces of pyridine were removed by addition of 0.1 N NH40H (1 ml) and reevaporation. The dried residues from each peak were dissolved and incubated in the buffer system described by Wu (7) with 1.2 units oaf spleen phosphodiesterase in final volumes of 200 pl for 2-4 hours at 37 . The incubation mixtures were then subjected to paper electrophoresis in the ammonium acetate system (pH 3.5) at 2000 volts for 120 minutes as described previously (2). The numbers at the right side of each diagram refer to the peak numbers (2-7) of figure 1. Paper strips (1 cm) were cut serially and counted. 433
Vol. 45, No. 2, 1971
BIOCHEMICAL
2, 3, 4, 5, 6 and 7 of figure ment appears
with
respectively. ginal
octanucleotide
observed all
the
tion
the
of the
presence
[32P]-labelled
is
amount
dCp in figure after
located
spleen
2f,
of the
from the
neighbouring
peaks
In these
1.
had to be pooled
material,
which
resulted
peak
the bulk
phosphodiesterase
is
2a and from
of
for
evident
of radioactivity
treatment
cases
1, and in
7; this
of 32P i in figure
is
in contamina-
from peak
peak 6 into
where
ori-
to be (C-A-T-C-C-A)d.
peaks
from
of a small
dCp, dCp and dAp
3'-terminus
[ 32 PI-phosphate
2 by some inorganic
treat-
respective
radioactive
from the
dTp,
2 and peak 7 of figure
to the
of (A-C-C-A-T-C-C)d*pAr
phosphodiesterase
at the
directly
from peak
trailing
expected
sequences
contamination
belonging
recovery of peak
base
of cross
materials
fractions
greater
spleen
dCp, dAp,
can be deduced
amount
with
1, after
the mononucleotides
From this
A small
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
some [as
(A-C-c-A-T-c-C-A)~$A~I
in dAp.
Analogous
results
sequence
to figures
was applied
1 and 2 were
to the
obtained
hexanucleotide
when the
(A-C-C-A-T-C)d
entire (not
reaction
shown).
Discussion One crucial for
the
point
partial
of the method digestion
diesterase.
In fact
encountered
which
Since
on the
addition (l),
reaction
the
ideal
of smaller molecules
other
consists
of the
oligonucleotide
a few previous apparently
were
condition
oligonucleotides remaining.
activity with would
experiments
with
chain
very
of digestion
extent
venom phospho-
of the material.
necessary the
conditions
had to be
for
the
length
little
terminal
of the primer
be the one in which
are produced
Thus the
proper
snake
due to overdigestion
rATP decrases
digestion
the
with
unsuccessful
hand the priming with
in meeting
more molecules
undigested
has to be balanced
carefully. For practical amounts than
1
usefulness,
of oligonucleotide A2Go-unit
the method material.
of oligonucleotide
should
According is 434
sufficient
be workable to the for
with
present the
small
results
sequence
less
Vol. 45, No. 2, 1971
BIOCHEMICAL
determination residues reduce
of a given towards
this
amount
by application and his
the
deoxynucleotide
with
recently
ation
5 '-end
determined
excluding
It
seems,
[oL,32 P]ATP finger
the two nucleotide
however,
of higher print
method
based
spleen
phosphodiesterase
oligonucleotide
of the
RESEARCH COMMUNICATIONS
possible
specific method,
to
further
activity
and
developed
by Sanger
(8).
was proposed
tides
5'-terminus.
by using
A complimentary
of both
oligonucleotide
of the nucleotide
coworkers
resulting
AND BIOPHYSICAL
by the the methods
species (8).
This
of a given method the
on the
with
partial
degradation followed
would
allow
oligodeoxynucleotide,
described complete
by labelling
polynucleotide
method
in this sequence
of an oligo-
kinase the
at the
sequence
which
of the 5'-ends
determin-
remains
un-
paper.
Thus by a combination
analysis
of deoxyoligonucleo-
seems possible. Acknowledgements
This work was supported by grants which are gratefully acknowledged. and E. HBrle for helpful criticism
-from the Deutsche Forschungsgemeinschaft, The authors are indebted to Drs. G. Feix of the manuscript.
References 1. 2.
z: 5. 6. 7. 8.
Roychoudhury, R. and Kijssel, H. Eur. J. Biochem., in KBssel, H and Roychoudhury, R. Eur. J. Biochem., in Yoneda M. and Bollum, F. J., J. Biol. Chem. 2& 3385 Asbeck' F Beyreuther, K., KBhler, H., v. Wettstein, BrauniCzei: G., Hoppe-Seylers 2. Phys. Chem. 350, 1047 Fischer, D. and K&ssel, H., in preparation Kdssel, H, Biichi, H. and Khorana, H. G., J. Am. Chem. wu, Ft., J. Mol. Biol. 2, 501 (1969) Szekely,M. and Sanger, F., J. Mol. Biol. 43, 607 (1969)
435
press. press (1965) G. and (1969) Sot.
&,2185(1967)