A new method for the sequence analysis of oligodeoxynucleotides

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)