 |
Description  |
|
|
BACKGROUND OF THE INVENTION
The detection of mutations in DNA is of importance in a variety of fields.
One such field is the diagnosis of genetically determined diseases and to
identify carriers of such diseases. It has been estimated that, in
Northern Europe, diseases caused by genetically determined defects may
affect 1% of all live births. In some Mediterranean countries, 20% of the
population are said to have genetic defects, associated with thalassaemia.
Conventional methods of gene analysis involve DNA isolation and restriction
digestion, gel electrophoresis and DNA blotting by the technique of Dr. E.
Southern, hybridization and washing, and finally autoradiography. A total
of 3-10 days are required and radioactive probes are used for
hybridization. Such methods are the subject of a review by P. F. R. Little
in "Genetic Engineering" volume 1, pages 61-102, published 1981 by
Academic Press.
Such methods can be used whether or not the DNA has been accurately
sequenced in the region of interest. But they have major disadvantages;
they are only effective to detect point mutations where these happen to be
present in a restriction enzyme cleavage site, and then only provided that
there are not other nearby cleavage sites for the same enzyme; they
require the tedious preliminary steps of DNA isolation, restriction, gel
electrophoresis, and Southern blotting; and they generally require the use
of radioactive labels. These disadvantages have inhibited the development
of genetic screening in clinical laboratories by these techniques.
When the DNA sequence in the region of interest is known, it is possible to
overcome some of these disadvantages. B. J. Conner et al (Proc. Natl.
Acad. Sci. U.S.A., 80, January 1983, 278-282) describe a method which does
not require the mutation to be at a restriction enzyme cleavage site. A
radioactively-labelled 19-base oligonucleotide probe is caused to
hybridize with the region of the DNA which includes the possible mutation.
The hybridization conditions are carefully chosen so that the probe does
or does not hybridize depending on whether the mutation is or is not
present. But the length of the probe and the hybridization conditions are
difficult to get right and are critical for success. The aforesaid tedious
preliminary steps are used, as is a radioactively labelled probe.
The method of the present invention generally requires a knowledge of the
nucleic acid sequence in the region of interest. But it does not require
the mutation to be at a restriction enzyme cleavage site. (In the case of
mutations within restriction sites, it may be possible to infer the base
change involved from a simple end-filling experiment, without needing to
know the exact nucleic acid sequence). The method is capable of giving
unambiguous results. In the preferred forms, it does not require the
tedious preliminary steps that characterize prior methods, and it may not
require the use of a radioactive label.
SUMMARY OF THE INVENTION
The present invention thus provides a method of detecting a mutation of a
specific nucleotide base in a target nucleic acid chain by providing a
linear probe complementary to a part of the nucleic acid chain extending
in one direction from the specific base,
(a) hybridizing the probe to the target to form a nucleic acid hybrid,
whereby one end of the probe becomes hybridized to the nucleic acid chain
substantially adjacent the specific base,
(b) admixing with the hybrid a nucleotide derivative, under conditions
appropriate for probe extension, so as to cause the nucleotide derivative
to join on to the end of the probe only if the specific base in the target
is, or is not, the mutation to be detected, a probe carrying said
nucleotide derivative being resistant to digestion under particular
conditions,
(c) subjecting the hybrid to digestion under the said particular conditions
whereby the double-stranded portion thereof is progressively digested
starting at the said end of the probe unless the end has had said
nucleotide derivative joined to it,
(d) removing portions of the probe which are no longer hybridized to the
nucleic acid chain,
(e) and using the presence or absence of the probe remaining after
digestion to detect a mutation of the specific nucleotide base, in the
target.
Crucial to the method is step (b) which involves the use of a nucleotide
derivative having a special property. When this nucleotide derivative is
joined to the end of the probe, the probe is then resistant to digestion
under particular conditions. In one alternative, a nucleotide derivative
is mixed with the hybrid under conditions to cause it to join on to the
end of the probe only if the specific base in the target is normal, i.e.
not the suspected mutation. In another alternative, a (different)
nucleotide derivative is mixed with the hybrid under conditions to cause
it to join on to the end of the probe only if the specific base in the
target is the suspected mutation. Various ways of achieving this are
possible, and there will be described, designated A, B and C, of which
embodiments A and B are preferred.
A. A probe is provided such that in step (a) one end becomes hybridized to
the nucleic acid chain immediately adjacent the specific base. It is then
possible to perform step (b) by admixing with the hybrid a nucleotide
derivative under conditions appropriate for probe extension so as to cause
the nucleotide derivative to join on to the end of the probe if it is
complementary to the specific base.
B. A probe is provided such that in step (a) one end becomes hybridized to
the nucleic acid chain a few bases away from the specific base. It is then
possible to perform step (b) by admixing with the hybrid a nucleotide
derivative, together with one or two other different nucleotides, under
conditions appropriate for probe extension so as to cause them to join on
to the end of the probe, including the nucleotide derivative if it is
complementary to the specific base.
It may be helpful to illustrate embodiments of the invention where 1, 2 and
3 nucleotides are used in step (b).
A. The probe becomes hybridized in step (a) to the nucleic acid chain (the
target) with its end base opposite the base immediately adjacent the
specific base being tested for. We can consider by way of example a target
having the sequence, in which * represents the point mutation
##STR1##
The probe contains the sequence
##STR2##
In step (a), the two become hybridized thus:
##STR3##
in this preferred embodiment a derivative of guanosine (G) is used in step
(b) without any other nucleotide and will be incorporated in one case but
not the other:
##STR4##
B. The probe becomes hybridized in step (a) to the target with its 3'-end a
few bases away from the specific base being tested for. With a target
sequence as in A, we can consider a probe containing the sequence
##STR5##
In step (a), the two become hybridized thus:
##STR6##
In this embodiment, the derivative of guanosine (G) is used together with
dATP and dTTP in step (b) to give the following
##STR7##
It is easy to envisage comparable situations where the nucleotide
derivative is used in admixture with one other nucleotide. It is however
necessary in this embodiment that the nucleotide derivative be
incorporated only opposite the specific base in the target.
C. A probe is provided such that in step (a) one end becomes hybridized to
the nucleic acid chain immediately adjacent the specific base (as in A.),
or a few bases away from the specific base (as in B). Step (b) can then be
performed by:
(b)(i) admixing with the hybrid a chain-terminating nucleotide compound,
optionally together with one or two other different nucleotides, under
conditions appropriate for probe extension so as to cause the
chain-terminating nucleotide compound to join on to the end of the probe
if it is complementary to the specific base,
(b)(ii) admixing with the resulting hybrid one or more nucleotide
derivatives under conditions appropriate for probe extension so as to
cause them to join on to the end of the probe if a chain-terminating
nucleotide compound is not already present, a probe carrying said one or
more nucleotide derivatives being resistant to digestion under particular
conditions.
We can consider the same target/probe hybrid that was formed in step (a) of
embodiment A above. A chain-terminating guanosine compound (G) is used in
step (b)(i) and will be incorporated in one case but not the other:
##STR8##
Then step (b)(ii) is performed with all four nucleotide derivatives (A, C,
G, T) which will be incorporated in one case but not the other:
##STR9##
Clearly step (b)(ii) could have been performed using A, alone or together
with C and optionally T. Suitable as chain-terminating nucleotide
compounds are dideoxynucleotides and also several other nucleotide
compounds which do not permit further addition of nucleotide to their 3'
(or alternatively 5') end. They do not, however, protect the probe from
digestion under chosen particular conditions.
It may be convenient to produce a probe as a restriction fragment when the
site of the restriction cut is not immediately adjacent the site of
mutation and one or two nucleotide types can be omitted to limit
elongation.
The target and the probe may both be of DNA. Alternatively, either or both
may be RNA.
In order to determine the presence or absence of probe in step (e) of the
method, the probe will generally be labelled, for example with a
radioactive isotope or with a group that takes part in an enzyme or
fluorescent or chemiluminescent reaction.
In the method, the target may be immobilised or in solution. The use is
preferred of an immobilised target, because that reduces the risk of
complementary target strands re-hybridizing in step (a) and facilitates
removal of unhybridized probe. However, use of a target in solution may be
preferred on some occasions.
Crucial to be invention is the nucleotide derivative used in step (b) and
the digestion conditions used in step (c). According to a preferred
embodiment, a thionucleotide is used as the nucleotide derivative in step
(b) and is caused to join on to the 3' end of the labelled probe. Then in
step (c), digestion is effected using Exonuclease III, an enzyme from E.
coli which digests double-stranded nucleic acid chains only from the 3'
end, releasing deoxynucleoside-5'-mono-phosphates. This enzyme, if it does
so at all, cleaves phospho-ester bonds when the phosphorus atom is linked
to sulphur only at reduced efficiency. Thus a chain terminated with a
thionucleotide at its 3' end is resistant to degradation by Exonuclease
III.
Thus, in embodiments A and B above, a single thionucleotide is used in step
(b). If that thionucleotide is complementary to the specific base of the
target, it will join to the 3' end of the probe, and the resulting hybrid
will be resistant to digestion in step (c) with Exonuclease III. If, on
the other hand, the thionucleotide is not complementary to the specific
base, it will not join the 3' end of the probe, and the hybrid will be
digested in step (c).
DETAILED DESCRIPTION
If the nucleic acid to be investigated (the target) is not single-stranded,
it must be made so. This can be done by conventional means such as heat
denaturation of DNA. The single-stranded target chains are preferably
immobilised e.g. on nitrocellulose. This pretreatment may be effected by
spotting purified DNA onto nitrocellulose filters and baking at 80.degree.
C. to fix the single-stranded target, or possibly by direct processing of
cells on nitrocellulose filters. It may not be necessary, though it may be
advantageous, to subject the target to restriction digestion, gel
electrophoresis and Southern blotting.
The linear probe may be of single- or double-stranded DNA; if
double-stranded, it is converted to single-stranded form at the time of
use. It is necessary that one end of a strand be complementary to a part
of the target extending in one direction from, but not including, the
specific base under investigation. Techniques for synthesising or
otherwise providing such linear probes are known to those skilled in the
field and will not be described here. The probe should be at least 10
nucleotides in length to ensure strong hybridization to the target, and
may be as long as desired. Longer probes may be advantageous as they
permit a larger amount of label per probe molecule and a higher degree of
specificity of hybridization.
The nature of the label used to label the probe is not critical, save only
that the label must not interfere with the digestion performed in step
(c). Radioactive labels will often be convenient. Clinical laboratories
will generally prefer non-radioactive labels, such as enzymes or
chemiluminescent or fluorescent materials, and in such cases direct
labelling may be possible, or labelling with a reporter molecule such as
biotin.
It may be useful to design a probe with two polynucleotide sequences, one
to hybridize to the target and the other to carry label. Provided that the
label sequence has not become hybridized to the target in step (a), it
does not matter whether or not the labelled bases are susceptible to
digestion in step (c). Thus, if Exonuclease III is the enzyme used for
digestion in step (c), a .sup.35 S-thionucleotide can be used as label
only in a part of the probe sequence that will not become hybridized in
step (a) to the target sequence. Similarly, label groups such as biotin or
proteins may conceivably inhibit digestion in step (c).
If the probe is double-stranded, both strands will hybridize to their
complementary strands of the target. Care must therefore be taken with
labelling of a double-stranded probe. There are three alternatives for
probe generation:
(i) A linear single-stranded uniformly labelled or end-labelled probe. This
can be prepared by synthesising an oligonucleotide. Alternatively,
labelled RNA probes can be prepared using phage SP6 RNA polymerase and a
suitable template.
(ii) A linear double-stranded probe labelled only on the strand which
hybridizes with its end adjacent the specific base under investigation.
Such probes can be prepared, uniformly labelled, from an M 13 clone. Or
they can be end-labelled in only one strand if the label intensity is
found to be adequate. Or they can be labelled using T4 DNA polymerase.
(iii) A linear double-stranded probe labelled in both strands. Such probes
can most conveniently be prepared but can give rise to problems of
interpretation. One end, for example the 3' end, of one strand anneals to
the target adjacent the specific base under investigation, and the 3' end
of the other strand anneals at some other region of the target adjacent
another base. It is preferable that this other base should be different
from both the specific base under investigation and its expected mutant.
When this other base is the same as either the specific base or its
mutant, the method can still give useful information, but of a
quantitative rather than a qualitative nature.
The labelled probe is first converted if necessary to a single-stranded
form, and is then hybridized with the target to form a hybrid. After
excess labelled probe has been removed by washing, the hybrid is
subjected, under conditions appropriate for probe extension, e.g.
polymerisation conditions, to reaction in embodiments A and B above with a
nucleotide derivative optionally in the presence of one or two other
different nucleotides, (or in embodiment C with a chain-terminating
nucleotide compound). Hybridization, washing, and polymerisation
conditions may be conventional.
However, any polymerase enzyme used must fulfil two requirements:
(i) The enzyme must be very faithful, i.e. must effect addition of one or
more nucleotides to the end of the probe sequence if those nucleotides are
complementary to the bases in the target sequence, but do so not at all or
only at a very low frequency if they are not.
(ii) The enzyme must be free of exonuclease activity, i.e. must not tend to
remove nucleotides from the end of the probe sequence.
One enzyme that meets these requirements is suitably purified calf thymus
DNA polymerase. Others could readily be found, particularly among
eukaryotic DNA polymerases, or among prokaryotic DNA polymerases that have
been modified to remove unwanted exonuclease activity. Usually the same
enzyme should be applicable, irrespective of whether the probe is of DNA
or RNA.
The nucleotide derivative must also fulfil two requirements:
(i) In embodiments A and B above, it must join to the desired end of the
labelled probe if, and only if, it is complementary to the specific
nucleotide base. Thus if the specific nucleotide base is adenine, a
derivative of thymidine or uridine would be suitable but a derivative of
adenosine, cytidine or guanosine wound not. (In embodiment C, the job of
detecting a mutation at the specific base of the target sequence is
performed, not by a nucleotide derivative but by a chain-terminating
nucleotide compound).
(ii) When joined to the end of the labelled probe, it must protect the
resulting hybrid from digestion under conditions effective to digest
hybrid not so protected.
The nucleotide derivative may in principle be a nucleotide which has been
modified in the sugar, or in the base, or in the phosphate group that
becomes involved in the phosphodiester bond. Many such modified
nucleotides have been described in the literature. The nucleotide
derivative needs to be chosen in conjunction with the exonuclease enzyme
that is to be used in step (c).
As noted above, a suitable nucleotide derivative in some circumstances is
one in which an oxygen atom attached to the alpha phosphorus atom has been
replaced by sulphur, for example alpha-S-deoxythymidine triphosphate
(alpha-SdTTP) or alpha-S-deoxyadenosine triphosphate (alpha-SdATP).
It may be possible to use a nucleotide derivative which is itself labelled.
If a sufficiently high label density can be incorporated in the nucleotide
derivative, then it may be possible to use a probe which has not been
previously labelled, but which becomes labelled by attachment to it of the
nucleotide derivative. This approach may be particularly useful when some
preliminary purification of the target has been carried out. Non-specific
label incorporation may occur in complex targets with palindromic regions.
In step (c), the resulting hybrid is subjected to digestion under
conditions which
(i) do not affect the labelled probe where this is protected at one end by
the nucleotide derivative, and
(ii) progressively digest the hybrid where the labelled probe is not so
protected so as to remove it from the nitrocellulose or other medium on
which the target has been immobilised.
The exonuclease enzyme is therefore one which: attacks double-stranded DNA,
or DNA/RNA hybrids, or double-stranded RNA progressively from the 3' end;
(alternatively, an enzyme could be used that attacks progressively from
the 5' end); and is inhibited by the nucleotide derivative used in step
(b).
As noted above an enzyme which can be used for the digestion is Exonuclease
III. This enzyme attacks double-stranded DNA from 3' end only or DNA/RNA
hybrid progressively from the 3' end of the RNA chain only. If there were
used an exonuclease enzyme that digests double-stranded DNA or a DNA/RNA
hybrid progressively from the 5' end, it would be necessary in step (b) to
attach a nucleotide derivative at the 5' end of the labelled probe.
Where digestion proceeds progressively along the chain, it must be
continued for long enough to remove most or all the label of the labelled
probe from the immobilised target. This factor may place a limit on the
maximum length of the labelled probe that is complementary to the target
sequence and so becomes hybridized to the target.
When the method of this invention is performed with the target sequence in
solution rather than immobilised, step (a) involves mixing the target
material, denatured if necessary to present it in single-stranded form,
with the probe material and then maintaining conditions, which effect
hybridization. An extra enzyme may be included in step (c) to digest any
single-stranded probe or target sequences present, but without digesting
double-stranded sequences; RNase enzymes are available for this purpose
when an RNA probe is used. It is then a simple matter in step (d) to
remove non-hybridized portions of probe, since they have been broken down
in step (c) to single nucleotides.
In all cases, after removal of non-hybridized portions of probe in step
(d), the label still attached to the target is determined. This may be
done by conventional methods depending on the nature of the label. If
desired, the label may be eluted from the target in order to assist
determination.
BRIEF DESCRIPTION OF THE DRAWINGS
Reference is directed to the accompanying drawings, in which:
FIGS. 1 to 3 are reaction schemes relating to Example 1 and showing
respectively target preparation, probe preparation, and hybridization and
test;
FIG. 4 is a representation of an autoradiograph showing the results
obtained in Example 1;
FIGS. 5 to 7 are reaction schemes relating to Example 2 and showing
respectively target preparation, probe preparation, and hybridization and
test; and
FIG. 8 is a representation of an autoradiograph showing the results
obtained in Example 2.
The following Examples 1 and 2 illustrate the invention. The description of
Examples 1 and 2 should be read in conjuction with, respectively, FIGS. 1
to 4 and FIGS. 5 to 8 of the drawings.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Example 1
Aims:
1. To demonstrate the ability of E. coli Exonuclease III to digest probe
which has been hybridised to an immobilised target.
2. To demonstrate inhibition of Exonuclease III by an incorporated
thionucleotide.
Method:
Target preparation (FIG. 1)
1. A sample of plasmid pAT153 was linearised by digestion with restriction
endonuclease Cla I, under the following conditions:
10 mM Tris HCl pH 7.4
10 mM MgCl.sub.2
50 mM NaCl
100 micrograms/ml bovine serum
albumin (BSA)
2. The linear double-stranded DNA was converted to single-stranded form by
heating at 100.degree. C. for 2 minutes.
3. 1 microliter aliquots (containing 10 ng of DNA) of the denatured linear
pAT153 solution were spotted in pairs in a grid pattern on a single sheet
of nitrocellulose membrane (Schleicher and Schull type BA85), such that
the sheet of membrane could be cut into identical 1.5 cm.sup.2 sections
each of which contained one pair of spots. The sheet containing spots was
air-dried and baked in vacuo at 80.degree. C. for 2 hours.
The sequence surrounding the BamHI site of plasmid pAT153 was the region to
which the probe was expected to hybridize.
Probe preparation (FIG. 2)
1. Plasmid pAT153 was digested with restriction endonuclease BamHI in 10 mM
Tris-HCl pH 7.4, 50 mM NaCl, 10 mM MgCl.sub.2, 1 mM dithiothreitol, 100
micrograms/ml BSA.
2. Both ends of digested molecules were expected to have recessed 3'
termini which would incorporate a deoxyguanosine nucleotide in the
presence of the "Klenow" fragment of E. coli DNA polymerase I. An
extension reaction was conducted by adding 10 units of Klenow polymerase
and 50 microcuries of (alpha-.sup.32 P)dGTP (3000 curies/mmole) to 100
microliters of the BamHI digestion mix from Step 1. This amount contained
4 micrograms of linear plasmid pAT153. The extension reaction was
incubated at 20.degree. C. for 15 minutes. Unlabelled dGTP was added to a
final concentration of 100 micromolar, and the reaction was allowed to
continue for a further 5 minutes at 20.degree. C. to ensure completion of
the extension. The mixture was then heated at 65.degree. C. for 10 minutes
to inactivate the Klenow polymerase.
3. The labelled DNA preparation was digested with restriction endonuclease
HaeIII by addition of 20 units of this enzyme to the mix from Step 2 and
incubation at 37.degree. C. for 30 minutes. This step reduced the chance
that the 5' end of a given probe molecule could obstruct extension of its
3' end by complete annealing to the target. The reaction was stopped by
extraction with 100 microliters of a 1:1 mixture of buffered phenol and
chloroform.
Unincorporated radioactivity was removed in the following way:
100 microliters of 4M ammonium acetate pH 4.5 and 400 microliters of
ethanol were added to the aqueous phase. The mixture was chilled at
-70.degree. C. for 10 minutes, warmed to 37.degree. C. for 2 minutes and
spun in a microcentrifuge at room temperature for 10 minutes.
Unincorporated nucleotide remained in the supernatant. The pellet was
washed twice in 66% ethanol containing 666 mM ammonium acetate pH 4.5, and
redissolved in 100 microliters of 10 mM Tris-HCl pH 7.5, 1 mM EDTA.
4. The probe preparation from Step 3 was denatured by heating at
100.degree. C. for 2 minutes. The single-stranded labelled DNA fragments
in the mixture were complementary to regions on either side of the BamHI
site of the target.
Hybridization and test (FIG. 3)
1. The sheet of nitrocellulose membrane, which carried 96 pairs of spots of
denatured target DNA, was shaken gently at 65.degree. C. for 2 hours in
30 ml of
6.times.standard saline citrate
5.times.Denhardt's solution
100 micrograms/ml yeast tRNA
0.1% sodium dodecyl sulphate (SDS)
1.times.standard saline citrate (SSC)=
0.15M NaCl
0.015M Na.sub.3 citrate
pH 7.0
1.times.Denhardt's solution=
0.02% (w/v) BSA
0.02% (w/v) Ficoll
0.02% (w/v)
polyvinylpyrrolidone
10 microliters of freshly boiled probe mix (which contained approximately
400 ng of DNA and 10.sup.6 dpm of .sup.32 P) was then added, and the
mixture was shaken gently at 65.degree. C. for 16 hours. The radioactive
mixture was then discarded, and the nitrocellulose membrane was washed by
gentle shaking at 65.degree. C. for 30 minutes in 50 ml of
6.times.SSC, 5.times.Denhardt's solution, 0.1% SDS
The membrane was then washed for 30 minutes at room temperature in the
following solutions: twice in 2.times.SSC and once in 0.1.times.SSC. The
membrane was stored at 4.degree. C. in 2.times.SSC. 1.5 cm.sup.2 sections
of membrane which contained pairs of spots were washed in 50 mM Tris-HCl
pH 7.8 prior to use.
2. Washed sections of membrane were placed in flat-bottomed cylindrical
test tubes of cross-sectional area 2.8 cm.sup.2. Calf thymus DNA
polymerase-catalysed extension reactions were conducted in
300 microliters of
50 mM Tris-HCl pH 7.8
10 mM Mg Cl.sub.2
1 mM dithiothreitol
500 micrograms/ml BSA
containing 37.5 units of calf thymus DNA polymerase-alpha (supplied by
Pharmacia P-L biochemicals). Nucleotides were present where applicable at
a final concentration of 100 micromolar. Reactions were incubated at
37.degree. C. for 2 hours.
dATP was supplied by Pharmacia P-L biochemicals. Alpha-SdATP was a mixture
of both A and B isomers and was prepared at Amersham.
Following the polymerase extension reaction probe molecules which initially
were labelled by addition of a .sup.32 P-deoxyguanosine nucleotide to the
BamHI-generated 3'-terminus were expected to have been extended by one "A"
residue if the reaction contained dATP or alpha-SdATP. Polymerase
reactions were terminated by addition of 5M NaCl to a final concentration
of 100 mM. (Calf thymus DNA polymerase is inhibited at high salt
concentration.)
3. 200 units Exonuclease III were added where applicable and the reaction
mixtures were incubated at 37.degree. C. for 2 hours. Membrane sections
were then washed separately in 30 ml 2.times.SSC, dried, and exposed to
X-ray film with an intensifying screen at -70.degree. C.
Following autoradiography, membranes were assessed for bound .sup.32 P by
liquid scintillation counting.
Results
FIG. 4 is a representation of an autoradiograph of four representative
pairs of spots obtained under the following conditions:
1. DNA polymerase plus dATP used in step (2). No exonuclease used in step
(3).
2. dATP used without polymerase in step (2). Exonuclease III used in step
(3).
3. DNA polymerase plus alpha-SdATP used in step (2). Exonuclease III used
in step (3).
4. DNA polymerase plus dATP used in step (2). Exonuclease III used in step
(3).
The results indicate clearly that:
1. Exonuclease III has removed terminal label in the absence of
incorporated thio-adenosine nucleotide.
2. Incorporation of thio-adenosine nucleotide has inhibited removal of
label by Exonuclease III.
Liquid scintillation counting showed that approximately 14% of the label
hybridized to control spots was retained following calf thymus DNA
polymerase-alpha-catalysed extension in the presence of alpha-SdATP and
digestion with Exonuclease III, compared to zero following extension in
the presence of dATP and digestion with Exonuclease III.
Example 2
Aims:
1. To demonstrate Exonuclease III-catalysed removal of 5'-end-labelled
probe from an immobilised target.
2. To demonstrate unequivocal discrimination of a "mutant" from a
"wild-type" sequence.
Rationale:
Plasmid pAT153 lacks the segment of plasmid pBR322 from position 1648 to
position 2353 bp (numbered from the EcoRI site). The base pair at position
1649 in pAT153 is A-T. That at position 1649 in pBR322 is G-C. Thus a
probe with its 3' end at position 1648 will anneal to both pAT153 and
pBR322, and will be adjacent to the point of "mutation".
Method:
Target preparation (FIG. 5)
1. Samples of plasmids pAT153 and pBR322 were digested separately with
restriction endonuclease BamHI under the following conditions.
10 mM Tris HCl pH 7.8
10 mM MgCl.sub.2
50 mM NaCl
100 micrograms/ml BSA
2. The linearised plasmids were heated to 100.degree. C. for 2 minutes to
separate their strands, and then transferred to an ice bath.
3. 0.5 microliter aliquots (containing 50 ng) of the denatured linear
plasmid solutions were spotted in pairs in a grid pattern on
nitrocellulose membrane (Schleicher & Schull type BA85), such that the
membrane could be cut into identical 1.0 cm.sup.2 sections, each of which
contained one pair of pAT153 spots and one pair of pBR322 spots. Each 1 cm
square was marked to distinguish the spots, and to permit cutting for
separate liquid scintillation counting of pAT153 and pBR322 spots. The
sheet containing spots was air-dried and baked in vacuo at 80.degree. C.
for 2 hours.
Probe preparation (FIG. 6)
An oligodeoxynucleotide was synthesised with the same sequence as one
strand of pAT153 and pBR322 from position 1629 to position 1648 reading in
the 5' to 3' direction. The solution phospho-triester method was used.
This oligonucleotide was expected to hybridize to both plasmids with its
3' end adjacent to the point of divergence.
The 20-nucleotide probe was labelled with .sup.32 P at its 5' end using T4
polynucleotide kinase and (.gamma.-.sup.32 P)ATP under standard
conditions. The reaction mix contained 100 ng of oligodeoxynucleotide and
100 microcuries of (.gamma.-.sup.32 P)ATP.
Unincorporated label was removed by selective precipitation of the
oligonuceotide, by the method described in Example I, except that
following addition of ammonium acetate and ethanol the mixture was chilled
at -20.degree. C. for 16 hours and at -70.degree. C. for 15 minutes.
Approximately 16% of applied label was incorporated.
Hybridization and Test (FIG. 7)
1. The membrane sheet containing 24 sets of spots of denatured target DNA
was shaken gently for 2 hours at 60.degree. C. in 15 ml of 6.times.SSC,
5.times.Denhardt's solution, 100 micrograms/ml yeast tRNA, 0.1% SDS. 10
microliters of .sup.32 P-labelled probe mix, which contained approximately
5 ng of DNA and 10.sup.6 dpm of .sup.32 P, was added, and the mixture was
shaken gently at 60.degree. C. for 16 hours.
The membrane was washed for 30 minutes at 60.degree. C. in 50 ml of
6.times.SSC, 5.times.Denhardt's solution, 0.1% SDS; then twice at room
temperature for 30 minutes in 100 ml of 2.times.SSC; and then once at room
temperature for 30 minutes in 50 ml of 50 mM Tris-HCl pH 7.8. Membrane
squares were cut from the sheet and used immediately, or stored at
4.degree. C. in 2.times.SSC and re-washed in 50 mM Tris pH 7.8 prior to
use.
2. Calf thymus DNA polymerase-alpha reactions were conducted in 300
microliters volume in flat-bottomed cylindrical polypropylene test tubes
of cross-sectional area 2.8 cm.sup.2. The reaction buffer was:
50 mM Tris-HCl pH 7.8
10 mM MgCl.sub.2
1 mM dithiothreitol
500 micrograms/ml BSA
Calf thymus DNA polymerase-alpha fraction C, generously provided by Dr. A.
M. Holmes of the Uniformed Services University of the Health Sciences,
Bethesda, Md, USA, was used at a final concentration of 43 units/ml.
Nucleotides were used when applicable at a concentration of 100
micromolar. The alpha-SdTTP stock contained approximately equal
proportions of A and B isomers. The alpha-SdCTP stock contained>90% A
isomer. Both stocks were prepared at Amersham.
Reactions were shaken at 37.degree. C. on an oscillating shaker at 160
excursions per minute for 2 hours. Polymerase reactions were terminated by
aspiration of the reaction mix and membrane squares were rinsed
individually in 30 ml of 50 mM Tris-HCl pH 7.8.
3. Exonuclease III reactions were conducted in 300 microliter volume in the
same tubes as those used for the polymerase reactions. The reaction
mixtures contained: 50 mM Tris-HCl pH 7.8, 75 mM NaCl, 10 mM MgCl.sub.2, 1
mM dithiothreitol, 500 micrograms/ml BSA, and 264 units/ml Exonuclease III
(Pharmacia P-L biochemicals).
Reactions were shaken at 37.degree. C. on an oscillating shaker at 160
excursions per minute for 30 minutes. Membrane squares were then washed in
30 ml 2.times.SSC, air dried, and exposed to X-ray film with an
intensifying screen at -70.degree. C. Following autoradiography, membrane
squares were cut for separate determination of .sup.32 P bound to pAT153
and pBR322 spots.
Results
FIG. 8 is a representation of an autoradiograph of nine representative sets
of spots obtained under the following conditions. In each case, the left
hand pair of spots is derived from pAT153 and the right hand pair from
pBR322.
1. No polymerase in step (2). No exonuclease in step (3).
2. No polymerase in step (2). Exonuclease III used in step (3).
3. DNA polymerase plus dTTP and dCTP used in step (2). No exonuclease in
step (3).
4. DNA polymerase plus dTTP and dCTP used in step (2). Exonuclease III used
in step (3). (Scheme (d) of FIG. 7).
5. DNA polymerase plus alpha-SdTTP used in step (2). Exonuclease III used
in step (3). (Scheme (a) of FIG. 7).
6. DNA polymerase plus dTTP used in step (2). Exonuclease III used in step
(3).
7. DNA polymerase plus alpha-SdCTP used in step (2). Exonuclease III used
in step (3). (Scheme (b) in FIG. 7).
8. DNA polymerase plus dCTP used in step (2). Exonuclease III used in step
(3).
9. DNA polymerase plus alpha-SdTTP and alpha-SdCTP used in step (2).
Exonuclease III used in step (3). (Scheme (c) of FIG. 7).
The results indicate clearly that:
1. Exonuclease III efficiently removes a 5'-end labelled 20-nucleotide
probe at moderate enzyme concentration in a relatively short time.
2. The probe can be protected from Exonuclease III digestion by calf thymus
DNA polymerase-catalysed incorporation of thionucleotide.
3. The method has unequivocally distinguished pAT153 and pBR322 on the
basis of their different sequences using only the first base of the
divergent sequence.
Liquid scintillation counting has shown that approximately 44% of .sup.32 P
label hybridized to control pAT153 spots was protected as a result of
extension in the presence of alpha-SdTTP, and approximately 28% of label
hybridized to control pBR322 spots was protected as a result of extension
in the presence of alpha-SdCTP. Less than 5% of .sup.32 P label hybridized
to any pair of spots was protected by the presence of non-complementary
thionucleotide.
* * * * *
|
|
 |
|
 |
Description |
|
