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Description  |
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This invention relates to a novel method for identifying a base in a target
position in a DNA sequence.
In the diagnostic or forensic use of DNA analysis, full sequencing of
target DNA may be unnecessary where the detection of a single base
variation or mismatch is sufficient to provide the required information.
Such a single base variation or mismatch may for example arise from a
point mutation or, in fact, any deletion or insertion of genetic material
where the detection of the first irregular base in the sequence will give
the required diagnostic information. Thus, Allelic Specific PCR has been
developed whereby PCR (polymerase chain reaction) is carried out on a
sample using a pair of primers for the target DNA one of which is
relatively short and will hybridise to one allelic locus of the DNA but
not to the other allelic sequence. Failure to amplify is thus indicative
that the non-hybridising allele of the DNA was present but unfortunately
the conditions required to obtain reliable hybridisation to the normal DNA
are difficult to achieve in practice.
It has been proposed to carry out PCR using probes hybridising to positions
away from the target mutation or area of allelic variation, followed by
use of a labelled probe which will not hybridise to the mutated region or
area of allelic variation. However, this also commonly gives false
negatives.
A method of detecting allele-specific DNA called the Ligase Chain Reaction
(LCR) has recently been developed and has been reviewed by F. Barang (PCR
Methods and Applications Vol.1, 5-16). Two different oligonucleotides,
which hybridise adjacent to each other on complementary DNA, are required
and the products of LCR need to be separated on a polyacrylamide gel
before a result can be determined.
Full length sequencing, particularly solid phase sequencing, as described
in WO 89/09282 gives accurate results but is more demanding and may thus
not be appropriate for diagnostic screening in some instances.
The present invention is based on the concept of using a polymerase
reaction on four aliquots of amplified and immobilised DNA of interest in
single stranded form. Each aliquot uses the same specific extension primer
and a different dideoxynucleotide but no deoxynucleotides so that only the
dideoxynucleotide complementary to the base in the target position is
incorporated; the target position being directly adjacent to the 3' end of
the specific extension primer hybridising to the DNA. Put another way, the
target position on the immobilised strand is immediately 5' of where the
specific primer hybridises to the DNA. Chain extension using normal
deoxynucleotides is then effected (a so-called chase reaction) using the
specific primer so that the dideoxy-blocked DNA will remain unreacted
while the un-blocked DNA will form double stranded DNA. Various methods
may then be used to distinguish double stranded DNA from non-extended DNA,
i.e. substantially single stranded DNA, and thus enable the base in the
target position to be identified.
The invention thus provides a method of identification of the base in a
target position in a DNA sequence wherein sample DNA is subjected to
amplification; the amplified DNA is immobilised and then subjected to
strand separation, the non-immobilised strand being removed and an
extension primer, which hybridises to the immobilised DNA immediately
adjacent to the target position, is provided; each of four aliquots of the
immobilised single stranded DNA is then subjected to a polymerase reaction
in the presence of a dideoxynucleotide, each aliquot using a different
dideoxynucleotide whereby only the dideoxynucleotide complementary to the
base in the target position becomes incorporated; the four aliquots are
then subjected to extension in the presence of all four deoxynucleotides,
whereby in each aliquot the DNA which has not reacted with the
dideoxynucleotide is extended to form double stranded DNA while the
dideoxy-blocked DNA remains as non-extended DNA; followed by
identification of the double stranded and/or non-extended DNA to indicate
which dideoxynucleotide was incorporated and hence which base was present
in the target position.
The term dideoxynucleotide as used herein includes all 2'-deoxynucleotides
in which the 3'-hydroxyl group is absent or modified and thus, while able
to be added to the primer in the presence of the polymerase, is unable to
enter into a subsequent polymerisation reaction.
Preferably, the sample DNA is amplified in vitro by PCR although
amplification by other methods may be used such as in vitro Self Sustained
Sequence Replication (3SR) or in vivo in a vector, and, if desired, in
vitro and in vivo amplification may be used in combination. Whichever
method of amplification is used it is desirable that the amplified DNA
becomes immobilised or is provided with means for attachment to a solid
support. For example, a PCR primer may be immobilised or be provided with
means for attachment to a solid support. Also, a vector may comprise means
for attachment to a solid support adjacent the site of insertion of the
sample DNA such that the amplified sample DNA and the means for attachment
may be excised together.
In the PCR method a pair of polymerisation primers specific to known
sequences of the target DNA are selected, one hybridising at or near the
5' end of one of the strands and the other at or near the 5' end of the
complementary strand such that in the presence of a polymerase, each
primer produces a DNA sequence extending the full length of the target DNA
template. If the DNA so produced is then subjected to strand separation,
typically by melting at a temperature of about 90.degree. C., the newly
formed single stranded DNA sequences will hybridise to excess primer
present in the mixture, usually after reducing the temperature to the
range suitable for annealing, whereupon in the presence of the polymerase,
further DNA strands are synthesised, this time extending only between the
termini of the two primers. The polymerase is preferably capable of
surviving the high temperature used in the strand separation step, a
suitable thermophilic polymerase, namely Taq, having recently become
available. If an excess of the two primers and of nucleotides needed for
DNA synthesis is maintained in the medium, it is possible to operate a
repeated cyclic process in which the separate strands are synthesised,
separated, annealed to primer and new strands synthesised, merely by
raising and lowering the temperature between the optimal temperatures for
each of the above stages. In this way, it is found that amplification of
the original target DNA can be exponential and million-fold increases of
concentration can be effected in a relatively short time.
It is desirable that when PCR is used its effectiveness is assessed, e.g.
to determine whether or not sufficient DNA has been formed to give clear
results with a relatively low level of background. Various tests are known
in the art but we prefer to use the solid phase approach we described
earlier for detection of immobilized amplified nucleic acids, designated
DIANA (PCT/EP90/00454 [WO90/11369]), which has been used for example in
its preferred embodiment in the colorimetric detection of in vitro
amplified DNA. The assay is based on the use of a biotinylated or
otherwise functionalised PCR primer, which is used to capture in vitro
amplified material on, for example, streptavidin-coated magnetic beads.
The other PCR primer contains a "handle", such as a lac operator sequence,
allowing colorimetric detection of the captured DNA using a LacI
repressor-.beta.-galactosidase fusion protein. (Wahlberg, J., Lundeberg,
J., Hultman, T. and Uhlen, M. (1990) "General colorimetric method for DNA
diagnostics allowing direct solid-phase genomic sequencing of the positive
samples." Proc. Natl. Acad. Sci U.S.A. 87, 6569-6573). The preferred form
of the qualitative DIANA assay combines the advantages of the PCR method
with the high specificity and stability of the biotin-streptavidin system
and the simplicity of a colorimetric detection based on
.beta.-galactosidase. The strong interaction between biotin and
streptavidin (K.sub.d =10.sup.-15 M.sup.-1) accentuates the efficiency of
the system. The magnetic beads as solid support ensure that no
centrifugations, filtrations or precipitations are needed (T. Hultman, S.
St.ang.hl, E. Hornes and M. Uhlen Nucl. Acids Res. 17, 4937 (1989)).
However, it is preferred in the method according to the present invention
to use the same PCR primer both as the means of immobilisation and for the
incorporation of the lac operator sequence.
A number of proteins are known which bind to specific DNA sequences and are
often involved in genetic processes such as switching operons on and off.
One such protein is the lac repressor LacI which reacts with the lac
operator (lacOP) to inhibit transcription. Thus, if the recognition site
is the DNA sequence lacOP, the label can be attached via the protein LacI.
It is particularly convenient to devise a fusion protein of a DNA binding
protein such as LacI with a further protein which can be subsequently used
for detection for example using methods based on colour fluorescence or
chemiluminescence. Examples of such proteins are .beta.-galactosidase,
alkaline phosphatase and peroxidase.
It is preferred to use as a label a LacI repressor-.beta.-galactosidase
fusion protein which recognises a 21 base pair lac operator sequence
introduced at the end of the amplified DNA. The lac operator sequence may
be introduced for example by one of the PCR primers if used, preferably
the immobilised primer, or the sequence may be in an amplification vector
in a suitable position for excision with the amplified sample DNA. The
fusion protein will bind to the lac OP sequence of the DNA and the
addition of ONPG (ortho-nitrophenyl-.beta.-D-galactoside will lead to a
colour formation which can be assessed spectrophotometrically. Use of this
fusion protein and ONPG (ortho-nitrophenyl-.beta.-D-galactoside) allows
for a fast simple colorimetric assay which does not have the safety
problems associated with using radiolabels. IPTG
(n-isopropyl-.beta.-D-thiogalactopyranoside) for example, can be added to
release the fusion protein from the DNA.
Two-stage PCR (using nested primers), as described in our co-pending
application PCT/EP90/00454 (WO90/11369), may be used to enhance the signal
to noise ratio and thereby increase the sensitivity of the method
according to the invention. By such preliminary amplification, the
concentration of target DNA is greatly increased with respect to other DNA
which may be present in the sample and a second-stage amplification with
at least one primer specific to a different sequence of the target DNA
significantly enhances the signal due to the target DNA relative to the
`background noise`.
Any suitable polymerase may be used, although it is preferred to use a
thermophilic enzyme such as Taq polymerase to permit the repeated
temperature cycling without having to add further polymerase, e.g. Klenow
fragment, in each cycle of PCR.
Regardless of whether one-stage or two stage PCR is performed, the
efficiency of the PCR is not critical since the invention relies on the
distinct difference between the aliquots. However, as mentioned above, it
is preferred to run an initial qualitative DIANA as a check for the
presence or absence of amplified DNA.
Immobilisation of the amplified DNA may take place as part of PCR
amplification itself, as where one or more primers are attached to a
support, or alternatively one or more of the PCR primers may carry a
functional group permitting subsequent immobilisation, e.g. a biotin or
thiol group. Immobilisation by the 5' end of a primer allows the strand of
DNA emanating from that primer to be attached to a solid support and have
its 3' end remote from the support and available for subsequent
hybridisation with the extension primer and chain extension by polymerase.
The present invention includes a particularly useful primer which
comprises, reading 5' to 3', means permitting immobilisation of said
primer, a sequence which is bound by a DNA binding protein, and a sequence
capable of hybridising at or near the 5' end of a strand of target DNA.
Use of such a primer allows for immobilisation and the ability to
determine whether or not double stranded DNA is formed in a polymerisation
step substantially up to the point of immobilisation. It will be clear
that several nucleotides may intervene between the means permitting
immobilisation and the sequence which is bound by a DNA binding protein or
between that sequence and the sequence capable of hybridising to target
DNA.
Preferably, the means permitting immobilisation is biotin although other
functional groups, such as thiol groups, may be used. However, biotin is
preferred because of its strong interaction with streptavidin and the
relative ease by which it can be incorporated into a primer. The sequence
which is bound by a DNA binding protein is preferably the lac operator
which is reversibly bound by the lac I repressor protein.
The solid support may conveniently take the form of microtitre wells, which
are advantageously in the conventional 8.times.12 format, or dipsticks
which may be made of polystyrene activated to bind the primer DNA (K
Almer, Doctoral Theses, Royal Institute of Technology, Stockholm, Sweden,
1988). The support may also comprise particles, fibres or capillaries
made, for example, of agarose, cellulose, alginate, Teflon.RTM. or
polystyrene. The support may also comprise magnetic particles e.g. the
superparamagnetic beads produced by Dynal AS (Oslo, Norway).
The solid support may carry functional groups such as hydroxyl, carboxyl,
aldehyde or amino groups, or other moieties such as avidin or
streptavidin, for the attachment of primers. These may in general be
provided by treating the support to provide a surface coating of a polymer
carrying one of such functional groups, e.g. polyurethane together with a
polyglycol to provide hydroxyl groups, or a cellulose derivative to
provide hydroxyl groups, a polymer or copolymer of acrylic acid or
methacrylic acid to provide carboxyl groups or an aminoalkylated polymer
to provide amino groups. U.S. Pat. No. 4,654,267 describes the
introduction of many such surface coatings.
The assay technique is very simple and rapid, thus making it easy to
automate by using a robot apparatus where a large number of samples may be
rapidly analysed. Since the preferred detection and quantification is
based on a colorimetric reaction a visual analysis is often sufficient for
evaluation.
The target DNA may be cDNA synthesised from RNA in the sample and the
method of the invention is thus applicable to diagnosis on the basis of
characteristic RNA. Such preliminary synthesis can be carried out by a
preliminary treatment with a reverse transcriptase, conveniently in the
same system of buffers and bases of subsequent PCR steps if used. Since
the PCR procedure requires heating to effect strand separation, the
reverse transcriptase will be inactivated in the first PCR cycle. When
mRNA is the sample nucleic acid, it may be advantageous to submit the
initial sample, e.g. a serum sample, to treatment with an immobilised
polydT oligonucleotide in order to retrieve all mRNA via the terminal
polyA sequences thereof. Alternatively, a specific oligonucleotide
sequence may be used to retrieve the RNA via a specific RNA sequence. The
oligonucleotide can then serve as a primer for cDNA synthesis, as
described in International Patent Application PCT/89EP/00304 (WO89/09282).
PCR has been discussed above as a preferred method of initially amplifying
target DNA although the skilled person will appreciate that other methods
may be used instead of in combination with PCR. A recent development in
amplification techniques which does not require temperature cycling or use
of a thermostable polymerase is Self Sustained Sequence Replication (3SR).
3SR is modelled on retroviral replication and may be used for
amplification (see for example Gingeras, T. R. et al PNAS (USA)
87:1874-1878 and Gingeras, T. R. et al PCR Methods and Applications Vol.
1, pp 25-33).
Advantageously, the extension primer is sufficiently large to provide
appropriate hybridisation with the immobilised strand immediately adjacent
the target position, yet still reasonably short in order to avoid
unnecessary chemical synthesis. It will be clear to persons skilled in the
art that the size of the extension primer and the stability of
hybridisation will be dependent to some degree on the ratio of A-T to C-G
base pairings, since more hydrogen bonding is available in a C-G pairing.
Also, the skilled person will consider the degree of homology between the
extension primer to other parts of the amplified sequence and choose the
degree of stringency accordingly. Guidance for such routine
experimentation can be found in the literature, for example, Molecular
Cloning: a laboratory manual by Sambrook, J., Fritsch, E. F. and Maniatis,
T. (1989). The extension primer is preferably added before the sample is
divided into four aliquots although it may be added separately to each
aliquot. It should be noted that the extension primer may be identical
with the PCR primer but preferably it is different, to introduce a further
element of specificity into the system.
The polymerase reaction in the presence of dideoxy nucleotides is carried
out using a polymerase which will incorporate dideoxynucleotides, e.g. T7
polymerase, Klenow or Sequenase.RTM. Ver. 2.0 (USB U.S.A.). However, it is
known that many polymerases have a proof-reading or error checking ability
and that 3' ends available for chain extension are sometimes digested by
one or more nucleotides. If such digestion occurs in the method according
to the invention the level of background noise increases. In order to
avoid this problem it is preferable to use a non proof-reading polymerase,
e.g. T7 polymerase or Sequenase. Otherwise it is desirable to add to-each
aliquot fluoride ions or nucleotide monophosphates which suppress 3'
digestion by polymerase.
Identification of the double stranded and/or non-extended DNA is possible
via a variety of means. With regard to the double stranded DNA,
conventional techniques such as radiolabel incorporation during chain
extension are possible but it is preferred to use the lac operator
sequence which is preferably incorporated into the DNA during
amplification, as discussed above. Full chain extension creates the double
stranded DNA sequence which is bound by the lac I
repressor-.beta.galactosidase fusion protein. Bound fusion protein can
then be identified colorimetrically as discussed above and this identifies
the three aliquots which have been extended, thereby identifying the
dideoxy base which was added in the remaining aliquot.
With regard to the non-extended DNA, where extension of the primer was
blocked by a dideoxynucleotide, again a number of means for identification
are possible and will be readily apparent to the skilled person.
Preferably, a probe which hybridises downstream of the 3' end of the
extension primer is used, i.e. the probe hybridises to the immobilised
strand between the site of hybridisation of the extension primer and the
5' end of the immobilised strand. The probe is suitably labelled or
provided with means for attaching a label. Such a probe will bind to the
single strand DNA but will not bind to the double stranded DNA.
If desired, both double and single stranded DNA can be identified and this
provides additional checking for the accuracy of the results. It will
usually be desirable to run a control with no dideoxynucleotides and a
`zero control` containing a mixture of all four dideoxynucleotides.
Another means of identification is that disclosed in our co-pending
application of even date (PCT/EP93/01205 [WO93/23564]) which relates to
detection of pyrophosphate released during chain extension. When each
nucleotide is incorporated, a pyrophosphate group is split off the
nucleotide triphosphate and the remaining nucleotide monophosphate is
incorporated at the end of the growing nucleic acid chain. In those
aliquots which have not incorporated a chain terminating dideoxynucleotide
there is extensive pyrophosphate release during chain extension. This
release of pyrophosphate can be measured using luciferin and luciferase
which emit light in substantially direct proportion to the amount of
pyrophosphate present.
In many diagnostic applications, for example genetic testing for carriers
of inherited disease, the sample will contain heterozygous material, that
is half the DNA will have one nucleotide at the target position and the
other half will have another nucleotide. Thus of the four aliquots used in
the method of the invention, two will show a positive signal and two will
show half the positive signal. It will be seen therefore that it is
desirable to quantitatively determine the amount of label detected in each
sample. In the case of a homozygous sample it will be clear that there
will be three negative and one positive signal of the four aliquots.
Advantageously, the method according to the present invention may be
combined with the method taught in our co-pending patent application of
even date (PCT/EP93/01204 [WO93/23563]) which uses PCR to introduce loop
structures which provide a permanently attached 3' primer at the 3'
terminal of a DNA strand of interest. For example, in such a modified
method, the extension primer is introduced as part of the 3'-terminal loop
structure onto a target sequence of one strand of double stranded DNA
which contains the target position, said target sequence having a region A
at the 3'-terminus thereof and there being optionally a DNA region B which
extends 3' from region A, whereby said double-stranded DNA is subjected to
polymerase chain reaction (PCR) amplification using a first primer
hybridising to the 3'-terminus of the sequence complementary to the target
sequence, which first primer is immobilised or provided with means for
attachment to a solid support, and a second primer having a 3'-terminal
sequence which hybridises to at least a portion of A and/or B of the
target sequence while having at its 5'-end a sequence substantially
identical to A, said amplification producing double-stranded target DNA
having at the 3'-end of the target sequence, in the following order, the
region A, a region capable of forming a loop and a sequence A'
complementary to sequence A, whereafter the amplified double-stranded DNA
is subjected in immobilised form to strand separation whereby the
non-immobilised target strand is liberated and region A' is permitted or
caused to hybridise to region A, thereby forming said loop. The 3' end of
region A' hybridises immediately adjacent the target position. The dideoxy
and extension reactions use the hybridised portion as a primer and the
base incorporated at the target position can be identified in any manner,
preferably by pyrophosphate release as taught by our co-pending
PCT/EP93/01205 (WO93/23564) application mentioned above.
The invention also comprises kits which will normally include at least the
following components:
(a) a test specific extension primer which hybridises to sample DNA so that
the target position is directly adjacent to the 3' end of the primer;
(b) a polymerase;
(c) deoxynucleotides and dideoxynucleotides; and
(d) optionally a solid support.
If the kit is for use with initial PCR amplification then it will also
normally include at least the following components:
(i) a pair of primers for PCR at least one primer having means permitting
immobilisation of said primer;
(ii) a polymerase which is preferably heat stable, for example Taq1
polymerase;
(iii) buffers for the PCR reaction; and
(iv) deoxynucleotides.
Where an enzyme label is used, the kit will advantageously contain a
substrate for the enzyme and other components of a detection system.
Preferably, one of the primers will include both means permitting
immobilisation of said primer and a sequence which is bound by protein. A
preferred form of primer comprises biotin to act as the means permitting
immobilisation, eg to an avidin or streptavidin coated surface, and the
lac operator as the means permitting labelling. The kit for carrying out
the invention using a preferred primer of the type described above would
preferably contain an enzyme label conjugated to the lac I repressor
protein; a preferred enzyme label being .beta.-galactosidase.
The invention will now be described by way of non-limiting examples with
reference to the drawings in which:
FIG. 1 shows a protocol for identifying a base in a single target position
using the method according to the invention;
FIG. 2 shows oligonucleotide primers (SEQ ID Nos. 4-7) used in Example 1
together with sample DNA for amplification (SEQ ID Nos. 1-3);
FIG. 3 shows further oligonucleotide primers (SEQ ID Nos. 8 and 9) used in
the Example together with the sample DNA (SEQ ID Nos. 1-3); and
FIG. 4 is a graph showing the results obtained in the Example of the method
according to the invention.
MATERIALS AND METHODS
Bacterial strains and plasmids. Escherichia coli RRI.DELTA.M15 (Ruther,
U.(1982). pUR 250 which allows rapid chemical sequencing of both strands
of its inserts (Nucl. Acids Res., 10, 5765-5722) was used as bacterial
host. The plasmid vector used was pRIT 28 (Hultman, T., Stahl, S., Moks,
T. and Uhlen, M. (1988) "Approaches to Solid Phase DNA Sequencing",
Nucleosides & Nucleotides. 7, 629-638).
Synthesis of oligonucleotides. 7 oligonucleotide primers (See FIGS. 2 and
3), RIT 135 (SEQ ID No:4), RIT 321 (SEQ ID No:6), RIT 322 (SEQ ID No:7),
RIT 331 (SEQ ID No:9), RIT 332 (SEQ ID No:5) and RIT 333 (SEQ ID No:8),
complementary to regions (SEQ ID Nos 1-3) encoding a part of the active
site of the HIV reverse transcriptase gene (RT) (bases 625 to 1165 Myers,
G., Korber, B., Berkovsky, J. A. Smith, R. F. and Pavlakis, G. N. Human
Retroviruses and AIDS 1991 (Los Alamos National Laboratory, New Mexico
1991)), were synthesized by phosphoramidite chemistry on an automated DNA
synthesis apparatus (Gene Assembler Plus.RTM., KABI-Pharmacia, Sweden) as
described by the manufacturer. RIT322 was biotinylated by using a biotin
phophoramidite (Clonetech, Calif., U.S.A.). Purification was performed on
a pepRPC 5/5 reversed phase column (KABI-Pharmacia, Sweden).
Enzymes and nucleotides. Restriction enzymes, T4 DNA ligase
(KABI-Pharmacia, Sweden), T7 DNA polymerase (KABI-Pharmacia, Sweden), Taq
DNA polymerase (Cetus, Calif. U.S.A.) and Sequenase.RTM. ver 2.0 (USB
U.S.A.) were used in accordance with the supplier's recommendations. Deoxy
and dideoxynucleotides were obtained from Boehringer Mannheim, Germany.
PCR cloning
The HIV RT fragment was cloned by amplification from a clinical sample
obtained from a patient with HIV-1 (Swedish Bacteriology Laboratory, SBL,
Stockholm, Sweden) using 5 pmol each of the oligonucleotides RIT331 and
RIT333 (FIG. 3) both containing "handles" in order to introduce an
upstream Bam HI and a downstream Eco RI recognition sites. The PCR
reaction mix contained 200 .mu.M dNTPs, 20.TM.(polyoxyethylene sorbitan
monolamate) mM Tris-HCl (pH 8.7), 2 mM MgCl.sub.2, 0.1% Tween.RTM. 20 and
0.5 units AmpliTaq.TM. resulting in a final volume of 50 .mu.l . The
temperature profile was set up by a denaturation step at 95.degree. C. for
0.5 min. followed by a primer annealing step at 55.degree. C. for 0.5 min.
and a final extension step at 72.degree. C. for 2 mins. These steps were
repeated 30 times using a Gene Amp PCR System, PE 9600 (Perkin Elmer,
Calif., U.S.A.). The PCR amplified HIV RT fragment and the pRIT 28 vector
were both restricted with Bam H1 and Eco R1, cut out and purified from
agarose and then ligated for 1 hour in room temperature. The construction
was transformed into competent RRI.DELTA.M15cells and spread on TBAB
(Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) loc.cit). plates
containing IPTG(n-isopropyl-.beta.-D-thiogalactopyranoside), X-gal
(5-bromo-4-chloro-3-indolyl-.beta.-D-galactoside) and ampicillin allowing
blue/white selection (Langley, E. K. Villarejo, M. R. Fowler, A. V.
Zamenhof, P. J. and Zabin, I. (1975). Proc. Natl. Acad. Sci. U.S.A. 72,
1254-1257). Five white colonies containing the plasmid with a correct
insert was confirmed by solid phase sequencing (Hultman, T., Bergh, S.,
Moks, T and Uhlen, M. (1991) "Bidirectional solid-phase sequencing of in
vitro-amplified plasmid DNA". Bio Techniques 10, 84-93.). One of those
clones was designated pRIT-RT and choosen for further studies. This clone
is stored at the Department of Biochemistry, Royal Institute of
Technology, Stockholm, Sweden.
Template preparation for DIANA detected Mini Sequencing
A colony harbouring pRIT28-RT was transferred to a vial and lysed at
99.degree. C. for 5 min. in 10 .mu.l 20 mM Tris-HCl (pH 8.7). 1 .mu.l
lysate was subsequently transferred to a PCR mixture of 5 pmol RIT135 and
RIT322 (biotinylated), 0.25 pmol RIT321, 200 .mu.M dNTPs, 20 mM Tris-HCl
(pH 8.7), 2 mM MgCl.sub.2, 0.1% Tween 20.TM. and 0.5 units AmpliTaq.TM. to
a final volume of 50 .mu.l. It will be noted that primer RIT322 comprises
a 5' Biotin, for subsequent attachment to a streptavidin coated solid
support, and the 21 bases which define the lac Op recognition sequence.
Amplification was performed as above and the resulting PCR product was
subsequently immobilized (Hultman, T. Stahl, S., Hornes, E. and Uhlen, M.
(1989) "Direct solid phase sequencing of genomic and plasmid DNA using
magnetic beads as solid support". Nucl. acids Res. 17, 4937-4946) on
prewashed streptavidin coated paramagnetic beads, (Lea, T., Vartdal, F.,
Nustad, K., et al. (1988). "Monosized, magnetic polymer particles: and
their use in separation of cells and subcellular components and in the
study of lymphocyte function in vitro". Journal of Molecular Recognition
1, 9-18) Dynabeads.RTM. M280-Streptavidin (Dynal AS, Norway), prewashed
with binding solution according to manufacturer. After immobilization, the
beads were rinsed with 50 .mu.l binding-washing solution and assayed for
bound DNA. The beads with the immobilized DNA were mixed with 50 .mu.l of
the fusion protein, lacI-.beta.-galactosidase (Dynal AS, Norway), and
incubated for 20 minutes. Excess of the fusion protein was removed by
washing the beads 4 times with DIANA buffer (Dynal AS, Norway) and
changing to new tubes in the last step in order to avoid background due to
coating of the walls. 100 .mu.l of chromogenic substrate,
ortho-nitrophenyl-.beta.-D-galactoside (ONPG, 1.25 mg/ml), was added and
after 6 min. the reaction was stopped by an addition of 100 .mu.l 1M
Na.sub.2 CO.sub.3 and the supernatant was analyzed in an EAR340AT ELISA
plate reader (SLT-Labinstruments, Austria) by measuring the absorbence at
405 nm. The strands were separated by melting by incubation with 20 .mu.l
0.1M NaOH for 5 min. generating single stranded immobilized DNA template,
which was once again washed with 50 .mu.l binding solution, 50 .mu.l
1.times.TE. The primer annealing was performed in 8 mM MgCl.sub.2 and 20
mM Tris-HCl (pH 7.5) with the use of 1 pmol RIT332 (FIG. 2) in a volume of
13 .mu.l by heating to 65.degree. C. for 5 min. and then placed in room
temperature for 10 min.
Mini Sequencing reactions
Six separate extension reactions with respect to the appropriate
dideoxynucleotide were set up (one with only ddATP, one with only ddCTP,
one with only ddGTP, one with only ddTTP, one with all four ddNTPS present
and one without any of ddNTPs) in a total of 10 .mu.l containing 2 .mu.l
of the annealing mixture, 17 mM Tris-HCl (pH7.5), 6 mM MgCl.sub.2, 1 mM
DTT, 1 .mu.M of the appropriate dideoxynucleotide and 0.13 units of
Sequenase.RTM. ver. 2. A schematic outline of the experiment is shown in
FIG. 1. The dideoxy incorporation was performed at room temperature for 5
mins. and stopped by adding 20 .mu.l 0.5M EDTA. Thereafter the beads were
washed twice with 30 .mu.l 10 mM Tris-HCl (pH 7.5). In the following
extension step 200 .mu.M dNTP concentration was used together with 25 mM
Tris-HCl (pH 7.5), 12.5 mM MgCl.sub.2, 1 mM DDT and 0.13 units
Sequenase.RTM. in a total of 10 .mu.l. In the aliquots where a
dideoxynucleotide had not been incorporated, the Sequenase.RTM. leads to a
chain extension and to full double stranded DNA being attached to the
beads. After a 5 min. incubation in room temperature 20 .mu.l 0.5M EDTA
was added and the beads were washed with 40 .mu.l DIANA buffer (Dynal AS,
Norway) (0.1M Tris-HCl (pH 7.5), 0.15M NaCl, 0.1% Tween 20, 1 mM
MgCl.sub.2 and 10 mM .beta.-mercaptoethanol).
Detection by DIANA
The results were detected by DIANA (Wahlberg, J., Lundeberg, J., Hultman,
T. and Uhlen, M. (1990) "General colorimetric method for DNA diagnostics
allowing direct solid-phase genomic sequencing of the positive samples."
Proc. Natl. Acad. Sci U.S.A. 87, 6569-6573). The beads with the
immobilized DNA were mixed with 50 .mu.l of the fusion protein,
lacI-.beta.-galactosidase (Dynal AS, Norway), and incubated for 20
minutes. Excess of the fusion protein was removed by washing the beads 4
times with DIANA buffer (Dynal AS, Norway) and changing to new tubes in
the last step in order to avoid background due to coating of the walls.
100 .mu.l of chromogenic substrate, ortho-nitrophenyl-.beta.-D-galactoside
(ONPG, 1.25 mg/ml), was added and after 6 min. the reaction was stopped by
an addition of 100 .mu.l 1M Na.sub.2 CO.sub.3 and the supernatant was
analyzed in an EAR340AT ELISA plate reader (SLT-Labinstruments, Austria)
by measuring the absorbence at 405 nm. The results are shown in FIG. 4.
The assay show that a low signal is obtained when all four
dideoxynucleotides (ddNTP) are used as well as when only ddATP is used.
Since the complementary base next to the 3'-end of the sequencing primer
is a dideoxythymidine, the result demonstrates that the assay can be used
to detect a base sequence at a specific point.
EXAMPLE 2
Template preparation
A HIV reverse transcriptase gene fragment from a patient showing AZT
resistance was PCR-cloned (Petterson, B, et al unpublished data) into the
vector pRIT 28 by using the primers RIT 331 and RIT 333. E.coli
RR1.DELTA.M15 was transformed and blue/white selectivity was used (Langley
E. K., et al (1975) loc. cit.) PCR amplification was carried out by lysing
a bacterial colony in 10 .mu.l 20 mM Tris-Cl (pH 8.7) at 99.degree. C. for
5 minutes. Then 1 .mu.l of the lysate was added to 5 pmol Primer set A,
200 .mu.M dNTP, 20 mM Tris-Cl, pH 8.7, 2 mM MgCl.sub.2, 0.1% Tween.RTM.
20.TM. and 0.5 units AmpliTaq DNA polymerase (Cetus, Calif., USA) making
up a total volume of 50 .mu.l. The temperature profile included a 0.5 min.
denaturation step at 95.degree. C. and a 1.5 min. annealing/extension step
at 70.degree. C., these steps were repeated 30 times. A GeneAmp PCR
System 9600 (Perkin Elmer, Calif., USA) was used for both lysing the
bacterial colony and running the reactions. The PCR product was
immobilized on paramagnetic beads (Lea, T., et al (1988) loc. cit.) with
covalently coupled streptavidin, Dynabeads.RTM. M280. The beads were used
as described by the manufacturer (Dynal AS, Norway). Single stranded DNA
was obtained by removing the supernatant after incubation of the
immobilized PCR product in 0.10M NaOH for 10 minutes. The immobilized
single stranded DNA was washed with 50 .mu.l 10 mM Tris-Cl (pH 7.5), 1 mM
EDTA, 2M NaCl followed by 50 .mu.l 10 mM Tris-Cl (ph 7.5). After washing,
20 mM Tris-Cl (pH 7.5), 8 mM MgCl.sub.2 and 1 pmol sequencing primer were
added to a final volume of 13 .mu.l. The mixture was incubated at
65.degree. C. for 5 minutes and then cooled to room temperature.
Mini-sequencing
The dideoxynucleotide incorporation reactions were performed in a mixture
of 1 .mu.l (1/13 of a 50 .mu.l PCR amplification reaction) of the
template/primer-fragment immobilized on paramagnetic beads, 0.13 units
Sequenase.RTM. version 2.0 (United States Biochemical, USA), 0.5 .mu.l 10
.mu.M of a single ddNTP, and a buffer containing 25 mM Tris-Cl (pH 7.5),
12.5 mM MgCl.sub.2 and 2.5 mM DTT in a final volume of 10 .mu.l. After
incubation at room temperature for 5 minutes, the beads were washed with
50 .mu.l 10 mM Tris-Cl (pH 7.5), 1 mM EDTA, 2M NaCl, 1% Tween 20.TM.
followed by 50 .mu.l 10 mM Tris-Cl (pH 7.5), 1 mM EDTA, 2M NaCl and
finally with 50 .mu.l 10 mM Tris-Cl (pH 7.5). The volume was adjusted to 5
.mu.l with 10 mM Tris-Cl (pH 7.5). Control fragments were incubated with
DNA polymerase in the absence of ddNTPS and zero control fragments in the
presence of all ddNTPS. The different samples were subsequently analyzed
with the ELIDA.
ELIDA
Samples from the above described mini-sequencing preincubation were assayed
for full primer extension by the ELIDA. The assay was performed using an
LKB 1250 luminometer and a potentiometric recorder. The luminometer was
calibrated to give a response of 10 mV for the internal light standard.
The luminescence output was calibrated by the addition of a known amount
of ATP or ppi. The reaction was carried out at room temperature. The
standard assay volume was 0.2 ml and contained the following components:
0.1M Tris-acetate (pH 7.75), 2 mM EDTA, 10 mM magnesium acetate, 0.1% BSA,
1 mM DTT, 0.4 mg/ml polyvinylpyrrolidone 360,000, 2 .mu.M dNTP, 100
.mu.g/ml D-luciferin (BioOrbit, Finland), 4 .mu.g/ml L-luciferin
(BioOrbit, Finland), 0.3 units/ml ATP-sulfurylase (Sigma, USA) and
purified luciferase (Enzymatix, UK). The amount of luciferase used gave a
response of 1 V for 100 pmol ATP in a volume of 1 ml. After five minutes
of preincubation, adenosine 5'-phosphosulfate, NaF and dNMP were added to
final concentrations of 2 .mu.M, 5 mM and 0.4 mM, respectively. The
reaction was started after the addition of 5 .mu.l of
template/primer-fragments, taken from the dideoxy incorporation, by the
addition of 0.13 units of Sequenase.RTM.. The reaction was completed
within 5 minutes.
RESULTS
Principle of the mini-sequencing method
The principle of the mini-sequencing method is outlined in FIG. 1 in which
the presence or absence of a T residue is investigated. The specific
DNA-fragment of interest is amplified by PCR with one of the primers
biotinylated in the 5' end. The PCR-amplified DNA is immobilized on
magnetic beads containing covalently coupled streptavidin and subsequently
converted into single stranded form by washing with NaOH, and a primer is
annealed to the single stranded DNA. The template/primer-fragments are
then divided into four different aliquots which are separately treated
with one of the four ddNTPs in the presence of the polymerase. After the
reaction, the resulting fragments are washed and used as substrate in a
primer extension reaction with all four dNTPs present (see FIG. 1). The
progress of the DNA-directed polymerisation reactions are monitored with
the ELIDA. Incorporation of a dideoxynucleotide in the first reaction will
prevent the formation of pyrophosphate during the subsequent "chase"
reaction. In contrast, no dideoxynucleotide incorporation gives extensive
pyrophosphate release during the "chase" reaction and this will lead to
generation of light through the ELIDA reactions. From the ELIDA results,
the first base after the primer is easily deduced. It is also possible to
include both a negative control, which is incubated with all ddNTPs, and a
positive control, which is incubated with DNA polymerase in the absence of
dNTPs.
Mini-sequencing of a specific DNA-fragment
Incorporation of a single ddNTP was observed only when the complementary
dideoxynucleotide (ddATP) was present during the polymerase reaction. No
incorporation of noncomplementary bases was observed under the conditions
used. The formation of ppi was detected by the ELIDA during the "chase"
reaction only when template/primer-fragments were incubated with
noncomplementary bases. When a complementary base was incorporated, no
extension of the DNA was possible due to the lack of a free 3' OH group.
The same result as above was obtained if the DNA-fragments (in the first
step) were incubated with four different mixtures of three ddNTPs (not
shown). It is important to note that a DNA polymerase lacking exonuclease
activity must be used to obtain clean signals, although it is known that
exonuclease activity of certain polymerases can be suppressed, e.g. by
fluoride ions. It is also important to use low concentrations of
nucleotides (0.05-5 .mu.M) to avoid incorporation of non-complementary
bases (data not shown).
Sensitivity
In the experiments presented above 1/13th of a 50 .mu.l PCR amplification
reaction was used per ELIDA test. However, both lower and higher amounts
can be used. The initial rate and the extent of ppi formation during
primer extension of a 161 bases long DNA-fragment as a function of DNA
concentration was determined. Both the initial rate and the extent of ppi
formed in the ELIDA are proportional to the DNA concentration in the
interval tested (1/130 to 2/13 of a 50 .mu.l PCR amplification reaction).
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