WO1996003525A1 - Method and reagents for the detection of target nucleic acid sequences - Google Patents

Method and reagents for the detection of target nucleic acid sequences Download PDF

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Publication number
WO1996003525A1
WO1996003525A1 PCT/EP1995/002944 EP9502944W WO9603525A1 WO 1996003525 A1 WO1996003525 A1 WO 1996003525A1 EP 9502944 W EP9502944 W EP 9502944W WO 9603525 A1 WO9603525 A1 WO 9603525A1
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Prior art keywords
vector
probe
nucleic acid
sequence
acid sequence
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PCT/EP1995/002944
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French (fr)
Inventor
Stanislavo Marcolini
Giorgio Martinazzo
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Raggio-Italgene S.P.A.
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Application filed by Raggio-Italgene S.P.A. filed Critical Raggio-Italgene S.P.A.
Priority to AU33424/95A priority Critical patent/AU3342495A/en
Publication of WO1996003525A1 publication Critical patent/WO1996003525A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6862—Ligase chain reaction [LCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

  • the present invention relates to a method and reagents for the detection and, optionally, amplification of a target nucleic acid sequence.
  • a target nucleic acid sequence may be any sequence of interest, for example, it may be a sequence present in a pathogenic organism and may be of interest for clinical diagnosis or in the context of food hygiene and public health. Other fields where detection of target nucleic acid sequences are of interest include forensic science.
  • WO85/04663 describes a process for detecting a specific (target) nucleotide sequence
  • DNA probe molecule is capable of transforming bacteria if and only if it is held in a circular configuration by base pairing to the target nucleotide sequence, said transformation resulting in the detection of a phenotype specified by the DNA probe molecule. Detection of the phenotype establishes the presence, absence or quantity of the target.
  • That method has the disadvantages that long sequences of target DNA are required to maintain the circular configuration; short sequences may not be detected. Furthermore the method does not allow discrimination between very similar sequences in such a way that small base variations, especially single point mutations, in a sequence can be discriminated. This will prevent, for example, the identification of a wild type from a mutated sequence of interest for clinical diagnosis.
  • the transformation is carried out without separating the target from the probe, thus increasing the risk of non-specific binding (increased background) reducing both the specificity and the sensitivity of the procedure.
  • the circularization reaction cannot be repeated, thus preventing the possibility of amplification of target sequences occurring at low level.
  • the present invention provides a single stranded linearisable circular vector-probe, said vector-probe comprising a nucleic acid sequence complementary to a target nucleic acid sequence, said complementary nucleic acid sequence preferably being present in a "polylinker" zone of the vector-probe, and said complementary nucleic acid sequence being in reading frame with a region of the vector- probe coding for a selectable or detectable marker, said vector-probe being linearisable by cleavage of a phosphodiester bond in the nucleic acid sequence complementary to the target sequence.
  • Said vector-probe in single stranded linear form that is to say, having been linearised by cleavage of a phosphodiester bond in the complementary sequence, or having appropriate parts of the complemetary sequence added to a cleaved circular vector not previously comprising the complementary sequence, is also part of the present invention.
  • the present invention also provides a method for the detection of a target nucleic acid sequence in a sample, which comprises
  • step (iii) separating the resulting circularized single stranded linear vector-probe from the annealed target; (iv) contacting the circularized single stranded linear vector-probe, optionally in the form of the reaction mixture resulting from step (ii) , with competent host bacteria that lack the marker present in the vector-probe under transformation conditions; and
  • the vector-probe is obtainable by cleavage of a single stranded circular vector-probe that comprises a nucleic acid sequence complementary to the target sequence, the cleavage site being within the complementary sequence, preferably at a phosphodiester bond.
  • the linear vector probe is also obtainable by cleaving a circular single stranded probe, adding to 5 '-end one part of the nucleic acid sequence complementary to the target sequence and adding to the 3'- end the other part of the complementary sequence.
  • the method of the present invention involves the detection of target nucleic acid sequences, for example, genomic sequences, by hybridisation with a linear single stranded vector-probe comprising a cleaved nucleic acid sequence complementary to the target nucleic acid sequence.
  • the linear vector-probe may be obtained from a circular vector- probe comprising a sequence complementary to the target sequence by cleavage in the complementary sequence.
  • a circular vector that does not already comprise the complementary sequence may be linearised then one part of the complementary sequence added to the 5'-end and the other part added to the 3 '-end.
  • the target sequence will hybridise to the linear vector-probe, forming a double stranded region with a "nick" in the vector-probe strand. That single stranded "nick" in the vector-probe can then be repaired using an appropriate enzyme.
  • the resulting circularized single stranded vector-probe is used to transform competent hosts that lack the marker present in the vector-probe, the presence of the marker in the resulting transformed bacteria indicates the presence of target nucleic acid sequence in the sample under investigation.
  • Single stranded circular vectors are well known and are generally derived from filamentous phage. Examples are M13mp derivatives, for example, as described by Messing J, "New M13 vectors for cloning" Meth. Enzymol. (1983) 101:20- 78; Deute L, Cesareni G & Cortese R, "pEMBL A new family of single-stranded plasmids" Nuc. Acids Res. (1983) 11:1645- 1655; Messing J, Groenborn B, Muller-Hill B & HofSchneider PH, "Single strand filamentous DNA phage as carrier for in vitro recombined DNA" Proc. Natl. Acad. Sci. U.S.A.
  • M13mp derivative is M13mpl8.
  • An M13mpl8/pUC polylinker is described by Yonisch-Petron C, Vieire J & Messing J (1985) "Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mpl8 and pUC19 vector-probes" Gene 33:109-119.
  • a commercially available circular vector is Bluescript (Trade Mark) SK +/- phagemid (Stratagene) .
  • Figure 1 of the accompanying drawings gives a map of the Bluescript phagemid showing various functional features and various restriction sites.
  • Figure 2 gives sequence information relating to the Bluescript phagemid. Various primers are shown, as are restriction sites.
  • An essential characteristic of a circular single-stranded vector-probe used according to the present invention is that it can be linearised, preferably in the central zone of the sequence complementary to the target sequence, by cleavage of a phosphodiester bond. Cleavage may be effected using an appropriate enzyme, for example, a restriction enzyme. Suitable enzymes are well known.
  • linear vector-probe may be obtained by cleavage of the complementary sequence already present in a circular vector
  • a linear vector-probe may be prepared by other methods.
  • a circular vector may be cleaved and one part of the desired complementary sequence added to the 5'-end and the other part added to the 3'-end.
  • Any linear vector that can form a circular single stranded vector capable of transforming competent bacteria may be modified by the addition of a complementary sequence.
  • linear and linearised vector-probes include all linear vector-probes that may be used according to the present invention, regardless of the method of their production.
  • the linear vector-probe In addition to the ability to form a circular single stranded vector that is capable of transforming competent bacteria, the linear vector-probe should generally have high efficiency, a strong promoter and must be functional only when in circular form. It should also have a selectable or detectable marker.
  • a linear vector-probe according to the invention has two termini, one of the 3 '-hydroxyl type and the other of the 5 '-phosphate type.
  • a sample under investigation that may comprise the target nucleic acid sequence (which sample has been treated to bring any nucleic acids present into single-stranded form) , for example, an extract of denatured DNA
  • a predetermined amount for example, a known surplus
  • the vector-probe of the present invention and to the action of a modification enzyme that repairs single-stranded breaks ("nicks") in a double-stranded nucleic acid molecule, generally from the ligase family
  • nicks single-stranded breaks
  • a characteristic feature of the repair enzyme used is that it requires as substrate two single-stranded fragments of nucleic acid, the 5' terminal of one having a phosphate group and the 3' terminal of the other having a hydroxyl group. Both fragments must be complementary, vicinal and contiguous to a second strand whose length is intact, and take the form of a "duplex" which presents a nick (ie. a hydrolysed phosphodiester bond) in one of the two strands.
  • This discriminating condition corresponds to that found when the linear vector-probe is hybridised with the single- stranded complementary target sequence: only under these circumstances will the enzyme, generally a ligase, be active and reconstitute the phosphodiester bond, thereby circularising the vector-probe.
  • the enzyme As the enzyme is inefficient in ligating single stranded DNA fragments with blunt ends, it will not circularise the linear vector-probe unless the latter has perfectly hybridised the two ends complementary to the target sequence in a contiguous, vicinal manner. If any mismatch is present at and/or near the 5' end or at and/or near the 3 ' end of the linear vector-probe, the enzyme will not be able to close the nick and to circularize the vector-probe.
  • Each circularised vector-probe will therefore correspond to the presence of a target nucleic acid sequence in the sample analyzed.
  • E. coli ligase or any other DNA ligase may be used when the target sequence is DNA. If the target sequence is RNA either an appropriate RNA ligase may be chosen or DNA complementary to the RNA may be produced and then subjected to the method of the invention.
  • the ligase may be a thermostable or thermolabile ligase.
  • a thermostable ligase is particularly useful if repeated cycles of ligation are desired.
  • a thermolabile ligase provides a simple method for denaturating the enzyme when the desired ligation reaction is complete.
  • a circular single stranded vector comprising a complementary sequence
  • a circular single stranded vector that is linearisable in another region of the vector for example, at one or both ends of the complementary sequence to give a linear vector-probe according to the present invention.
  • Such circular vectors and linear vector-probes and their use are also part of the present invention.
  • it is preferable to cleave a circular single stranded vector at a phosphodiester bond it is possible to design vectors having cleavage sites at other bonds, for example, using specially-designed polylinker sequences.
  • Such circular vectors, the resulting linear vector-probes, and their use are all part of the present invention.
  • the second part of the method of the present invention involves replicating the now-circularised single stranded vector-probe so as to produce an easily detectable signal which can be correlated with the presence or absence of the target sequence, and optionally with its quantification.
  • the repair enzyme is generally inactivated and/or, if desired, the resulting circularised vector-probe may be isolated from the reaction mixture.
  • the circularised vector and the annealed target sequence should be separated from one another.
  • suitable markers are known in the art, for example, antibiotics-resistance markers, for example, ampicillin resistance gene.
  • a further type of marker that may be used in the present invention is one whose gene product is needed by the cell for the chromogenic catalysis of a suitable substrate, or a gene whose product is a substance characteristic of the vector-probe.
  • the marker gene should be expressible and so is generally, but not necessarily, in-frame with the target nucleic and sequence. Only circular vector-probes carrying the marker and will be able to confer the marker characteristic, for example, confer antibiotics resistance or induce a chromogenic reaction (in the form of a coloured solution or coloured plaque to the transformed bacteria) .
  • One method of amplification is to carry out a plurality of cycles of hybridisation of the target sequence and the linearised vector-probe, repair of the resulting "nick" to circularise the vector-probe, dissociation of the target sequence and the circularised vector-probe and removal of the circularised vector-probe.
  • This may be achieved by denaturing and subsequent addition of ligase, and may be carried out particularly efficiently by using thermostable ligase.
  • a plurality of circular vector-probes are therefore produced per target sequence, which is thereby amplified.
  • the second way of amplifying the target sequence is indirectly by amplifying the vector-probe.
  • Both the vector- probe and the infected host cells may be expanded several times in an appropriate culture medium until a detectable signal is obtained. For example, at the end of the expansion, after centrifugation, there will be an accumulation of particles containing the single stranded circular vector-probe in the supernatant, and infected cells containing the double-helix circular vector-probe in the precipitate. If LB agar plates are used to expand the host cells, then visible and/or coloured plagues will be detected onto the agar medium.
  • Either of the two methods of amplification may be used alone, or both may be used to maximise the amplification.
  • the accumulation of the vector-probe or the gene product associated with the vector-probe can be used in further methods of detection.
  • the gene product is the detectable marker.
  • expansion can only be performed with a circularised vector-probe, the findings obtainable from the possible sources of detection will all be directly associated with the presence or absence of the target sequence in the original sample analyzed.
  • the method of the present invention may be used for either the detection or the determination of a target sequence, for example, the method may be used qualitatively, quantitatively or semi-quantitatively.
  • the method of the present invention can also be used to detect and/or identify mutations in a target nucleic acid sequence. If the position of a mutation in the target sequence corresponds to the 5' or 3 ' end of the complementary sequence in the vector-probe, circularisation ⁇ with a vector-probe comprising a nucleic acid sequence complementary to that of the unmutated version of the target sequence will not take place as hybridisation is not perfect. Circularisation will only take place if the vector-probe also contains the mutation. Accordingly, the use of a vector comprising a sequence complementary to a mutated target sequence enables detection of that mutated target sequence.
  • the sample under investigation may be, for example, any material that comprises the target nucleic acid either naturally or by contamination, for example, a sample of a human or animal body fluid or tissue for clinical diagnosis or forensic investigation or a sample of food that may be contaminated with pathogenic bacteria.
  • the target nucleic acid sequence may therefore be a sequence derived from a pathogenic organism or may relate to a genetic disorder or any other disease state, for example, an autoimmune disease. It may be a nucleic acid sequence for so-called "DNA fingerprinting" for forensic investigation.
  • the method of invention may be used in clinical diagnosis of both disease and carrier states when the disease results from a mutation especially from a point mutation, for example cystic fibrosis. If, for example, a sample gives a positive result with a vector-probe comprising a sequence complementary to the unmutated target sequence ("normal" vector) and gives a negative result with a vector-probe comprising a sequence complementary to the mutated target sequence ("mutated" vector) , that indicates that the patient from whom the sample was obtained is unaffected by the mutation. If a sample gives a positive result with both normal and unmutated vector-probe, that indicates that the patient is heterozygous for the mutation. A negative result with the normal vector-probe and a positive result for the mutated vector-probe indicates that the patient is homozygous for the mutation.
  • the present invention provides a kit which comprises a linear vector-probe according to the present invention, for example, as described above, and an enzyme capable of repairing a single-stranded break in a double-stranded nucleic acid structure, for example, a ligase.
  • the present invention further provides a kit which comprises (i) a linear vector-probe according to the present invention, for example, as described above, in which the complementary nucleic acid sequence is complementary to a target unmutated sequence and
  • the present invention has the advantages that short target sequences can be detected, and that mutations, especially point mutations, can be detected.
  • the ability to carry out repeated cycles of hybridication and ligation provide an effective amplification system for the detection of target sequences that occur at low levels.
  • the vector-probe used was derived from the Bluescript (Trade Mark) SK (+/-) phagemid (Stratagene Cloning Systems, 11099
  • the Bluescript SK (+/-) phagemid is a 2958 basepair phagemid derived from pUC19.
  • the SK designation indicates the polylinker is oriented such that lacZ transcription proceeds from Sac 1 to Kpm 1.
  • Figure 1 of the accompanying drawings is a map of the Bluescript phagemid : fl (+) origin (6-462 bp) fl filamentous phage origin of replication allowing recovery of the sense strand of the lacZ gene when a host strain containing the Bluescript phagemid is co-infected with a helper phage.
  • fl (-) origin (6-462 bp) fl filamentous phage origin of replication allowing recovery of the anti-sense strand of the lacZ gene when a host strain containing the Bluescript phagemid is co-infected with a helper phage.
  • ColEl origin (1032-1912 bp) Plasmid origin of replication used in the absence of helper phage.
  • LacZ (Lac promoter: 816-938 bp) This portion of the lacZ gene provides a complementation for blue/white color selection of recombinant phagemids.
  • An inducible lac promoter upstream from the lacZ gene permits fusion protein expression with the 3-galaclosidase gene product.
  • MCS (657-759 bp) Multiple cloning site flanked by T3 and T7 promoters.
  • Ampicillin (1975-2832 bp) A picillin resistance gene for antibiotic selection of the phagemid vector-probe.
  • Figure 2 of the accompanying drawings gives sequence ⁇ information on the Bluescript phagemid; various primers and restriction sites are indicated.
  • the upper strand is the (+) strand, the lower strand is the (-) strand.
  • the Hind II restriction site is marked.
  • the two arrows indicate the sequence used to produce the oligonucleotide "Oligo N" (see below) .
  • the Bluescript sequence has been deposited at GenBank under accession numbers 52325 (SK+) and 52324 (SK-) .
  • the (+) strand of the Bluescript phagemid was obtained in single stranded form using an M13 helper phage, giving a single stranded circular vector-probe DNA (2.96Kb).
  • the single strand circular vector-probe DNA (1.2 micrograms) , dissolved in water, was mixed in the presence or absence of a 33-mer oligonucleotide (12 micrograms), synthesized as a complementary sequence to the single strand circular vector-probe DNA in the region flanking the recognition sequence of the restriction enzyme Hind II, which generates blunt ends (see Figure 2).
  • the oligonucleotide (Oligo N) corresponds to the sequence from base 663 to base 695 of the negative strand of the Bluescript phagemid.
  • the 33-mer Oligo N has the following sequence (SEQ.ID. No.l) :
  • the annealing reaction was carried out in a final volume of 100 microlitres (25 mM Tris-HCl pH 7.5; 5mM MgCl 2 ; 5mM
  • the tubes in which the reaction was carried out were incubated at 65 ° C for 10 minutes, at 37 ° c for 30 minutes and then cooled at room temperature.
  • RECTIFIED SHEET (RULE 91 ) ISA/ ⁇ F (b) Ligation of linear vector-probe in the presence of target seguence After ethanol precipitation of the annealing reaction mixture (0.3 M NaAc pH 5.2; 75% EtOH absolute), the resulting single strand, linear vector-probe DNA functions as a pair of primers in a type of LCR (Ligase Chain Reaction).
  • the ligase enzyme used (Ampligase, obtainable from Epicenter Technologies, 1202 Ann Street, Madison, Wisconsin 53713, U.S.A.), can operate only when the primers are perfectly complementary to the template at the ligase site junction. Even a single base mismatch of the ends of the linear probe primersat the junction site makes the ligase inactive.
  • the target sequence is the 33-mer Oligo N described above.
  • the single strand linear vector-probe DNA (0.140 micrograms) obtained as described above was reacted with the 33-mer oligonucleotide of SEQ. ID.No.l described above (Oligo N; 0.96 pmol) or with another oligonucleotide (Oligo M; 0.96 p ol) , which had a single base mutated (G->T at position 15) with respect to Oligo N as shown in SEQ. ID.No.2 below:
  • reaction was also carried out in absence of both oligonucleotides.
  • reaction was carried out using single stranded circular DNA.
  • the ligation reactions were carried out in a final volume of 100 microlitres (20 mM Tris-HCl pH7.6 ; 25 mM KAc; lOmM MgAc; 10 mM DTT; 0.6 mM NAD and 0.1% Triton x-100) with 10U of Ampligase enzyme per reaction tube.
  • the reaction mixtures were incubated for 30 seconds at 94 ° C, then for 30 minutes at 45 C.
  • Single strand linear DNA (0.18 micrograms) prepared from Bluescript (Trade Mark) SK +/- phagemid according to Example 1 was reacted with Oligo N (SEQ.ID. No.l) described in Example 1, (0.96 pmoles) , or with Oligo M (SEQ.ID. No.2) as described in Example 1, (0.96 pmoles), or in absence of both oligonucleotides.
  • the reactions were carried out in a final volume of 100 ⁇ l (66mM Tris-HCl pH 7.5; 5 mM MgCl 2 1 mM DTT; 1 mM ATP) with 2 units/reaction mixture of T4 DNA ligase enzyme (Boeheringer) , which is not thermostable.
  • the reaction mixtures were incubated for 30 seconds at 94°C then overnight at 37'C.
  • reaction mixtures were then heated for 10 minutes at 65°C to denature the enzyme and, after butanol purification, the reaction products were resuspended in 10 ⁇ l water and boiled.
  • the reaction mixtures were then added to 50 ⁇ l of E. coli supercompetent cells (Stratagene) , incubated for 5 minutes in ice, for 3 minutes at 42 * C and then at room temperature for 5 minutes, to transform the bacteria.
  • the cells were incubated at 37°C for 30 minutes (with agitation) with 50 ⁇ l of LB medium and then spread on agar plates containing nutrients and ampicillin (100 ⁇ g/ml) ; the plates were incubated at 37°C overnight.
  • NAME RAGGIO-ITALGENE SpA (for all designated states except USA)
  • NAME MARTINAZZO Giorgio (for USA only)
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

Abstract

A method for the detection of a target nucleic acid sequence in a sample, which comprises: (i) contacting the sample, which has been treated to bring any nucleic acids present into single-stranded form, (a) with a linear single stranded vector-probe capable of transforming competent bacteria when in circular form, said vector-probe comprising a nucleic acid sequence complementary to the target nucleic acid sequence, said nucleic acid sequence being present in two parts, one part of said nucleic acid being present at the 5'-end of the vector-probe, the other part being present at the 3'-end, said vector-probe also comprising a region encoding a selectable or detectable marker, under conditions that permit hybridisation of any target sequence present in the sample to the complementary sequence, and (b) with an enzyme capable of repairing a single-stranded break in a double-stranded nucleic acid structure; (ii) inactivating the repair enzyme; (iii) separating any resulting circularized single stranded linear vector-probe from the annealed target; (iv) contacting the circularized single stranded linear vector-probe, optionally in the form of the reaction mixture resulting from step (ii), with competent host bacteria that lack the marker present in the vector-probe under transformation conditions; (v) culturing the resulting transformed bacteria and investigating the presence of the marker; and a vector for use in the method. The method may also be used for the detection and/or identification of mutations in a target nucleic acid sequence.

Description

METHOD AND REAGENTS FOR THE DETECTION OF TARGET NUCLEIC ACID SEQUENCES
The present invention relates to a method and reagents for the detection and, optionally, amplification of a target nucleic acid sequence.
A target nucleic acid sequence may be any sequence of interest, for example, it may be a sequence present in a pathogenic organism and may be of interest for clinical diagnosis or in the context of food hygiene and public health. Other fields where detection of target nucleic acid sequences are of interest include forensic science.
WO85/04663 describes a process for detecting a specific (target) nucleotide sequence DNA probe molecule is capable of transforming bacteria if and only if it is held in a circular configuration by base pairing to the target nucleotide sequence, said transformation resulting in the detection of a phenotype specified by the DNA probe molecule. Detection of the phenotype establishes the presence, absence or quantity of the target.
That method, however, has the disadvantages that long sequences of target DNA are required to maintain the circular configuration; short sequences may not be detected. Furthermore the method does not allow discrimination between very similar sequences in such a way that small base variations, especially single point mutations, in a sequence can be discriminated. This will prevent, for example, the identification of a wild type from a mutated sequence of interest for clinical diagnosis. After the cicularization with the target sequence the transformation is carried out without separating the target from the probe, thus increasing the risk of non-specific binding (increased background) reducing both the specificity and the sensitivity of the procedure. In addition the circularization reaction cannot be repeated, thus preventing the possibility of amplification of target sequences occurring at low level.
The present invention provides a single stranded linearisable circular vector-probe, said vector-probe comprising a nucleic acid sequence complementary to a target nucleic acid sequence, said complementary nucleic acid sequence preferably being present in a "polylinker" zone of the vector-probe, and said complementary nucleic acid sequence being in reading frame with a region of the vector- probe coding for a selectable or detectable marker, said vector-probe being linearisable by cleavage of a phosphodiester bond in the nucleic acid sequence complementary to the target sequence. Said vector-probe in single stranded linear form that is to say, having been linearised by cleavage of a phosphodiester bond in the complementary sequence, or having appropriate parts of the complemetary sequence added to a cleaved circular vector not previously comprising the complementary sequence, is also part of the present invention.
The present invention also provides a method for the detection of a target nucleic acid sequence in a sample, which comprises
(i) contacting the sample, which has been treated to bring any nucleic acids present into single-stranded form, (a) with a linear single stranded vector-probe capable of transforming competent bacteria when in circular form, said vector-probe comprising a nucleic acid sequence complementary to the target nucleic acid sequence, said nucleic acid sequence being present in two parts, one part of said nucleic acid being present at the 5'-end of the vector-probe, the other part being present at the 3 '-end, said vector-probe also comprising a region encoding a selectable or detectable marker, under conditions that permit hybridisation of any target sequence present in the sample to the complementary sequence, and (b) with an enzyme capable of repairing a single-stranded break in a double-stranded nucleic acid structure; (ii) inactivating the repair enzyme;
(iii) separating the resulting circularized single stranded linear vector-probe from the annealed target; (iv) contacting the circularized single stranded linear vector-probe, optionally in the form of the reaction mixture resulting from step (ii) , with competent host bacteria that lack the marker present in the vector-probe under transformation conditions; and
(v) culturing the resulting transformed bacteria and investigating the presence of the marker.
The vector-probe is obtainable by cleavage of a single stranded circular vector-probe that comprises a nucleic acid sequence complementary to the target sequence, the cleavage site being within the complementary sequence, preferably at a phosphodiester bond. The linear vector probe is also obtainable by cleaving a circular single stranded probe, adding to 5 '-end one part of the nucleic acid sequence complementary to the target sequence and adding to the 3'- end the other part of the complementary sequence.
The method of the present invention involves the detection of target nucleic acid sequences, for example, genomic sequences, by hybridisation with a linear single stranded vector-probe comprising a cleaved nucleic acid sequence complementary to the target nucleic acid sequence. The linear vector-probe may be obtained from a circular vector- probe comprising a sequence complementary to the target sequence by cleavage in the complementary sequence. Alternatively, a circular vector that does not already comprise the complementary sequence may be linearised then one part of the complementary sequence added to the 5'-end and the other part added to the 3 '-end.
If present in a sample under investigation, the target sequence will hybridise to the linear vector-probe, forming a double stranded region with a "nick" in the vector-probe strand. That single stranded "nick" in the vector-probe can then be repaired using an appropriate enzyme. When the resulting circularized single stranded vector-probe is used to transform competent hosts that lack the marker present in the vector-probe, the presence of the marker in the resulting transformed bacteria indicates the presence of target nucleic acid sequence in the sample under investigation.
Single stranded circular vectors are well known and are generally derived from filamentous phage. Examples are M13mp derivatives, for example, as described by Messing J, "New M13 vectors for cloning" Meth. Enzymol. (1983) 101:20- 78; Deute L, Cesareni G & Cortese R, "pEMBL A new family of single-stranded plasmids" Nuc. Acids Res. (1983) 11:1645- 1655; Messing J, Groenborn B, Muller-Hill B & HofSchneider PH, "Single strand filamentous DNA phage as carrier for in vitro recombined DNA" Proc. Natl. Acad. Sci. U.S.A. (1977) 74:3642-3646. An example of an M13mp derivative is M13mpl8. An M13mpl8/pUC polylinker is described by Yonisch-Petron C, Vieire J & Messing J (1985) "Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mpl8 and pUC19 vector-probes" Gene 33:109-119. A commercially available circular vector is Bluescript (Trade Mark) SK +/- phagemid (Stratagene) .
Figure 1 of the accompanying drawings gives a map of the Bluescript phagemid showing various functional features and various restriction sites. Figure 2 gives sequence information relating to the Bluescript phagemid. Various primers are shown, as are restriction sites.
An essential characteristic of a circular single-stranded vector-probe used according to the present invention is that it can be linearised, preferably in the central zone of the sequence complementary to the target sequence, by cleavage of a phosphodiester bond. Cleavage may be effected using an appropriate enzyme, for example, a restriction enzyme. Suitable enzymes are well known.
As indicated above, although the linear vector-probe may be obtained by cleavage of the complementary sequence already present in a circular vector, a linear vector-probe may be prepared by other methods. For example, a circular vector may be cleaved and one part of the desired complementary sequence added to the 5'-end and the other part added to the 3'-end. Any linear vector that can form a circular single stranded vector capable of transforming competent bacteria may be modified by the addition of a complementary sequence.
References in the present specification to "linear" and "linearised" vector-probes include all linear vector-probes that may be used according to the present invention, regardless of the method of their production.
In addition to the ability to form a circular single stranded vector that is capable of transforming competent bacteria, the linear vector-probe should generally have high efficiency, a strong promoter and must be functional only when in circular form. It should also have a selectable or detectable marker. A linear vector-probe according to the invention has two termini, one of the 3 '-hydroxyl type and the other of the 5 '-phosphate type. Accordingly, if a sample under investigation that may comprise the target nucleic acid sequence (which sample has been treated to bring any nucleic acids present into single-stranded form) , for example, an extract of denatured DNA, is exposed to a predetermined amount, for example, a known surplus, of the vector-probe of the present invention and to the action of a modification enzyme that repairs single-stranded breaks ("nicks") in a double-stranded nucleic acid molecule, generally from the ligase family, the only vector-probes to be circularised will be those which perfectly hybridise both their ends with the target sequence.
A characteristic feature of the repair enzyme used is that it requires as substrate two single-stranded fragments of nucleic acid, the 5' terminal of one having a phosphate group and the 3' terminal of the other having a hydroxyl group. Both fragments must be complementary, vicinal and contiguous to a second strand whose length is intact, and take the form of a "duplex" which presents a nick (ie. a hydrolysed phosphodiester bond) in one of the two strands.
This discriminating condition corresponds to that found when the linear vector-probe is hybridised with the single- stranded complementary target sequence: only under these circumstances will the enzyme, generally a ligase, be active and reconstitute the phosphodiester bond, thereby circularising the vector-probe.
As the enzyme is inefficient in ligating single stranded DNA fragments with blunt ends, it will not circularise the linear vector-probe unless the latter has perfectly hybridised the two ends complementary to the target sequence in a contiguous, vicinal manner. If any mismatch is present at and/or near the 5' end or at and/or near the 3 ' end of the linear vector-probe, the enzyme will not be able to close the nick and to circularize the vector-probe.
Each circularised vector-probe will therefore correspond to the presence of a target nucleic acid sequence in the sample analyzed.
E. coli ligase or any other DNA ligase may be used when the target sequence is DNA. If the target sequence is RNA either an appropriate RNA ligase may be chosen or DNA complementary to the RNA may be produced and then subjected to the method of the invention.
The ligase may be a thermostable or thermolabile ligase. A thermostable ligase is particularly useful if repeated cycles of ligation are desired. A thermolabile ligase provides a simple method for denaturating the enzyme when the desired ligation reaction is complete.
In a circular single stranded vector comprising a complementary sequence, it is preferable to have a cleavage site in the nucleic acid sequence complementary to the target sequence, especially in the central region of that sequence. However, it is also possible to design a circular single stranded vector that is linearisable in another region of the vector, for example, at one or both ends of the complementary sequence to give a linear vector-probe according to the present invention. Such circular vectors and linear vector-probes and their use are also part of the present invention. Furthermore, although it is preferable to cleave a circular single stranded vector at a phosphodiester bond, it is possible to design vectors having cleavage sites at other bonds, for example, using specially-designed polylinker sequences. Such circular vectors, the resulting linear vector-probes, and their use are all part of the present invention.
The second part of the method of the present invention involves replicating the now-circularised single stranded vector-probe so as to produce an easily detectable signal which can be correlated with the presence or absence of the target sequence, and optionally with its quantification.
The repair enzyme is generally inactivated and/or, if desired, the resulting circularised vector-probe may be isolated from the reaction mixture. The circularised vector and the annealed target sequence should be separated from one another. However, it is not generally necessary to isolate the vector-probe, and the inactivated, optionally denatured hybridisation mixture may be admixed as such under conditions suitable for transformation with a medium containing bacterial host cells which are competent but lack a gene fragment (present in the vector-probe) encoding the marker of choice. Many suitable markers are known in the art, for example, antibiotics-resistance markers, for example, ampicillin resistance gene.
A further type of marker that may be used in the present invention is one whose gene product is needed by the cell for the chromogenic catalysis of a suitable substrate, or a gene whose product is a substance characteristic of the vector-probe. The marker gene should be expressible and so is generally, but not necessarily, in-frame with the target nucleic and sequence. Only circular vector-probes carrying the marker and will be able to confer the marker characteristic, for example, confer antibiotics resistance or induce a chromogenic reaction (in the form of a coloured solution or coloured plaque to the transformed bacteria) .
It is well known that one of the basic problems in detecting target nucleic acid sequences is that the target sequence is often present in very small amounts in the sample under investigation, and that it is correspondingly difficult to detect. Using the method of the present invention, there are two potential means for amplifying the target sequence, which means may be used individually or in combination.
One method of amplification is to carry out a plurality of cycles of hybridisation of the target sequence and the linearised vector-probe, repair of the resulting "nick" to circularise the vector-probe, dissociation of the target sequence and the circularised vector-probe and removal of the circularised vector-probe. This may be achieved by denaturing and subsequent addition of ligase, and may be carried out particularly efficiently by using thermostable ligase. A plurality of circular vector-probes are therefore produced per target sequence, which is thereby amplified.
The second way of amplifying the target sequence is indirectly by amplifying the vector-probe. Both the vector- probe and the infected host cells may be expanded several times in an appropriate culture medium until a detectable signal is obtained. For example, at the end of the expansion, after centrifugation, there will be an accumulation of particles containing the single stranded circular vector-probe in the supernatant, and infected cells containing the double-helix circular vector-probe in the precipitate. If LB agar plates are used to expand the host cells, then visible and/or coloured plagues will be detected onto the agar medium.
Either of the two methods of amplification may be used alone, or both may be used to maximise the amplification.
The accumulation of the vector-probe or the gene product associated with the vector-probe (capsidic proteins, etc) can be used in further methods of detection. In such a case the gene product is the detectable marker. As expansion can only be performed with a circularised vector-probe, the findings obtainable from the possible sources of detection will all be directly associated with the presence or absence of the target sequence in the original sample analyzed.
The method of the present invention may be used for either the detection or the determination of a target sequence, for example, the method may be used qualitatively, quantitatively or semi-quantitatively.
The method of the present invention can also be used to detect and/or identify mutations in a target nucleic acid sequence. If the position of a mutation in the target sequence corresponds to the 5' or 3 ' end of the complementary sequence in the vector-probe, circularisation ■with a vector-probe comprising a nucleic acid sequence complementary to that of the unmutated version of the target sequence will not take place as hybridisation is not perfect. Circularisation will only take place if the vector-probe also contains the mutation. Accordingly, the use of a vector comprising a sequence complementary to a mutated target sequence enables detection of that mutated target sequence. The sample under investigation may be, for example, any material that comprises the target nucleic acid either naturally or by contamination, for example, a sample of a human or animal body fluid or tissue for clinical diagnosis or forensic investigation or a sample of food that may be contaminated with pathogenic bacteria.
The target nucleic acid sequence may therefore be a sequence derived from a pathogenic organism or may relate to a genetic disorder or any other disease state, for example, an autoimmune disease. It may be a nucleic acid sequence for so-called "DNA fingerprinting" for forensic investigation.
The method of invention may be used in clinical diagnosis of both disease and carrier states when the disease results from a mutation especially from a point mutation, for example cystic fibrosis. If, for example, a sample gives a positive result with a vector-probe comprising a sequence complementary to the unmutated target sequence ("normal" vector) and gives a negative result with a vector-probe comprising a sequence complementary to the mutated target sequence ("mutated" vector) , that indicates that the patient from whom the sample was obtained is unaffected by the mutation. If a sample gives a positive result with both normal and unmutated vector-probe, that indicates that the patient is heterozygous for the mutation. A negative result with the normal vector-probe and a positive result for the mutated vector-probe indicates that the patient is homozygous for the mutation.
The present invention provides a kit which comprises a linear vector-probe according to the present invention, for example, as described above, and an enzyme capable of repairing a single-stranded break in a double-stranded nucleic acid structure, for example, a ligase. The present invention further provides a kit which comprises (i) a linear vector-probe according to the present invention, for example, as described above, in which the complementary nucleic acid sequence is complementary to a target unmutated sequence and
(ii) a linear vector-probe according to the present invention, for example, as described above, in which the complementary nucleic acid sequence is complementary to a mutated form of the target sequence.
The present invention has the advantages that short target sequences can be detected, and that mutations, especially point mutations, can be detected. The ability to carry out repeated cycles of hybridication and ligation provide an effective amplification system for the detection of target sequences that occur at low levels.
The following non-limiting Examples illustrate the invention:
EXAMPLE
(a) Generation of single strand linearised vector-probe
The vector-probe used was derived from the Bluescript (Trade Mark) SK (+/-) phagemid (Stratagene Cloning Systems, 11099
North Torray Pines Road, La Jolla, California 92037,
U.S.A. ) .
The Bluescript SK (+/-) phagemid is a 2958 basepair phagemid derived from pUC19. The SK designation indicates the polylinker is oriented such that lacZ transcription proceeds from Sac 1 to Kpm 1.
Figure 1 of the accompanying drawings is a map of the Bluescript phagemid : fl (+) origin (6-462 bp) fl filamentous phage origin of replication allowing recovery of the sense strand of the lacZ gene when a host strain containing the Bluescript phagemid is co-infected with a helper phage.
fl (-) origin (6-462 bp) fl filamentous phage origin of replication allowing recovery of the anti-sense strand of the lacZ gene when a host strain containing the Bluescript phagemid is co-infected with a helper phage.
ColEl origin (1032-1912 bp) Plasmid origin of replication used in the absence of helper phage.
LacZ (Lac promoter: 816-938 bp) This portion of the lacZ gene provides a complementation for blue/white color selection of recombinant phagemids. An inducible lac promoter upstream from the lacZ gene permits fusion protein expression with the 3-galaclosidase gene product.
MCS: (657-759 bp) Multiple cloning site flanked by T3 and T7 promoters.
Ampicillin: (1975-2832 bp) A picillin resistance gene for antibiotic selection of the phagemid vector-probe.
Figure 2 of the accompanying drawings gives sequence ■information on the Bluescript phagemid; various primers and restriction sites are indicated. The upper strand is the (+) strand, the lower strand is the (-) strand. The Hind II restriction site is marked. The two arrows indicate the sequence used to produce the oligonucleotide "Oligo N" (see below) .
The Bluescript sequence has been deposited at GenBank under accession numbers 52325 (SK+) and 52324 (SK-) . The (+) strand of the Bluescript phagemid was obtained in single stranded form using an M13 helper phage, giving a single stranded circular vector-probe DNA (2.96Kb).
The single strand circular vector-probe DNA (1.2 micrograms) , dissolved in water, was mixed in the presence or absence of a 33-mer oligonucleotide (12 micrograms), synthesized as a complementary sequence to the single strand circular vector-probe DNA in the region flanking the recognition sequence of the restriction enzyme Hind II, which generates blunt ends (see Figure 2). The oligonucleotide (Oligo N) corresponds to the sequence from base 663 to base 695 of the negative strand of the Bluescript phagemid. The 33-mer Oligo N has the following sequence (SEQ.ID. No.l) :
CCC CCC TCG AGG TCG ACG GTA TCG ATA AGC TTG (SEQ. ID. o.1)
In this way a partial double strand region of DNA was created. (Restriction enzymes recognise single strand DNA only with very great difficulty) .
The annealing reaction was carried out in a final volume of 100 microlitres (25 mM Tris-HCl pH 7.5; 5mM MgCl2; 5mM
NaCl) : the tubes in which the reaction was carried out were incubated at 65°C for 10 minutes, at 37°c for 30 minutes and then cooled at room temperature.
After the annealing reaction, 20 units of the restriction enzyme Hind II were added per reaction tube and the tubes were incubated overnight at 37 C. As shown by analysis on an agarose gel (0.7% in TEBlx: 0.09M Tris Dorate, 0.002M EDTA) , only those react ons performed in presence of the complementary 33-mer oligonucleotide resulted in single strand linear DNA.
RECTIFIED SHEET (RULE 91 ) ISA/ΞF (b) Ligation of linear vector-probe in the presence of target seguence After ethanol precipitation of the annealing reaction mixture (0.3 M NaAc pH 5.2; 75% EtOH absolute), the resulting single strand, linear vector-probe DNA functions as a pair of primers in a type of LCR (Ligase Chain Reaction). The ligase enzyme used (Ampligase, obtainable from Epicenter Technologies, 1202 Ann Street, Madison, Wisconsin 53713, U.S.A.), can operate only when the primers are perfectly complementary to the template at the ligase site junction. Even a single base mismatch of the ends of the linear probe primersat the junction site makes the ligase inactive. The target sequence is the 33-mer Oligo N described above.
The single strand linear vector-probe DNA (0.140 micrograms) obtained as described above was reacted with the 33-mer oligonucleotide of SEQ. ID.No.l described above (Oligo N; 0.96 pmol) or with another oligonucleotide (Oligo M; 0.96 p ol) , which had a single base mutated (G->T at position 15) with respect to Oligo N as shown in SEQ. ID.No.2 below:
CCC CCC TCG AGG TCT ACG GTA TCG ATA AGC TTG (SEQ. ID.No.2)
As a negative control, the reaction was also carried out in absence of both oligonucleotides. As a positive control, the reaction was carried out using single stranded circular DNA.
The ligation reactions were carried out in a final volume of 100 microlitres (20 mM Tris-HCl pH7.6 ; 25 mM KAc; lOmM MgAc; 10 mM DTT; 0.6 mM NAD and 0.1% Triton x-100) with 10U of Ampligase enzyme per reaction tube. The reaction mixtures were incubated for 30 seconds at 94 °C, then for 30 minutes at 45 C.
RECTIFIED SHEET (RULE 91) ISA/EP (c) Transformation of competent bacteria and detection of transfor ants Each ligation reaction mixture was purified with butanol, re-suspended in 10 microlitres of water and boiled, then was added to 50 microlitres of E. coli XIl-Blue supercompetent cells (Stratagene) , incubated for 5 minutes in ice, for 3 minutes at 42 °C and then at room temperature for 5 minutes. After the transformation the cells were incubated at 37 C for 30 minutes with 50 microlitres of LB medium and then spread on agar plates containing nutrients and ampicillin (100 micrograms/ml) . The plates were incubated overnight at 37°C.
The reaction performed in presence of Oligo N (SEQ.ID. o.1) gave 102 ampicillin resistant colonies i.e. 1 x 103 transformants per microgram of linear single strand DNA vector.
In contrast, the reactions performed in presence of Oligo M (SEQ.ID.No.2) or in the absence of any oligomer gave no colonies.
When the transformation was carried out using a single strand circular DNA as positive control, 3700 ampicillin resistance colonies i.e. 6 x 104 transformants per microgram of DNA were obtained.
The results show that the method of the present invention may be used to detect a target sequence and also to detect mutations, in particular a point mutation, in a target sequence. EXAMPLE 2
Single strand linear DNA (0.18 micrograms) prepared from Bluescript (Trade Mark) SK +/- phagemid according to Example 1 was reacted with Oligo N (SEQ.ID. No.l) described in Example 1, (0.96 pmoles) , or with Oligo M (SEQ.ID. No.2) as described in Example 1, (0.96 pmoles), or in absence of both oligonucleotides. The reactions were carried out in a final volume of 100 μl (66mM Tris-HCl pH 7.5; 5 mM MgCl2 1 mM DTT; 1 mM ATP) with 2 units/reaction mixture of T4 DNA ligase enzyme (Boeheringer) , which is not thermostable. The reaction mixtures were incubated for 30 seconds at 94°C then overnight at 37'C.
The reaction mixtures were then heated for 10 minutes at 65°C to denature the enzyme and, after butanol purification, the reaction products were resuspended in 10 μl water and boiled. The reaction mixtures were then added to 50 μl of E. coli supercompetent cells (Stratagene) , incubated for 5 minutes in ice, for 3 minutes at 42*C and then at room temperature for 5 minutes, to transform the bacteria.
After transformation, the cells were incubated at 37°C for 30 minutes (with agitation) with 50 μl of LB medium and then spread on agar plates containing nutrients and ampicillin (100 μg/ml) ; the plates were incubated at 37°C overnight.
The reaction performed in presence of Oligo N (SEQ. ID. No.l) gave 310 colonies/180 ng of linear probe (1.7 x 103 transformant/μg of the linear single strand DNA probe) .
The reaction performed in presence of Oligo M (SEQ. ID. No.2) or in absence of oligonucleotides gave no colonies. These results show that the method of the present invention may be used to detect a target sequence and also to detect mutations, in particular a point mutation, in a target sequence.
SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT:
(A) NAME: RAGGIO-ITALGENE SpA (for all designated states except USA)
(B) STREET: Via delle Antille 29
(C) CITY: Pomezia (RM)
(E) COUNTRY: Italy
(F) POSTAL CODE (ZIP) : 00040
(A) NAME: MARCOLINI Stanislao (for USA only)
(B) STREET: c/o RAGGIO-ITAGENE SpA
(C) CITY: Via delle Antille 29
(D) STATE: Pomezia (RM)
(E) COUNTRY: Italy (F) POSTAL CODE (ZIP) : 00040
(A) NAME: MARTINAZZO Giorgio (for USA only)
(B) STREET: c/o RAGGIO-ITALGENE SpA
(C) CITY: Via delle Antille 29
(D) STATE: Pomezia (RM) (E) COUNTRY: Italy
(F) POSTAL CODE (ZIP) : 00040
(ii) TITLE OF INVENTION: METHOD AND REAGENTS FOR THE DETECTION OF TARGET NUCLEIC ACID MOLECULES
(iii) NUMBER OF SEQUENCES: 2
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version £1.25 (EPO) (vi ) PRIOR APPLICATION DATA :
( A ) APPLICATION NUMBER : GB 94 14934 . 1
( B ) FI LING DATE : 25 -JUL- 1994
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
CCCCCCTCGA GGTCGACGGT ATCGATAAGC TTG 33
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: CCCCCCTCGA GGTCTACGGT ATCGATAAGC TTG 33

Claims

CLAIMS :
1. A method for the detection of a target nucleic acid sequence in a sample, which comprises:
(i) contacting the sample, which has been treated to bring any nucleic acids present into single-stranded form,
(a) with a linear single stranded vector-probe capable of transforming competent bacteria when in circular form, said vector-probe comprising a nucleic acid sequence complementary to the target nucleic acid sequence, said nucleic acid sequence being present in two parts, one part of said nucleic acid being present at the 5 '-end of the vector-probe, the other part being present at the 3'- end, said vector-probe also comprising a region encoding a selectable or detectable marker, under conditions that permit hybridisation of any target sequence present in the sample to the complementary sequence, and
(b) with an enzyme capable of repairing a single-stranded break in a double-stranded nucleic acid structure;
(ii) inactivating the repair enzyme;
(iii) separating any resulting circularized single stranded linear vector-probe from the annealed target;
(iv) contacting the circularized single stranded linear vector-probe, optionally in the form of the reaction mixture resulting from step (ii) , with competent host bacteria that lack the marker present in the vector-probe under transformation conditions;
(v) culturing the resulting transformed bacteria and investigating the presence of the marker.
2. A method as claimed in claim 1, wherein the vector- probe is obtainable by cleavage of a single stranded circular vector-probe that comprises a nucleic acid sequence complementary to the target sequence, the cleavage site being within the complementary sequence.
3. A method as claimed in claim 2, wherein the cleavage is at a phosphodiester bond.
4. A method as claimed in claim 1, wherein the linear vector probe is obtainable by cleaving a circular single stranded probe, adding to 5 '-end one part of the nucleic acid sequence complementary to the target sequence and adding to the 3 '-end the other part of the complementary sequence.
5. A method as claimed in any one of claim 1 to 4 , wherein the marker is an antibiotics resistance gene, a gene whose product catalyses a chromogenic reaction, or a gene whose product is a substance characteristic of the vector-probe.
6. A method as claimed in any one of claims 1 to 5, wherein the linear vector-probe is derived from vector M13mp or from a derivative thereof.
7. A method as claimed in claim 6, wherein the vector- probe is derived from vector M13pml8 or from vector pUC19.
8. A method as claimed in any one of claims 1 to 7 , wherein the target nucleic acid sequence is associated with a pathogenic organism, with a genetic disorder, or autoimmune disease, or is a sequence under forensic investigation.
9. A method as claimed in any one of claims 1 to 8, wherein the target nucleic acid sequence is a mutated sequence.
10. A method as claimed in any one of claims 1 to 9, wherein step (i) is repeated one or more times using the same sample and further amounts of vector-probe.
11. A method as claimed in any one of claims 1 to 10, wherein the culture of the host bacteria is repeated one or more times.
12. A method as claimed in any one of claims 1 to 11, wherein the target nucleic acid sequence is a mutated sequence and the vector-probe comprises a nucleic acid sequence complementary to the mutated target nucleic acid sequence.
13. A method as claimed in claim 12, wherein the mutation is a point mutation.
14. A kit which comprises a vector-probe as defined in any one of claims 1 to 7 and an enzyme capable of repairing a single-stranded break in a double-stranded nucleic acid structure.
15. A kit which comprises
(i) a vector-probe as defined in claim 1 in which the complementary nucleic acid sequence is complementary to a target unmutated sequence and
(ii) a vector-probe as defined in claim 1 in which the complementary nucleic acid sequence is complementary to a mutated form of the target sequence.
16. A vector-probe as defined in any one of claims 1 to
7.
17. A vector-probe as claimed in claim 16, in circular form.
PCT/EP1995/002944 1994-07-25 1995-07-25 Method and reagents for the detection of target nucleic acid sequences WO1996003525A1 (en)

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