EP0686200A1 - Amplification de sequences d'acides nucleiques - Google Patents

Amplification de sequences d'acides nucleiques

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Publication number
EP0686200A1
EP0686200A1 EP94907278A EP94907278A EP0686200A1 EP 0686200 A1 EP0686200 A1 EP 0686200A1 EP 94907278 A EP94907278 A EP 94907278A EP 94907278 A EP94907278 A EP 94907278A EP 0686200 A1 EP0686200 A1 EP 0686200A1
Authority
EP
European Patent Office
Prior art keywords
primer
nucleic acid
target nucleic
acid sequence
primers
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP94907278A
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German (de)
English (en)
Other versions
EP0686200A4 (fr
Inventor
Satish K. Bhatnagar
Albert L. George, Jr.
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Oncor Inc
Original Assignee
Oncor Inc
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Filing date
Publication date
Application filed by Oncor Inc filed Critical Oncor Inc
Publication of EP0686200A1 publication Critical patent/EP0686200A1/fr
Publication of EP0686200A4 publication Critical patent/EP0686200A4/fr
Withdrawn legal-status Critical Current

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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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • 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
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • 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
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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]

Definitions

  • the present invention relates to a process for amplifying nucleic acid sequences. More specifically, it relates to an improved process for producing nucleic acid sequences from a DNA or RNA template which may be purified, or may exist in a mixture of nucleic acids. The resulting nucleic acid sequences may be exact copies of the template, or may be modified.
  • the primers are extended from the 3 ' end in a 5' - ⁇ 3' direction by a DNA polymerase which incorporates free nucleotides into a nucleic acid sequence complementary to each strand of the target nucleic acid. After dissociation of the extension products from the target nucleic acid strands, the extension products become target sequences for the next cycle. In order to obtain satisfactory amounts of the amplified DNA, repeated cycles must be carried out. between which cycles, the complementary DNA strands must be denatured under elevated temperatures.
  • thermostable DNA polymerases have been discovered and isolated from thermophilic organisms such as Thermu ⁇ aquaticus. Such thermostable polymerases make it possible to add enzyme at the beginning of a series of synthesis and denaturation steps, without the need to add a new aliquot of enzyme after each denaturation step.
  • a potential problem associated with PCR is the hybridization of a primer sequence to regions of the DNA molecule not intended to be amplified. Generally these undesired hybridizations occur because the target sample contains, in addition to the target sequence itself, other sequences with some complementarity to the primer sequences.
  • primer extension may be successfully initiated by the polymerase enzyme, leading to the generation of an extension product different from the desired target sequence. Under some circumstances, this extension product will undergo exponential amplification, and be erroneously thought- to be the desired target sequence.
  • LCR ligase chain reaction
  • the probes Upon hybridization, the probes are ligated to form detectable fused probes complementary to the original target nucleic acid.
  • the fused probes are disassociated from the nucleic acid and serve as a template for further hybridizations and fusions of, the probes, thus amplifying geometrically the nucleic acid to be detected.
  • the method does not use DNA polymerase.
  • LCR has disadvantages due to the need for at least four separate oligonucleotide probes for amplification. It also requires that the entire sequence of the target nucleic acid be known. Further, background signal can be caused by target independent ligation of the probes. Since the third probe hybridizes to the first probe and the fourth probe hybridizes to the second probe, the probes, when added in excess, can easily form duplexes among themselves which can be ligated independently of the target nucleic acid.
  • European Application No. 0 439 182 which is incorporated herein in its entirety by reference discloses a method of improving LCR amplification by providing probes/primers which hybridize to the target nucleic acid wherein one end is modified such that ligation cannot occur until the modified end is corrected.
  • One such modification is the placement of a small gap between the probes preventing ligation of the probes.
  • the gap sequence of the target nucleic acid must be selected such that the DNA sequence is comprised of three or less different nucleotides from the four possible nucleotides.
  • the fourth nucleotide must be the first base complementary to the 5' end of the adjacent probe.
  • the gap is then filled using a DNA polymerase or reverse transcriptase to extend one or more of the probes in a 5' to 3 ' direction in a target dependent manner to render the probes ligatable.
  • the reaction mixture omits the fourth deoxynucleoside triphosphate complementary to the base at the 5' end of the adjacent probe. Because this method requires that the gap chosen in the target nucleic acid only contains bases which are complementary to a maximum of three of the deoxynucleoside triphosphates, the method limits the location of the gap on the target nucleic acid and also limits the size of the gap. Further, the method requires four primers.
  • the application also discloses a method of PCR amplification wherein one end of the primer is modified such that the primer is not extendable by a polymerase enzyme. When this modification is removed in a template dependent manner, the primer can be extended. However, this type of PCR requires an additional step of removal of the modification before extension can occur.
  • the present invention is based on the discovery that certain aspects of LCR and PCR can be used in combination to detect and amplify a target nucleic acid sequence with increased fidelity. Accordingly, in one of its process aspects, the present invention relates to a process for amplifying enzymatically a target nucleic acid sequence - contained in a nucleic acid or a mixture of nucleic acids, comprising the steps of: a. selecting the target nucleic acid sequence; b.
  • primers comprising a first primer which is substantially complementary to a first segment at a first end of the target nucleic acid sequence and a second primer which is substantially complementary to a second segment at a second end of the" target nucleic acid sequence and whose 3' end is adjacent to the 5'end of the first primer and a third primer which is similar to the first end of the target nucleic acid sequence and which is substantially complementary to at least a portion of said first primer; c. providing at least four different nucleotide bases; d. hybridizing said first and second primers to the target nucleic acid sequence in a target dependent manner to form a primer-target complex; e.
  • the present invention relates to a process for detecting enzymatically a point mutation or allele of ⁇ a target nucleic acid sequence - contained in a nucleic acid or a mixture of nucleic acids using the method disclosed above.
  • One of said primers is comprised of a number of similar oligonucleotide sequences. one of which is exactly complementary to the possible allele or point mutation and each of which oligonucleotides is labeled with a different label.
  • the allele is determined by detecting which labeled oligonucleotide is contained within the resulting amplification products.
  • the present invention relates to a process for amplifying enzymatically a target nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids comprising the steps of: a. selecting the target nucleic acid sequence; b. providing primers, said primers comprising a first primer which is substantially complementary to a first segment at a first end of the target nucleic acid sequence and a second primer which is substantially complementary to a second segment at a second end of the target nucleic acid sequence said second segment being spaced from said first segment and a third primer which is similar to the first end of the target nucleic acid sequence and which is substantially complementary to a portion of said first primer; c.
  • nucleic acid sequence providing at least four different nucleotide bases; d. hybridizing said first and second primers to the target nucleic acid sequence in a target dependent manner to form a primer-target complex; e. ( extending the 3 ' end of the second primer in the presence of the nucleotide bases under conditions such that an extended second primer is formed wherein the 3 ' end of the extended second primer.terminates at a base adjacent to the 5" end of the first primer; f. ligating the ends of the first and second extended primers under conditions such that said first and said extended second primers will form a fused amplification product substantially complementary to said target nucleic acid sequence; g. dissociating said fused amplification product from said target nucleic acid sequence; h.
  • the present invention relates to a kit for amplifying a target nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids comprising: first, second, and third primers and optionally a fourth primer; a ligating enzyme; a polymerizing enzyme; and at least four nucleotides.
  • Figure 1 depicts one embodiment of the method of DNA amplification/detection as set forth herein.
  • Figure 2 is a printout from a Phosphor Imager of a scanned acrylamide gel. The arrow indicates the resulting higher molecular weight amplification products.
  • Figure 3 depicts another embodiment of the method of DNA amplification/detection as set forth herein.
  • Figure 4 shows a portion of the sequence of the multidrug resistance gene (MDR-1) (SEQ ID NO:l).
  • Figures 5-11 are printouts from a Phosphor Imager of a scanned acrylamide gel which show amplification achieved with various embodiments of the present invention.
  • Figure 12 depicts another embodiment of the method of DNA amplification/detection as set forth herein.
  • target nucleic acid or “target nucleic acid sequence” suitable for use in the present invention may be taken from prokaryotic or eukaryotic DNA or RNA, including from plants, animals, insects, microorganisms, etc., and it may be isolated or present in samples which contain nucleic acid sequences in addition to the target nucleic acid sequence to be amplified.
  • the target nucleic acid sequence may be located on a nucleic acid strand which is longer than the target nucleic acid sequence. In this case, the ends of the target nucleic acid sequence are the boundaries with the unselected nucleic acid sequence and the target nucleic acid sequence.
  • the target nucleic acid sample may be obtained synthetically, or can be isolated from any organism by methods well known in the art.
  • nucleic acid particularly useful sources of nucleic acid are derived from tissues or blood samples of an organism, nucleic acids which are present in self-replicating vectors, and nucleic acids derived from viruses and pathogenic organisms such as bacteria and fungi. Also particularly useful for the present invention are target nucleic acid sequences which are related to disease states, such as those caused by chromosomal rearrangement, insertions, deletions, translocations and other mutations, those caused by oncogenes, and those associated with cancer.
  • selected means that a target nucleic acid sequence having the desired characteristics is located ' and probes are constructed around appropriate segments of the target sequence.
  • probe or “primer” has the same meaning herein, namely, an oligonucleotide fragment which is single stranded.
  • oligonucleotide means DNA or RNA.
  • a probe or primer is "substantially complementary" to the target nucleic acid sequence if it hybridizes to the sequence under renaturation conditions so as to allow target dependent ligation or extension. Renaturation depends on specific base pairing between A-X (where X is T or U) and G-C bases to form a double stranded duplex structure. Therefore, the primer sequence need not reflect the exact sequence of the target nucleic acid sequence. However, if an exact copy of the target nucleic acid is desired, the primer should reflect the exact sequence.
  • a “substantially complementary” primer will " " contain at least 70% or more bases which are complementary to the target nucleic segment. More preferably 80% of the bases are complementary and most preferably 90% of the bases are complementary. Generally, the primer must hybridize to the target nucleic acid sequence at the end to be ligated or extended to allow target dependent ligation or extension.
  • the primers may be RNA or DNA, and may contain modified nitrogenous bases which are analogs of the normally incorporated bases, or which have been modified by attaching labels or linker arms suitable for attaching labels. Inosine may be used at positions where the target sequence is not known, or where it may be degenerate.
  • the oligonucleotides must be sufficiently long to allow hybridization of the primer to the target nucleic acid and to allow amplification to proceed. They are preferably 15 to 50 nucleotides long, more preferably 20 to 40 nucleotides long, and most preferably 25 to 35 nucleotides long. The nucleotide sequence, content and length will vary depending on the sequence to be amplified.
  • a primer may comprise one or more oligonucleotides which comprise substantially complementary sequences to the target nucleic acid sequence.
  • each of the oligonucleotides would hybridize to the same segment of the target nucleic acid.
  • oligonucleotide sequence which is most complementary to the target nucleic acid sequence will hybridize.
  • the stringency of conditions is generally known to those in the art to be dependant on temperature, solvent and other parameters. Perhaps the most easily controlled of these parameters is temperature and since the conditions here are similar to those of PCR, one skilled in the art could determine the appropriate conditions required to achieve the level of stringency desired.
  • Oligonucleotide primers or oligonucleotide probes suitable for use in the present invention may be derived by any method known in the art, including chemical synthesis, or by cleavage of a larger nucleic acid using non-specific nucleic acid-cleaving chemicals or enzymes, or by using site-specific restriction endonucleases.
  • the primers may be prepared using the ⁇ -cyanoethyl- phosphoramidite method or other methods known in the ar .
  • a preferable method for synthesizing oligonucleotide primers is conducted using an automated DNA synthesizer by methods known in the art. Once the desired oligonucleotide primer is synthesized, it is cleaved from the solid support on which it was synthesized, and treated, by methods known in the art, to remove any protecting groups present.
  • the oligonucleotide primer may then be purified by any method known in the art, including extraction and gel purification. The concentration and purity of the oligonucleotide primer may be examined on an acrylamide gel, or by measuring the optical densities at 260 and 280 nm in a spectrophotometer.
  • the primers used in the present invention are preferably phosphorylated at their 5' ends. This may be achieved by any method known in the art, but is preferably conducted with the enzyme T4 polynucleotide kinase.
  • the oligonucleotides can be phosphorylated in the presence of unlabeled or labeled ATP. In order to monitor the amplification process,' labeled ATP may be used to phosphorylate the primers. Particularly preferable is [ ⁇ - 32p] ATP.
  • the oligonucleotide primers may alternatively be labeled with any detectable marker known in the art, including other radioactive nuclides such as 35s or 3 H and the like, fluorescent markers such as fluorescein, rhodamine, Texas red, Lucifer yellow, AMCA blue and the like, or with - - enzymatic markers which may produce detectable signals when a particular chemical reaction is conducted, such as alkaline phosphatase or horseradish peroxidase. Such enzymatic markers are preferably heat stable, so as to survive the denaturation steps of the amplification process.
  • Primers may be indirectly labeled by incorporating a nucleotide covalently linked to a hapten or other molecule such as biotin to which a labeled avidin molecule may be bound, or digoxygenin, to which a labeled anti-digoxygenin antibody may be bound.
  • Primers may be labeled during chemical synthesis or the label may be attached after synthesis by methods known in the art.
  • the method of labeling and the type of label is not critical to this invention.
  • the probes or primers may be modified.
  • the hydrolysis of a primer by 5' to 3 ' exonuclease associated with polymerase may be prevented by placing a phosphorothioate group between the last nucleotides of the 5' end of the primer.
  • the extension of a primer by polymerase can be blocked by placing a dideoxynucleotide, an amino group, a cordycepin, or a phosphate group at the 3' end.
  • the extension of a primer may be blocked by placing an arabinosyl nucleotide at the 3 ' end of the primer which blocks extension by polymerase but allows ligation of the primer to another primer.
  • the four different nucleotide bases shall refer to deoxythymidine triphosphate (dTTP) ; deoxyadenosine triphosphate (dATP) ; deoxyguanosine triphosphate (dGTP) ; and deoxycytidine triphosphate (dCTP) , when the context is DNA, but shall refer to uridine triphosphate (UTP) ; adenosine triphosphate (ATP) ; guanosine triphosphate (GTP) ; and cytidine triphosphate (CTP) when the context is RNA.
  • dTTP deoxythymidine triphosphate
  • dATP deoxyadenosine triphosphate
  • dGTP deoxyguanosine triphosphate
  • CTP deoxycytidine triphosphate
  • dUTP, dITP, rITP or any other modified base may replace one of the four nucleotide bases or may be included along with the four nucleotide bases in the reaction mixture so as to be incorporated into the amplified strand.
  • the amplification steps are conducted in the presence of at least the four deoxynucleoside triphosphates (dATP, dCTP, dGTP and dTTP) or a modified nucleoside triphosphate to produce a DNA strand, or in the presence of the four ribonucleoside triphosphates (ATP, CTP, GTP and UTP) or a modified nucleoside triphosphate to produce an RNA strand from extension of the oligonucleotide which acts as a primer.
  • deoxynucleoside triphosphates dATP, dCTP, dGTP and dTTP
  • ATP deoxynucleoside triphosphates
  • CTP CTP
  • GTP GTP
  • UTP ribonucleoside triphosphate
  • one of the oligonucleotide primers may comprise a set of oligonucleotide fragments, each differing in sequence and each labeled by a different method. That oligonucleotide fragment which is exactly complementary to the target DNA sequence will be detected by the presence of that label in the amplification products.
  • each oligonucleotide fragment may be labeled as described above.
  • the target nucleic acid is described as single stranded. However, this should be understood to include the case where the target is actually double stranded but is simply separated from its complementary strand prior to hybridization with probes/primers.
  • Primers one and two, together are substantially complementary to the target nucleic acid sequence and hybridize to adjacent regions of the target nucleic acid strand such that upon hybridization of the two primers to the target nucleic acid strand the 5 ' end of the first primer is adjacent to the 3' end of the second primer.
  • the 3' end of the first primer is substantially complementary to the 5' end of the target nucleic acid sequence and the 5' end of the second primer is substantially complementary to the 3' end of the target nucleic acid sequence.
  • the 5' end of the first primer is ligated to the 3 ' end of the second primer using ligase to create a fused amplification product in a double stranded complex.
  • the fused primer is dissociated from the target nucleic acid.
  • the third primer is substantially complementary to all or at least a portion of the first primer and is similar to the 5' end of the target nucleic acid.
  • the third primer should be complementary to enough of the first primer so that specific hybridization is achieved under the conditions used.
  • the third primer may be smaller than the first primer or it may be larger than the first primer and also be substantially complementary to a portion of the second primer.
  • the third primer is hybridized to the fused amplification product and extended by polymerase in the presence of at least four different nucleotide bases to form an extended amplification product which is substantially complementary to the fused amplification product. This comprises the first cycle.
  • oligonucleotide primers (1 and 2) are hybridized to the target nucleic acid sequence and the extended amplification product from the first cycle.
  • Primer 3 is hybridized to the fused amplification product. Extension and ligation occur as before and the process can be repeated.
  • the 3 ' end of the second primer may be modified to block the extension of the second primer by polymerase while still allowing ligation of the 3' end of the second primer to the 5' end of the first primer.
  • modification may be, for example, the placement of an arabinosyl nucleotide at the 3 1 end of the second primer.
  • Methods for the chemical synthesis of DNA oligomers containing cytosine arabinoside are known in the art (Beardsley, Nucl. Acid. Res. (1988) 16:9165-9176). Such” a modification does not need to be removed prior to the ligation of the first and second primers.
  • the 5' end of the first primer can be modified to prevent the hydrolysis of the primer by a 5' to 3' exonuclease associated with a polymerase.
  • a modification may be, for example, the placement of a phosphorothioate group between the last nucleotides of the 5' end of the first primer.
  • Methods for the chemical synthesis of phosphorothioate containing primers is known in the art (Ott and Eckstein, Biochemistry, (1987) 26:8237-8241) . Such a modification does not need to be removed prior to ligation of the first and second primers.
  • extension of the first primer can be prevented without affecting the ligation of this primer by modifying the 3' end of the primer with a dideoxynucleotide or a phosphate group.
  • the method for producing this modification is known in the art (Markiewicz and Wyrzykiewicz Nucl. Acid. Res. (1989) 17:7149-7158). It has been found that the process can be conducted sequentially without isolation or purification of the products or removal of the excess reagents. Accordingly, this will allow the entire process to be conducted in a single reaction medium (e.g. a test tube) . It is understood that the single strand variation is a more specialized version of the double strand variation.
  • the target nucleic acid is double stranded some of the third primers will hybridize to the second complementary strand and the first and second primers will hybridize to the first strand.
  • the extension and ligation from the first strand will proceed as described above.
  • Some of the third primers will also be extended in a target specific manner complementary to the second strand. After dissociation of the extended third primer and the second strand, at least some of the first and second primers will hybridize to the extended third primer and at least some" of the third primer will again hybridize to the second strand.
  • the target nucleic acid amplified by ligation of the first and second primers and extension of the third primer may be labeled using a marker as described above to render the amplified target nucleic acid detectable or by conducting the extension of the third primer in the presence of a labeled base, or a base which is activated for labeling.
  • both amplified strands may be labeled with different detectable markers: the first strand may be labeled by labeling the third primer with a particular marker; and the second strand may be labeled by labeling the first and/or second primer.
  • one primer comprising a mixture of oligonucleotides is added to the nucleic acid sample.
  • Each oligonucleotide may be labeled with different, separately detectable markers, so that information regarding the presence of a particular mutation or allele may be obtained in a single step.
  • the oligonucleotide which is exactly complementary to the target sequence will be included in the amplification product whereas the other oligonucleotides will not and its presence detected by determining which label is included in the product.
  • the amplification reaction is optimally conducted with an excess of primers at a ratio of oligonucleotide primers:target of approximately 10 7 to 10 3 :1, more preferably approximately 104:1. It is contemplated that adjusting the molarity of the primers will maximize the efficiency of the process.
  • the buffer used for amplification is preferably in a pH range of about 7.5-8.5, more preferably about 8-8.5, and most preferably about 8.0.
  • the target nucleic acid is described to be single stranded. However, this should be understood to include the case where the target is actually double stranded, but is simply separated from its complementary strand prior to hybridization with the probes/primers.
  • the target nucleic acid is hybridized to two primers.
  • the first primer is substantially complementary to the 5' end of the target nucleic acid sequence and the second primer is substantially complementary to the 3 ' end of the target nucleic acid sequence.
  • the primers (primers one and two) hybridize to regions of the target nucleic acid strand such that upon hybridization of the two primers to the target nucleic acid strand the 5' end of the first primer is spaced from the 3' end of the second primer.
  • the size of the space or gap between the primers is determined by the ability of a polymerase or transcriptase to extend the second primer such that the newly added 3 ' end of the second primer is directly adjacent to the 5' end of the first primer.
  • the size of the gap or space is sufficiently long such that at least four different nucleotides would be required by the polymerase or transcriptase in order to extend the second primer to "fill in" the gap.
  • the 3 ' end of the second primer is extended by polymerase or transcriptase in the presence of the four nucleotide bases.
  • the 5' end of the first primer is then ligated to the new 3 ' end of the second extended primer to form a double-stranded complex comprising the target nucleic acid and an extended fused primer.
  • the double stranded complex is dissociated and a third primer is hybridized to the extended fused primer.
  • the third primer is substantially complementary to all or a portion of the first primer and is similar to• the 5' end of the target nucleic acid sequence.
  • the 3' end of the third primer is extended by polymerase or transcriptase to form a double-stranded complex and complete the cycle.
  • the double-stranded complex is dissociated and the cycle repeated until the target nucleic acid is amplified. It will be understood that when the target sequence is part of a double stranded nucleic acid as shown in Fig. 12, some of the third primers present will hybridize to the second strand complementary to the target sequence, and will be extended to form an amplification product.
  • the process can be conducted sequentially without isolation or purification of the products or removal of the excess reagents. Accordingly, this will allow the entire process to be conducted in a single reaction medium (e.g. a test tube) .
  • a single reaction medium e.g. a test tube
  • the gap between the primers can be any size as discussed above, the method is not limited to a particular DNA sequence and extension of the third primer can proceed in the presence of four nucleotides.
  • the single strand variation is a more specialized case of the double strand variation wherein there are four primers and the first and second primers are substantially complementary to the first strand of the target nucleic acid and the third and fourth primers are substantially complementary to the second strand of the target nucleic acid.
  • the third primer is substantially complementary to at least a portion of the first primer and the fourth primer is substantially complementary to at least a portion of the second primer.
  • the extension and ligation of the third and fourth primers occurs as described above for the first and second primers. It will be understood that at least some of the third and fourth primers will hybridize to the extended fused primer (first and second primers) .
  • the third primer is then extended and ligation to the fourth primer occurs.
  • the 5' end of the first primer (and the 5' end of the fourth primer, where the nucleic- acid is double stranded) can be modified to prevent the hydrolysis of the primer by a 5' to 3' exonuclease associated with the polymerase.
  • a modification may be, for example, the placement of a phosphorothioate group between the last nucleotides of the 5' end of the first or fourth primers.
  • Methods for the chemical synthesis of phosphorothioate containing primers is known in the art (Ott and Eckstein, Biochemistry, (1987) 26:8237-8241). Such a modification does not need to be removed prior to ligation of the first and second primers.
  • DNA polymerases possess a DNA polymerising associated strand displacement activity. It is preferable to reduce or eliminate that activity, as it could lead to displacement of the nonextended primer. For example, referring to Figure 3, unless this activity is reduced or eliminated, the extension of oligo 2 or oligo 3 could result in the displacement of oligo 1 or oligo 4, respectively.
  • Strand displacement activity is related to the processivity of the DNA polymerase. Processivity of DNA polymerase is defined as the number of nucleotide residues added per enzyme binding event. A DNA polymerase enzyme with a high degree of processivity will show a strong strand displacement activity. The processivity of the enzyme is affected by factors such as 1) salt concentration, 2) nucleotide concentration, and 3) divalent cations. We have found that:
  • extension of the first and fourth primers can be prevented without affecting the ligation of these primers by modifying the 3 ' end of the primers with a dideoxynucleotide or a phosphate group. This method of producing this modification is known in the art (Markiewicz and Wyrzykiewicz Nucl. Acid. Res. (1989) 17:7149-7158).
  • the target nucleic acid amplified is to be detected, one or all of these primers may be labeled as described above to render the amplified strand detectable.
  • the strand may be labeled by conducting the extension of the second or third primer in the presence of a labeled base, or a base which is activated for labeling.
  • each of the oligonucleotides may be labeled with different, separately detectable markers, so that information regarding each mutation may be obtained in a single step.
  • the amplification reaction is optimally conducted with an excess of primers at a ratio of oligonucleotide primers:target of approximately 10 7 to 10 3 :1, more preferably approximately 10 4 :1. It is contemplated that adjustment of the molarity of the primers will maximize the efficiency of the process.
  • the buffer used for amplification is preferably in a pH range of about 7.5-8.5, more preferably about 8-8.5, and most preferably about 8.0.
  • the strands should be separated so that they can be used individually. This separation can be accomplished by any suitable denaturation method including physical, chemical or enzymatic means, each of which are well known in the art.
  • the amplification reaction will involve a series of steps.
  • the reaction may be either a two step process [i.e. 1) hybridization/extensio ligation followed by 2) denaturation] or a three step process [1) hybridization; 2) extension/ligation and 3) denaturation) .
  • These steps may be carried out manually, but they are preferably conducted in an automated thermal cycler.
  • Hybridization is generally conducted at a temperature of approximately 50-75°C for a period of 0.5-2 minutes, more preferably at 60-70°C for a period of 1-1.5 minutes, and most preferably at about 63-68°C for about 1 minute.
  • the extension/ligation or the hybridization/extension/ligation steps are generally conducted at a temperature of approximately 60-80°C for a period of 0.5-5 minutes, more preferably at 68-78°C for a period of 2-4 minutes.
  • the conditions and reagents which make possible the preferred enzymatic ligation step are generally known to those of ordinary skill in the art and depend directly on the type of ligase used.
  • the "ligating enzyme” may be any enzyme known in the art to ligate nucleic acid sequences, including T4 ligase, but it is preferably a ligase stable at temperatures of approximately 0-95°C, such as AMPLIGASE (Epicentre Technologies, Madison Wisconsin), Taq ligase (New England Biolabs, Beverly, Massachusetts) and Pfu ligase (Stratagene, La Jolla, California) . Absent a thermally stable ligase, the ligase must be added again each time the cycle is repeated.
  • ligating enzyme/picomole of oligonucleotide Approximately at least 5 units of ligating enzyme/picomole of oligonucleotide is used. One unit is defined as the amount required to catalyze the ligation of 50% of the cos sites in one microgram of BstE II digested bacteriophage ⁇ DNA in a total volume of 50 ⁇ l in fifteen minutes at 45°C.
  • the "polymerase” may be any enzyme capable of polymerizing an RNA or DNA strand, including E. coli DNA polymerase I, the Klenow fragment of E. coli DNA polymerase I, AmpliTaq DNA polymerase Stoffel fragment, T4 DNA polymerase, RNA polymerase or reverse transcriptase.
  • the primer is extended by the polymerase in a target dependent manner, for example, under conditions such that a nucleic acid strand is formed complementary to the nucleic acid sequence to which the primer is hybridized.
  • the polymerizing enzyme is stable at temperatures of approximately 0-95°C, such as Tag DNA polymerase (Perkin-Elmer Corporation, Norwalk,
  • Extension of a primer by polymerase or transcriptase proceeds in a 5' to 3' direction and requires the addition in adequate amounts of at least the four nucleotide bases in the reaction mixture.
  • the strand separation can be accomplished by any suitable denaturing method including well-known physical, chemical or enzymatic means.
  • one physical method of separating the strands of the nucleic acid involves heating the nucleic acid until it is completely denatured. Typical heat denaturation is generally conducted at a temperature of approximately 85-110°C, more preferably at 90-100°C, and most preferably at about 2-96°C for a period of at least about 0.5 minutes.
  • the reaction is stopped by any method known in the art, such as with a buffer containing a high percentage of denaturant such as formamide, EDTA or by freezing.
  • the products can then be analyzed by any method, but electrophoresis on a polyacrylamide gel is preferable.
  • the samples are boiled before loading on the gel to eliminate any secondary structures.
  • the gel may then be dried and placed against autoradiographic film or phosphor screen when the oligonucleotides or amplified strands contain radioactive nuclides.
  • the gel may also be blotted and probed with a probe specific to the region amplified.
  • the primer may be labeled with a detectable marker by any method known in the art.
  • a preferred method for labeling primers is by end labeling.
  • Primers may be labeled during chemical synthesis by substitution of the 3 ⁇ p atoms in the phosphate groups with 32p.
  • the substituted nucleotide may be directly labeled or contain a linker arm for attaching a label, or may be attached to a hapten or other molecule to which a labeled binding molecule may bind (Boehringer Mannheim, Indianapolis, Indiana) .
  • Suitable direct labels include -radioactive labels such as 32 , . 3H, and 35s and non-radioactive labels such as fluorescent markers, such as fluorescein, Texas Red, AMCA blue, lucifer yellow, rhodamine, and the like; cyanin dyes which are 210
  • Fluorescent markers may alternatively be attached to nucleotides with activated linker arms.
  • Primers may be indirectly labeled by the methods disclosed above, by incorporating a nucleotide covalently linked to a hapten or other molecule such as biotin or digoxygenin, and performing a sandwich hybridization with a labeled antibody directed to that hapten or other molecule, or in the case of biotin, with avidin conjugated to a detectable label.
  • Antibodies and avidin may be conjugated with a fluorescent marker, or with an enzymatic marker such as alkaline phosphatase or horseradish peroxidase to render them detectable.
  • Conjugated avidin and antibodies are commercially available from companies such as Vector Laboratories (Burlingame, California) and Boehringer Mannheim (Indianapolis, Indiana) .
  • the enzyme can be detected through a colorimetric reaction by providing a substrate and/or a catalyst for the enzyme. In the presence of various catalysts, different colors are produced by the reaction, and these colors can be visualized to separately detect multiple probes. Any substrate and catalyst known in the art may be used.
  • Preferred catalysts for alkaline phosphatase include 5-bromo-4-chloro-3-indolylphosphate (BCIP) and nitro blue tetrazolium (NBT) .
  • the preferred substrate for horseradish peroxidase is diaminobenzoate (DAB) .
  • dGTP deoxyguanosine triphosphate dTTP thymidine triphosphate UTP uridine triphosphate NTP nucleoside triphosphate n ole (nM) nanomole pmole (pM) picomole mmole (mM) millimole ( ⁇ M) micromole ng nanogram ⁇ g microgram bis bisacrylamide (N, N'-methylenebis- acrylamide)
  • MDR-1 multidrug resistance gene
  • Figure 4 SEQ ID NO:l
  • the target DNA was prepared by the standard polymerase chain reaction with
  • Primer A (SEQ ID. NO:2) 5'-AGGTTAGTACCAAAGAGGCTCTGG-3 ' and Primer B (SEQ ID NO:3) 5'-ACTAACAGAACATCCTCAAAGCTC-3' based on the known sequence of the gene.
  • the PCR reaction mixture comprised ImM Tris HCl (pH 8.4), 5 mM KCl, 1.2 mM MgCl 2 , 0.8 mM of each dNTP, 1 ⁇ M of Primer A, 1 ⁇ M of
  • Primer B 1 ng of template DNA, 2.5 units of AmplitaqTM DNA polymerase (Perkin Elmer Cetus Corporation, .Norwalk, Connecticut) .
  • the reaction mixture was heated at 94°C for 6 min. , and then put through the following cycle 30 times: 94°C for 1 min, 65°C for 45 sec, and 72°C for 3 min. " The final polymerization was done at 72°C for 10 min.
  • DNA 20 ⁇ g was digested with 40 units of - sal restriction endonuclease at 37°C for 2 hours under the conditions recommended for the enzyme. An aliquot was run on an agarose gel to confirm that the DNA was completely digested. DNA was then extracted sequentially with equal volumes of phenol, phenol-chloroform (1:1) and chloroform, and then precipitated with two volumes of ethanol. The " DNA pellet was suspended in deionized water and the concentration determined by measuring the optical density at 260 nm.
  • oligonucleotide oligomers were synthesized on Milligen/Biosearch Cyclone Plus DNA Synthesizers [Millipore Corporation, Bedford, Massachusetts] using beta-cyanoethyl phosphoramidite chemistry. All reagents for oligonucleotide synthesis were purchased from Millipore Corporation [Bedford, Massachusetts] .
  • Oligonucleotides having the following sequences were synthesized: Oligo 1 (SEQ ID NO. 4):
  • a 60 minute room temperature treatment with ammonium hydroxide was used to cleave the oligonucleotide from the support.
  • the oligonucleotide was incubated with ammonium hydroxide at 55°C overnight treatment to remove the protecting groups.
  • Ammonium hydroxide was evaporated to dryness in a Speedvac Concentrator [Savant Instruments, Inc., Farmingdale, New York]'.
  • the oligonucleotide was suspended in deionized water and extracted three times with an equal volume of water-saturated N-butanol. Any traces of
  • N-butanol left were removed by evaporation in a Speedvac Concentrator.
  • concentration of oligonucleotide was determined by measuring optical density at 260 nm in a spec rophotometer.
  • Each oligonucleotide was phosphorylated at the 5' end with ATP and T4 polynucleotide kinase.
  • the reaction mixture (100 ⁇ l) contained 2 nmoles of each oligonucleotide 50 mM Tris HCl pH 7.6, 10 mM MgCl 2 , 5 mM DTT, 0.1 mM spermidine hydrochloride, 0.1 mM EDTA, 1 mM ATP and 50 units of T4 polynucleotide kinase (GIBCO BRL, Gaithersburg, Maryland) . After 1 hour at 37°C, the enzyme was inactivated by heating at 65°C for 10 minutes.
  • Oligonucleotides (20 pmoles) were labeled with 3 P at their 5' end in 60 ⁇ l of 50 mM Tris HCl pH 7.6, 10 mM MgCl 2 , 5 mM DTT, 0.1 mM spermidine hydrochloride, 0.1 mM
  • the stock solution of dNTP's was maintained at -20°C.
  • Figure 2 is a printout from a Phosphor Imager scan-of the samples amplified by the method described in Example 5.
  • the reaction mixture contained labeled Oligo 1 and unlabeled Oligo 2 and 3.
  • the reaction 10 In Lane 2 the reaction 10
  • the reaction mixture was the same as in Lane 1 with the addition of target DNA.
  • the amplified DNA band is indicated with an arrow.
  • Lane 3 the reaction contained labeled Oligo 2 and unlabeled Oligo 1 and 3.
  • Lane 4 the reaction mixture was the same as for Lane 3 with the addition of target DNA.
  • Lane 5 the reaction mixture contained unlabeled Oligo 1 and 2 and labeled Oligo 3.
  • Lane 6 the reaction mixture was the same as in Lane 5 with the addition of target DNA. It can be seen that amplification does not occur in the absence of the target DNA and that amplification can be detected by labeling any of the oligonucleotides.
  • Deoxynucleotides are synthesized on Milligen/Biosearch Cyclone PlusTM DNA synthesizers (Millipore Corporation, Bedford Massachusetts) using beta-cyanoethyl phosphoramidite chemistry as described in Example 2. The synthesis of oligonucleotides 1 and 3 was previously described in Example 2.
  • Oligonucleotides having the following sequences are synthesized: Oligo 4 (SEQ ID NO:7) 5' GTTCGGAAGT TTTCTATTGC TTCAGTAGCG 3' Oligo 5 (SEQ ID NO:8) 5' CTACTGAAGC AATAGAAAAC TTCCGAAC 3'
  • the oligonucleotides are either phosphorylated at the 5' end with ATP and T4 polynucleotide kinase as described in Example 3 or labeled with 32 P at their 5' end as described in Example 4.
  • the reaction is stopped by adding 13 ⁇ l of stop solution (95% v/v formamide, 20 mM EDTA, 0.05% w/v bromophenol blue, 0.05% w/v xylene cyanol FF) . Samples are stored at -20°C until analyzed by electrophoresis.
  • EXAMPLE 8 This example shows the Figure 12 embodiment of the present invention which uses three primers, with a gap between the first and second primers.
  • Oligo 5 (second primer) (SEQ ID NO:9) : 5' CGCCAGGGTT TTCCCAGTCA CGAC 3'
  • Oligo 6 (first primer) (SEQ ID NO:10): 5' CGTAATCATG GTCATAGCTG TTTCCTG-NH 2 3'
  • Oligo 7 (third primer) (SEQ ID NO:11) 5' GGAAACAGCT ATGACCATGA TTACGA 3'
  • Ml3mpl8 phage single and double stranded DNA was obtained from New England Biolabs, Beverly, MA and used as a target in this example. Its sequence is well known, " and the relevant portion from nucleotides 6201 to 6340 is as follows (SEQ ID NO:12):
  • Oligo 5 is complementary to nucleotides 6311-6334; oligo 6 is complementary to nucleotides 6205-6231.
  • Oligos 5 and 7 at a final concentration of 0.25 ⁇ M, and phosphorylated oligo 6 at a final concentration of 0.5 ⁇ -M were incubated in the presence of Ml3mpl8 double stranded DNA (present at 2.5 x 10-13 M) in 20 ⁇ l of 50 mM Tris HCl pH 8.0, 10 mM DTT, 2 mM NAD, 10 mM KCl, 4 mM MgCl 2 , 20 ⁇ M dATP, dGTP, dTTP and 5 ⁇ M dCTP.
  • Ml3mpl8 double stranded DNA present at 2.5 x 10-13 M
  • oligo 5 was labelled.
  • oligo 6 was labelled.
  • oligo 7 was labelled.
  • a negative control was run using the same reagents except that target DNA was absent.
  • Five units of AmpliTaq Stoffel fragment and 30 units of Taq ligase were added. Reaction tubes were incubated in GeneAmpTM PCR system 9600 thermal cycler (Perkin Elmer Cetus) at 94°C for 2 minutes (1 cycle) and then 94°C for 1 minute and 55°C for 2..5.minutes (30 cycles) .
  • the reaction was stopped by adding stop solution (95% formamide, 20 mM EDTA] 0.05% bromophenol blue, 0.05% xylene cyanol ' FF) .
  • the products of the amplification reaction were analyzed on an 8% polyacrylamide denaturing gel.
  • the results are shown in Figure 5, which is a printout from a were analyzed on an 8% polyacrylamide denaturing gel.
  • the results are shown in Figure 5, which is a printout from a Phosphor Imager scan of the samples amplified in this example.
  • the reaction mixture contained 32 P- labeled oligo 5 and unlabelled oligo 6 and 7.
  • the reaction mixture was the same as in lane 1 with the addition of target DNA.
  • the amplified DNA band is indicated with an arrow.
  • the reaction mixture contained 32 P- labeled oligo 6 and unlabelled oligo 5 and 7.
  • the reaction mixture was the same as in lane 3 with the addition of target DNA.
  • the reaction mixture contained 32 P- labeled oligo 7 and unlabelled oligo 5 and 6.
  • the reaction mixture was the same as in lane 5 with the addition of target DNA. It can be seen that the amplification does not occur in the absence of the target DNA and that amplification product can be detected by labeling any one of the three oligonucleotides.
  • EXAMPLE 9 This example shows the Figure 3 embodiment of the present invention which uses four primers, two of which are extended.
  • Oligos 5 and 7 (the second and third primers, respectively) at a final concentration of 0.025 ⁇ M and phosphorylated oligos 6 and 8 (first and fourth primers, respectively) at a final concentration of 0.05 ⁇ M were incubated in presence of Ml3mpl8 phage double stranded DNA (2.5 x lO-" M) in 20 ⁇ l of 50 mM TrisCl pH 8.0, 10 mM DTT, 2 mM NAD, 10 mM KCl, 4 M MgCl 2 , 20 ⁇ M dATP, dGTP, dTTP and 5 ⁇ M dCTP.
  • Ml3mpl8 phage double stranded DNA 2.5 x lO-" M
  • the reaction was stopped by adding stop solution as in Example 8.
  • the products of the amplification reaction were analyzed on an 8% polyacrylamide denaturing gel.
  • Figure 6, is a printout from a Phosphor Imager scan of the samples amplified in this example.
  • the reaction mixture contained 32p- labeled oligo 5 and unlabelled oligo 6, 7 and 8.
  • the reaction mixture was the same as in lane 1 with the addition of target DNA.
  • the amplified DNA band is indicated with an arrow.
  • the reaction mixture contained 32 P- labeled oligo 6 and unlabelled oligo 5, 7 and 8.
  • lane 4 the reaction mixture was the same as in lane
  • the reaction mixture contained 32 p- labeled oligo 7 and unlabelled oligo 5, 6 and 8.
  • the reaction mixture was the same as in lane 5 with the addition of target DNA.
  • the reaction mixture contained 32 P- labeled olig 8 and unlabelled oligo 5, 6 and 7.
  • the reaction mixture was the same as in lane 7 with the addition of target DNA. It can be seen that the amplification does not occur in the absence of the target DNA and that amplification product can be detected by labeling any one of the four oligonucleotides.
  • oligo 5 was extended by DNA polymerase until it reached the 5' end of oligo 6.
  • the extended products accumulated for some time and then the extension continued beyond the blocking primer (oligo 6).
  • 5'-32P end labelled primer (oligo 5) (15nM) and blocking primer (oligo 6) (300nM) were simultaneously annealed to Ml3mpl8 phage single stranded DNA (26nM) .
  • Annealing was performed in lOmM TrisCl pH 7.5, lO M mgCl 2 and 50 mM NaCl, by heating at 95°C for 3 minutes followed by slow cooling to room temperature.
  • Strand displacement by AmpliTaq DNA polymerase was assayed by diluting the primer template complex ten fold in OCR buffer (50mM Tris HCl pH 8.0, lOmM DTT, 2mM NAD), lOOmM KCl, 2mM MgCl 2 , 25 units/ml of AmpliTaq DNA polymerase and appropriate concentrations of deoxynucleotide triphosphates as described below.
  • Assays using AmpliTaq DNA polymerase Stoffel fragment were performed under same conditions except lOmM KCl and 50 units/ml of enzyme. The reaction was incubated at 55°C and aliquots were taken after 30 seconds, 1 and 2 minutes. Stop solution was added and samples were analyzed on 8% polyacrylamide denaturing gel.
  • the assay were performed as in part A above except that the blocking primer was phosphorylated at the 5' end and 1500 units/ml of Taq ligase (New England Biolabs) were also added.
  • the results of both assays A and B are shown in Figures 7-10.
  • Fig. 7 shows the strand displacement by AmpliTaq DNA polymerase in presence of 100 mM KCl.
  • the lanes are as follows:
  • Lanes B1-B3 20 ⁇ M dA, dG, dT and 4 ⁇ M dC
  • Fig. 8 shows the strand displacement by AmpliTaq Stoffel fragment DNA polymerase in presence of 10 mM KCl.
  • the lanes are as follows: Lanes A1-A3 20 ⁇ M all four dNTP'.s . Lanes B1-B3 20 ⁇ M dA, dG, dT and 10 ⁇ M dC Lanes C1-C3 20 ⁇ M dA, dG, dT and 5 ⁇ M dC Lanes A, C, G, T display the dideoxy sequencing pattern from the oligo 5 primer and Ml3mpl8 phage DNA. The arrow points to the position where the 5' end of blocking primer (oligo 6) binds. Lanes 1, 2, 3 are 30 seconds, 1 minute and 2 minute time points, respectively.
  • Fig. 9 shows the strand displacement by AmpliTaq DNA polymerase in presence of 100 mM KCl and Taq ligase.
  • the lanes are as follows:
  • Fig. 10 shows the strand displacement by AmpliTaq Stoffel fragment DNA polymerase in presence of 10 mM KCl and Taq ligase.
  • the lanes are as follows: Lanes A1-A3; B1-B3: 20 ⁇ -M all four dNTP's
  • Lanes A, C, G, T display the dideoxy sequencing pattern from the oligo 5 primer and Ml3mpl8 phage DNA.
  • the arrow points to the position of the product formed by the ligation of extended primer (oligo 5) and oligo 6.
  • Lanes 1, 2, 3 are 30 seconds, 1 minute and 2 minute time points, respectively.
  • oligo 5 was extended by DNA polymerase until it reached the 5' end of oligo 6.
  • the extended products accumulated- for long enough time to be ligated to oligo 6 by DNA ligase.
  • AmpliTaq polymerase is about ten times more processive than Stoffel fragment. Therefore it requires higher salt concentration and lower dCTP concentration to achieve a similar degree of processivity as compared to Stoffel fragment.
  • This example shows the Figure 1 embodiment of the present invention which uses three primers and no gap.
  • oligonucleotide 3 was previously described in Example 2. Oligonucleotide having the following sequences are synthesized:
  • Oligo 10 (SEQ ID NO:15) 5' TTTCTTATCT TTCAGTGCTT GTCCAGA- araC 3'
  • Oligonucleotides 3 and 10 at a final concentration of 0.025 ⁇ M and oligo 9 phosphorylated at the 5' end at a final concentration of 0.05 ⁇ M were incubated in the presence of target DNA (5xl0-i2M, -RsaJ digested MDR-1 DNA) in 20 ⁇ l of 50mM Tris HCl pH 8.0, 10 mM DTT, 2mM NAD, 10 mM KCl, 4mM MgCl 2 , 20 ⁇ M of dATP, dCTP, dGTP, dTTP.
  • Three different sets of assays were set up. In each case, only one 5' - 32p labelled oligonucleotide was added.
  • oligo 9 was labelled.
  • oligo 10 was labelled.
  • oligo 3 was labelled.
  • a negative control was run using the same reagents except that target DNA was absent.
  • Five units of AmliTaq Stoffel fragment and 30 units of Taq ligase were added. Reaction tubes were incubated in GeneAmpTM PCR system 9600 thermal cycler (Perkin Elmer Cetus) at 94°C for 2 minutes (1 cycle) and then 94?C for 1 minute and 55°C for 2.5 minutes (30 cycles) .
  • the reaction was stopped by adding stop solution as in example 5.
  • the products of the amplification reaction were analyzed on an 8% polyacrylamide denaturing gel. The results are shown in figure 11, which is a printout from a Phosphor Imager scan of the samples amplified in this example.
  • the reaction mixture contained 32 p- labeled oligo 9 and unlabelled oligo 3 and 10.
  • the reaction mixture was the same as in lane 1 with the addition of target DNA.
  • the amplified DNA band is indicated with an arrow.
  • the reaction mixture contained 32p- labeled oligo 10 and unlabelled oligo 3 and 9.
  • the reaction mixture was the same as in lane 3 with the addition of target DNA.
  • the reaction mixture contained 3 2p- labeled oligo 3 and unlabelled oligo 9 and 10.
  • the reaction mixture was the same as in lane 5 with the addition of target DNA. It can be seen that the amplification does not occur in the absence of the target DNA and that amplification product can be detected by labeling any one of the three oligonucleotides. AraC at the 3' end of oligo 10 does get ligated to the 5' end of oligo 9.
  • NAME KARTA, GLENN E.

Abstract

Un procédé permet d'amplifier des séquences d'acides nucléiques provenant d'une matrice d'ADN ou d'ARN qui peuvent être purifiées ou exister sous forme de mélange d'acides nucléiques. Les séquences d'acides nucléiques résultantes peuvent être des copies exactes de cette matrice ou être modifiées. Ce procédé présente, par rapport à ceux utilisés à ce jour pour l'amplification, l'avantage d'accroître la fidélité pour la copie d'une séquence spécifique d'acides nucléiques et de faciliter la détection d'une mutation en un point particulier en un seul titrage (fig. 1).
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Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU1911395A (en) * 1994-02-04 1995-08-21 Beckman Instruments, Inc. Method, reagent and kit for the detection and amplification of nucleic acid sequences
US6852487B1 (en) 1996-02-09 2005-02-08 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
EP1736554B1 (fr) 1996-05-29 2013-10-09 Cornell Research Foundation, Inc. Detection de differences dans des sequences d'acides nucleiques utilisant une combinaison de la detection par ligase et de reactions d'amplification en chaine par polymerase
US6808880B2 (en) 1996-10-01 2004-10-26 Geron Corporation Method for detecting polynucleotides encoding telomerase
US6261836B1 (en) 1996-10-01 2001-07-17 Geron Corporation Telomerase
US6475789B1 (en) 1996-10-01 2002-11-05 University Technology Corporation Human telomerase catalytic subunit: diagnostic and therapeutic methods
US6093809A (en) 1996-10-01 2000-07-25 University Technology Corporation Telomerase
DE69739497D1 (de) 1996-10-01 2009-08-27 Geron Corp Menschlische Telomerase katalytische Untereinheit
US7585622B1 (en) 1996-10-01 2009-09-08 Geron Corporation Increasing the proliferative capacity of cells using telomerase reverse transcriptase
AUPO524897A0 (en) 1997-02-21 1997-03-20 Johnson & Johnson Research Pty. Limited Method of amplifying specific nucleic acid target sequences
AU728342B2 (en) * 1997-02-21 2001-01-04 Johnson & Johnson Research Pty. Limited Selective ligation and amplification method
US7413864B2 (en) 1997-04-18 2008-08-19 Geron Corporation Treating cancer using a telomerase vaccine
US7622549B2 (en) 1997-04-18 2009-11-24 Geron Corporation Human telomerase reverse transcriptase polypeptides
US6506594B1 (en) 1999-03-19 2003-01-14 Cornell Res Foundation Inc Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
US7014994B1 (en) 1999-03-19 2006-03-21 Cornell Research Foundation,Inc. Coupled polymerase chain reaction-restriction-endonuclease digestion-ligase detection reaction process
CA2405412A1 (fr) 2000-04-14 2001-10-25 Cornell Research Foundation, Inc. Procede de conception d'un reseau adressable dans la detection de differences de sequences d'acides nucleiques, au moyen d'une reaction de detection de ligase
EP2570487A1 (fr) * 2011-09-16 2013-03-20 Lexogen GmbH Procédé de transcription d'acide nucléique
EP2756098B1 (fr) 2011-09-16 2018-06-06 Lexogen GmbH Methode de fabrication d'une banque de molecules d'acide nucleique

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993000447A1 (fr) * 1991-06-28 1993-01-07 Abbott Laboratories Amplification d'acides nucleiques cibles par la reaction de la ligase comblant des espaces vides
WO1993024656A1 (fr) * 1992-05-29 1993-12-09 Abbott Laboratories Reaction en chaine de ligase commençant avec des sequences d'arn
WO1994003630A2 (fr) * 1992-08-04 1994-02-17 Beckman Instruments, Inc. Procede, reactif et trousse de detection et d'amplification de sequences d'acides nucleiques
WO1994017206A1 (fr) * 1993-01-27 1994-08-04 Oncor, Inc. Procede d'amplification de sequences d'acides nucleiques
EP0439182B1 (fr) * 1990-01-26 1996-04-24 Abbott Laboratories Procédé amélioré pour amplifier d'acides nucléiques cibles applicable à la réaction en chaîne de polymérase et ligase

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683195A (en) * 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
JP2955759B2 (ja) * 1988-07-20 1999-10-04 セゲブ・ダイアグノスティックス・インコーポレイテッド 核酸配列を増幅及び検出する方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0439182B1 (fr) * 1990-01-26 1996-04-24 Abbott Laboratories Procédé amélioré pour amplifier d'acides nucléiques cibles applicable à la réaction en chaîne de polymérase et ligase
WO1993000447A1 (fr) * 1991-06-28 1993-01-07 Abbott Laboratories Amplification d'acides nucleiques cibles par la reaction de la ligase comblant des espaces vides
WO1993024656A1 (fr) * 1992-05-29 1993-12-09 Abbott Laboratories Reaction en chaine de ligase commençant avec des sequences d'arn
WO1994003630A2 (fr) * 1992-08-04 1994-02-17 Beckman Instruments, Inc. Procede, reactif et trousse de detection et d'amplification de sequences d'acides nucleiques
WO1994017206A1 (fr) * 1993-01-27 1994-08-04 Oncor, Inc. Procede d'amplification de sequences d'acides nucleiques

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF VIROLOGICAL METHODS, vol. 35, no. 2, 1 November 1991, pages 117-126, XP000571446 BIRKENMEYER L G ET AL: "DNA PROBE AMPLIFICATION METHODS" *
See also references of WO9417210A1 *

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