WO1994002648A1 - Amplification et detection d'acides nucleiques de remplissage de breches - Google Patents

Amplification et detection d'acides nucleiques de remplissage de breches Download PDF

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Publication number
WO1994002648A1
WO1994002648A1 PCT/US1993/007066 US9307066W WO9402648A1 WO 1994002648 A1 WO1994002648 A1 WO 1994002648A1 US 9307066 W US9307066 W US 9307066W WO 9402648 A1 WO9402648 A1 WO 9402648A1
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oligonucleotide
target
oligonucleotides
sequence
pair
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PCT/US1993/007066
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Imclone Systems Incorporated
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Priority to AU47884/93A priority Critical patent/AU4788493A/en
Publication of WO1994002648A1 publication Critical patent/WO1994002648A1/fr

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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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • 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 is directed to an improved process for amplifying and detecting existing nucleic acid sequences if they are present in a test sample.
  • the present invention is an improvement of the repair chain reaction (RCR) process.
  • RCR was discovered by David Segev and is described in PCT application US89/03125, which is
  • RCR involves detecting the presence of at least one single or double stranded target molecule in a sample.
  • a single stranded target molecule comprises a nucleic acid target
  • a double stranded target molecule comprises a nucleic acid target sequence and a nucleic acid target complementary sequence.
  • the RCR process of gap-filling plus ligation is analogous to the repair of mismatched bases, and other errors that occur during DNA replication, repair of UV damage, and other processes in vivo. The process comprises the steps of:
  • each pair of oliyonucleotides is selected to hybridize either to the target sequence or the target complementary sequence
  • step (b) filling the gaps formed in step (a) with less than all four different types of bases complementary to the base or bases in the gap and joining the bases filling the gap to each other and to both adjacent hybridized oligonucleotides, thereby forming joined oligonucleotide products,
  • step (c) treating the hybridized joined oligonucleotide products of step (b) under denaturing conditions to separate the joined oligonucleotide products from the target sequence and the target complementary sequence to produce single-stranded
  • each pair of oligonucleotides comprises two oligonucleotides selected so as to be sufficiently complementary to the target sequence or to the target
  • step (e) filling the gaps formed in step (d) with less than all four different types of bases complementary to the base or bases in the gap and joining the base or bases filling the gap to each other and to both adjacent hybridized
  • oligonucleotide products resulting in the amplification of the target sequence
  • step (f) treating the hybridized oligonucleotides of step (e) under denaturing conditions to separate the hybridized oligonucleotides and produce single-stranded molecules, wherein steps (d), (e) and (f) are repeated a desired number of times; and
  • both members of the pair In a nucleic acid amplification and detection method, such as RCR, in which the members of an oligonucleotide pair are the same size or nearly the same size, both members of the pair generally have the same or very similar melting temperatures (Tm's).
  • Tm's melting temperatures
  • oligonucleotide and its complementary sequence The number of hydrogen bonds that an oligonucleotide can form is largely a function of its length. Oligonucleotide pairs of similar size are supplied in great excess in the reaction mixture of a process such as RCR. A problem with nucleic acid amplification reactions in which the oligonucleotides are of similar size is target independent hybridization of complementary
  • oligonucleotide probes and the formation of false product by blunt end ligation, especially at the beginning of the reaction. Formation of false product by blunt end ligation results in the loss of reliability and reproducibility.
  • the problem to be solved by the present invention is the improvement of the reliability and reproducibility of RCR.
  • RCR prior nucleic acid amplification and detection methods, such as RCR, the advantage of using oligonucleotide pairs with different Tm's to selectively control the hybridization of the oligonucleotides was not recognized.
  • the improvement of the present invention comprises, prior to step (g), :
  • step (h) selecting the pairs of oligonucleotides for step (a) and step (d) wherein,
  • one member of each pair of oligonucleotides has a melting temperature, with respect to the target sequence or the target complementary sequence, that is sufficiently higher than the melting temperature, with respect to the target sequence or target complementary sequence, of the other member of the pair of oligonucleotides to cause selective hybridization of both oligonucleotides in each pair to the target sequence or target complementary sequence,
  • step (i) selectively hybridizing the oligonucleotide pairs from step (h) to the target sequence or target complementary sequence by conducting steps (d), (e) and (f) under conditions alternating between high stringency, middle stringency and low stringency wherein, (1) under conditions of high stringency,
  • oligonucleotide member in each pair of oligonucleotides both hybridize to the target sequence or the target complementary sequence; and (j) treating the hybridized oligonucleotides of step
  • steps (d)-(f) and (h)-(j) are repeated a desired number of times.
  • Figure 1 illustrates the steps of the process of the present invention as disclosed in Example 1.
  • Figure 2 is a photograph of an electrophoretic gel showing the experimental results of Method I in Example 1.
  • Figure 3 is a bar chart showing the experimental results of Method II in Example 1.
  • Figure 4 illustrates the steps of the capture assay
  • the present invention relates to an improved process for amplifying and detecting a target nucleic acid molecule in a test sample.
  • the process of the present invention can produce geometric amplification of a target nucleic acid molecule, provided that at least part of the nucleotide sequence is known in sufficient detail that complementary oligonucleotide pairs can be
  • the target molecule can be in purified or non-purified form, and can be single stranded or double stranded DNA, single stranded or double stranded RNA or a DNA-RNA hybrid.
  • the target nucleic acid molecule contains the specific nucleotide sequence that hybridizes to members of the
  • target sequence oligonucleotide pairs. This sequence is called the target sequence. If a target nucleic acid molecule is double stranded, it will contain a target sequence and its complement called the target complementary sequence.
  • the target sequence comprises at least as eight nucleotides, preferably at least twelve
  • nucleotides and more preferably at least sixteen nucleotides. There is no maximum number of nucleotides in the target sequence or target complementary sequence, which can constitute either a portion of the target molecule or the entire target molecule.
  • DNA or RNA isolated from bacteria, viruses, algae, protozoans, yeast, fungi, plasmids, cells in tissue culture and higher organisms such as plants or animals can be amplified with the process of the present invention.
  • DNA or RNA from these sources may, for example, be found in samples of a bodily fluid from an animal, including a human, such as, but not limited to, blood, urine, lymphatic fluid, synovial fluid, bile, phlegm, saliva, aqueous humor, lacrimal fluid, menstrual fluid and semen.
  • samples of a bodily fluid from an animal including a human, such as, but not limited to, blood, urine, lymphatic fluid, synovial fluid, bile, phlegm, saliva, aqueous humor, lacrimal fluid, menstrual fluid and semen.
  • DNA or RNA may, for example, be found in fluids from a plant, such as, but not limited to, xylem fluid, phloem fluid and plant exudates. Samples containing DNA or RNA may, for example, also be found in non-living sources such as, but not limited to, food, sewage, forensic samples, wet lands, lakes, reservoirs, rivers and oceans.
  • the improved process of the present invention is based on the fact that a longer oligonucleotide generally has a higher melting temperature (Tm) than a shorter oligonucleotide with respect to a target sequence or a target complementary sequence.
  • Tm melting temperature
  • the oligonucleotide members hybridize to the target sequence or the target complementary sequence permits the order in which the oligonucleotide members hybridize to the target sequence or the target complementary sequence to be selectively controlled.
  • melting temperature refers to the temperature at which an oligonucleotide hybridizes to a complementary nucleic acid sequence to form a stable complex.
  • Tm melting temperature
  • the Tm of a given oligonucleotide is a function of various properties of the oligonucleotide, such as the size and composition of the oligonucleotide, as well as of the reaction medium, such as the concentration of the oligonucleotide, and the composition and pH of the reaction solvent.
  • the oligonucleotide pairs of the present invention comprise one member that is longer than the other member.
  • the longer member of each oligonucleotide pair generally has a higher Tm, with respect to its complementary sequence, than the shorter member of the oligonucleotide pair, with respect to its complementary sequence.
  • the longer member of the oligonucleotide pair is usually "the higher Tm oligonucleotide” and the shorter member of the oligonucleotide pair is usually "the lower Tm oligonucleotide,” the relationship between length and Tm is not a necessary one.
  • the shorter Tm oligonucleotide is the higher Tm oligonucleotide.
  • the members of an oligonucleotide pair are selected so that, under the conditions used in the amplification reaction, the Tm's are sufficiently different to cause selective
  • hybridize or “selective hybridization” as used herein refers to the ability to control the order in which oligonucleotide members with different Tm's hybridize to the target sequence or the target complementary sequence in order to effect a
  • Selective hybridization is primarily a function of the different melting temperatures of each member of an oligonucleotide pair with respect to the portion of the target sequence or the target complementary sequence to which it hybridizes. Shorter oligonucleotides generally require a smaller difference in length to effect selective hybridization than longer oligonucleotides. For example, when the lower Tm oligonucleotide has nine to twelve nucleotides, the higher Tm nucleotide should be at least two, preferably at least three and more preferably at least four nucleotides longer than the lower Tm oligonucleotide. When the lower Tm oligonucleotide has twelve to eighteen nucleotides, the higher Tm nucleotide should be at least three, preferably at least four and more preferably at least five nucleotides longer than the lower Tm
  • melting temperatures of the members of an oligonucleotide pair are sufficiently different to cause
  • the higher Tm oligonucleotide and its complementary oligonucleotide constitute an oligonucleotide complement pair.
  • the lower Tm oligonucleotide and its complementary oligonucleotide constitute an oligonucleotide complement pair.
  • the following generalized illustration depicts a higher Tm oligonucleotide with its complementary oligonucleotide and a lower Tm oligonucleotide with its complementary oligonucleotide.
  • oligonucleotides of the present invention may be characterized in two ways. One characterization of the
  • oligonucleotides is the grouping of the higher Tm and lower Tm oligonucleotides as an oligonucleotide pair, the members of which either both hybridize to a target sequence or both hybridize to a target complementary sequence.
  • the other characterization of the oligonucleotides is the grouping of the higher Tm oligonucleotide with its complementary oligonucleotide and the lower Tm oligonucleotide with its complementary
  • oligonucleotide as oligonucleotide complement pairs.
  • the oligonucleotide pairs of the present invention are designed so that a gap of at least one nucleotide base is formed between the members of the
  • oligonucleotide pair when both members are hybridized to the target sequence or target complementary sequence.
  • the space that is created between the higher Tm and the lower Tm members of an oligonucleotide pair when they hybridize to a target sequence or to a target complementary sequence is referred to as a gap.
  • the oligonucleotide pairs are selected so that there is a gap in the nucleotide sequence of at least one base,
  • the gap may be filled before the lower Tm oligonucleotide hybridizes to the target strand, so that the gap may not be actually formed.
  • the gap More than two oligonucleotide pairs, resulting in more than two gaps, may be used in the process of the present invention.
  • the gap is filled with less than all four types of
  • nucleotide bases are nucleotide bases.
  • the term "less than all four nucleotide bases” as used herein refers to any combination of the
  • complementary deoxyribonucleotide bases adenosine, thymidine, guanine or cytosine wherein fewer than all four types of bases, i.e. one, two or three, but not all four types of bases, are used in the method to fill the gap.
  • the ribonucleotide uracil can be substituted for thymidine in the method of the present invention.
  • the term "gap-filling" as used herein refers to the
  • oligonucleotide pair one member acts as a primer in the
  • the member that acts as a primer is designated the priming
  • oligonucleotide member the member that becomes ligated to the polymerization extension product is designated the ligation oligonucleotide member.
  • oligonucleotide member is phosphorylated at its 5' end.
  • the gap-filling process begins with the addition by polymerization of a nucleotide that is complementary to the first base in the gap of a target sequence, or target complementary sequence, to the priming oligonucleotide member. If the gap is larger than one nucleotide base, additional nucleotide bases are added by polymerization. The addition of nucleotides ends when a base on the target sequence, or target complementary sequence, is reached that does not have a complementary base supplied in the reaction mixture.
  • the ligation member of the oligonucleotide pair hybridizes to the target sequence or target complementary sequence, if present, immediately adjacent to the gap.
  • the last nucleotide used to fill the gap and the ligation oligonucleotide member are then ligated to form a single stranded nucleic acid termed a "joined oligonucleotide product.”
  • joined oligonucleotide product refers to the single stranded nucleic acid sequence that is formed when the nucleotides that fill the gap are joined to the oligonucleotides that define the gap by the process of the present invention.
  • the joined oligonucleotide product that hybridizes to the target sequence comprises the sequence of the target complementary sequence.
  • complementary sequence comprises the sequence of the target sequence. Accordingly, reference to the target sequence or the target complementary sequence in the specification and the claims of the present invention also includes joined
  • oligonucleotide products that have hybridized to and been formed from the target or target complementary sequences.
  • the gap-filling and ligation steps occur in a buffered aqueous solution, preferably at a pH of 7-9, most preferably at pH 7.5.
  • the oligonucleotide complement pairs will be present in molar excess of about 10 5 -10 18 , preferably 10 9 -10 16 , pairs per nucleic acid target sequence.
  • the exact amount of the pairs to be used in diagnostic purposes may not be known due to uncertainty as to the amount of the nucleic acid target in a sample. However, using an average amount of 10 15 oligonucleotide complement pairs is applicable in a typical diagnosis assay format. A large molar excess is preferred in any case to improve the efficiency of the process of the invention.
  • the lower Tm members of the oligonucleotide complement pairs hybridize not only to the target nucleic acid molecule or joined oligonucleotide product, but to any unhybridized higher Tm members of the oligonucleotide complement pairs as well. Hybridization of the lower Tm members of the oligonucleotide complement pairs to the higher Tm members of the pairs reduces the number of lower Tm oligonucleotides that are available to hybridize to nucleic acid molecules having the target sequence or target complementary sequence, as the case may be.
  • the lower Tm oligonucleotide members are provided at a higher concentration than the higher Tm oligonucleotide members.
  • the lower Tm oligonucleotides are preferably provided at a
  • concentration at least 1.1 times greater than the higher Tm oligonucleotides, more preferably at least 1.5 times greater than the higher Tm oligonucleotides and most preferably at least 1.75 times greater than the higher Tm oligonucleotides.
  • concentration of the lower Tm members can be increased relative to the concentration of the higher Tm members for a given oligonucleotide complement pair.
  • the maximum increase in concentration of the lower Tm members relative to the higher Tm members is dependent primarily on the differential between the melting temperatures of the lower Tm and higher Tm members of an oligonucleotide complement pair.
  • the differential in melting temperature between members of an oligonucleotide complement pair is dependent on factors such as, but not limited to, the length of the members, the composition of the members and the composition of the reaction mixture.
  • the maximum increase in concentration of the lower Tm oligonucleotides relative to the higher Tm oligonucleotides is preferably at most ten times greater than the concentration of the higher Tm oligonucleotides, more preferably at most six times greater than the concentration of the higher Tm
  • oligonucleotides even more preferably at most four times greater than the concentration of the higher Tm oligonucleotides and most preferably at most two times greater than the
  • overhang as defined herein is used to describe that portion of an oligonucleotide complement pair wherein one member of the pair has several bases that protrude beyond the other member of the pair.
  • An overhang can be at the 3' or 5' end of the particular oligonucleotide member, or at both the 3' and the 5' ends. For example:
  • the bases in the overhang may protrude into the area of the gap, or into the area of an oligonucleotide on the side away from the gap.
  • the bases in the overhang may protrude into the area of the gap, or into the area of an oligonucleotide on the side away from the gap.
  • overlapping gap and non-overlapping gap refers to the embodiment in which two
  • oligonucleotide pairs are selected so that the position of the gap on one target strand relative to the gap on the
  • complementary target strand can result in an overlap of the gaps or a non-overlap of the gaps.
  • gaps For example:
  • the number of bases in the overhang of the oligonucleotide pairs is sufficient to cause a sufficient difference in the Tm of each member of an oligonucleotide pair so that the hybridization of each member of the pair to its respective complementary sequence on the target sequence or the target complementary sequence is selectively controlled.
  • the overhang has at least two bases, more preferably at least six bases and most
  • temperatures of the two complementary nucleotide pairs, A-T and C-G are important elements to consider in the design of the oligonucleotide pairs.
  • the embodiment of the present invention wherein two oligonucleotide complement pairs each have 3 ' overhangs that form a non-overlapping gap will now be illustrated.
  • oligonucleotide complement pairs are illustrated as follows:
  • a double stranded nucleic acid target sequence and the oligonucleotide complement pairs are denatured under high stringency conditions.
  • the process proceeds in the presence of about 10 5 -10 18 , preferably 10 9 -10 16 molar excess of the two oligonucleotide complement pairs per initial nucleic acid target sequence, a supply of less than all four types of nucleotide bases (A, C, G), a heat-stable
  • the process is begun by subjecting a single or double stranded target molecule and the oligonucleotide complement pairs to conditions of high stringency, usually high
  • the higher Tm members of each oligonucleotide complement pair, A and D selectively hybridize to the target sequence and target complementary sequence, respectively.
  • the gap filling process is then initiated by a polymerase at the 3' end of the hybridized oligonucleotides.
  • the lower Tm members of each oligonucleotide complement pair, B and C selectively hybridize to the target sequence and target complementary sequence, respectively, at the site where the gap, which will now usually be filled, ends.
  • oligonucleotide complement pairs with 5' overhangs form a non-overlapping gap.
  • oligonucleotide complement pairs are illustrated as follows:
  • a double stranded nucleic acid target sequence and the oligonucleotide complement pairs are denatured under conditions of high
  • the process proceeds in the presence of about 10 5 -10 18 , preferably 10 9 -10 16 molar excess of the two oligonucleotide complement pairs per initial nucleic acid target sequence, a supply of less than all four types of
  • nucleotide bases A, C, G
  • a heat-stable polymerase A, C, G
  • heat-stable ligase A, C, G
  • the higher Tm members of each oligonucleotide complement pair, F and G selectively hybridize to the target sequence and target complementary sequence, respectively.
  • the lower Tm members of each oligonucleotide complement pair, E and H selectively hybridize to the target sequence and target complementary sequence, respectively.
  • the gap filling process is then initiated by a polymerase at the 3' end of the lower Tm oligonucleotides, E and H. The sequence that fills the gap and the higher Tm
  • oligonucleotides are then ligated to form joined oligonucleotide products. The process is repeated to achieve the desired level of amplification.
  • one member of an oligonucleotide pair has a 3' overhang and the equivalent member of the other pair has a 5' overhang that form an
  • oligonucleotide complement pairs are illustrated as follows:
  • a double stranded nucleic acid target sequence and the oligonucleotide complement pairs are denatured under high stringency conditions, i.e. high temperature.
  • the process proceeds in the presence of about 10 5 -10 18 , preferably 10 9 -10 16 , molar excess of the two oligonucleotide complement pairs per initial nucleic acid target sequence, a supply of less than all four types of nucleotide bases (A, C, T), a heat-stable polymerase and heat-stable ligase.
  • the higher Tm members of each oligonucleotide complement pair, J and K selectively hybridize to the target sequence.
  • the gap filling process is then initiated by the polymerase at the 3' end of the oligonucleotide J.
  • the lower Tm members of each oligonucleotide complement, L and M selectively hybridize to the target complementary sequence.
  • the gap filling process is then initiated by a polymerase at the 3' end of the
  • oligonucleotide M The sequence that fills the gaps and the respective oligonucleotides are then ligated to form joined oligonucleotide products. The process is repeated to achieve the desired level of amplification.
  • one member of the pair has a 5' overhang and the equivalent member of the other pair has a 3' overhang that form an overlapping gap.
  • oligonucleotide complement pairs are illustrated as follows :
  • a double stranded nucleic acid target sequence and the oligonucleotide complement pairs are denatured under high stringency conditions, i.e. high temperature.
  • the process proceeds in the presence of about 10 5 -10 18 , preferably 10 9 -10 16 molar excess of the two oligonucleotide complement pairs per initial nucleic acid target sequence, a supply of less than all four types of nucleotide bases (A, C, T), a heat-stable polymerase and heat-stable ligase.
  • the polymerase at the 3' end of the oligonucleotide Q.
  • the lower Tm members of each oligonucleotide complement pair, N and O selectively hybridize to the target sequence.
  • the gap filling process is then initiated by the polymerase at the 3' end of the
  • oligonucleotide N The sequence that fills the gaps and the respective oligonucleotides are then ligated to form joined oligonucleotide products. The process is repeated to achieve the desired level of amplification.
  • both members, R and T, of one oligonucleotide complement pair are higher Tm oligonucleotides and both members, S and U, of another oligonucleotide complement pair are lower Tm oligonucleotides.
  • oligonucleotide complement pairs are illustrated as follows:
  • a double stranded nucleic acid target sequence and the oligonucleotide complement pairs are denatured under high stringency conditions, i.e. high temperature.
  • the process proceeds in the presence of about 10 5 -10 18 , preferably 10 9 -10 16 molar excess of the two oligonucleotide complement pairs per initial nucleic acid target sequence, a supply of less than all four types of nucleotide bases, a heat-stable polymerase and heat-stable ligase.
  • the higher Tm members of each oligonucleotide complement pair, R and T selectively hybridize to the target sequence and the target complementary sequence, respectively.
  • the gap filling process is then initiated by the polymerase at the 3' end of the oligonucleotide R. As the temperature is further reduced to effect conditions of low stringency, the lower Tm members of each oligonucleotide complement pair, S and U, selectively hybridize to the target sequence and the target complementary sequence, respectively. The gap filling process is then initiated by the polymerase at the 3' end of the
  • oligonucleotide U The sequence that fills the gaps and the respective oligonucleotides are then ligated to form joined oligonucleotide products. The process is repeated to achieve the desired level of amplification.
  • thermostable polymerase and ligase a heat-stable polymerase and ligase could be extracted from Thermus
  • thermophilus Such a heat-stable polymerase is described by A.D. Kaledin, et al., Biokhimiya, 45, 644-51 (1980).
  • a heatstable ligase is described by M. Takahashi et al., J. Biol.
  • This example illustrates the gap filing-ligation embodiment to amplify and detect target DNA sequences of hepatitis B virus (HBV) using oligonucleotide complement pairs having 3' overhangs that form a non-overlapping gap. (See Figure 1)
  • the first source is a 3.2 Kb nucleic acid sequence containing the HBV-ayw genome.
  • the second source is a series of clinical serum samples suspected to contain HBV.
  • the HBV target source is the region of the genome spanning nucleotides 2429-2480 having the
  • the two oligonucleotide complement pairs used in this embodiment, A-C and B-D each have one member of the pair that has a Tm that is different than the other member of the pair.
  • Oligonucleotides A and D have higher Tm's than oligonucleotides
  • Tm melting temperature
  • each of the complementary oligonucleotide pairs there are 3' overhung bases.
  • the 5' and 3' ends of oligonucleotides D and C, respectively, are protected with an amino linker arm.
  • the 5' end of oligonucleotide A is labeled with Fluorescein.
  • the 3' end of oligonucleotide B is labeled with Biotin.
  • the 5' ends of oligonucleotides B and C are phosphorylated.
  • the construction of the oligonucleotides is as follows:
  • reaction mixture contains the following:
  • Method I Amplification of the HBV samples for detection on an electrophoretic gel.
  • Clinical serum samples suspected of containing HBV are treated with proteinase K prior to analysis with the method of the present invention as follows: 10 ⁇ l of proteinase K at 10 mg/ml is added to 90 ⁇ l of each serum sample for a final proteinase K concentration of 1mg/ml. The serum samples are incubated for three (3) hours at 37°C, followed by an incubation for ten (10) minutes at 95°C. The serum samples are spun down in a microfuge for ten (10) minutes. The supernatant from each tube is collected and used as the source of HBV target DNA for amplification.
  • microfuge tubes 500 ⁇ l size.
  • Method II Titration of HBV samples for detection with a capture assay.
  • reaction mixture 25 ⁇ l is added to each of 5 microfuge tubes (500 ⁇ l size).
  • Each of the tubes is covered with 25 ⁇ l of light mineral oil to prevent evaporation.
  • the tubes are placed in a DNA Thermal Cycler (Perkin Elmer Model 4800).
  • the tubes are incubated in cycles as follows:
  • the set of tubes from Method I are cycled for 32 cycles.
  • the set of tubes from Method II are cycled for 27 cycles. All tubes are then placed at 4°C until ready for
  • reaction products are separated by size using non-denaturing polyacrylamide gels. 8 ⁇ l aliquots of solution from each tube is loaded on a 12% polyacrylamide gel (6 cm. long) using Tris-borate buffer (pH 8.5). Electrophoresis is carried out at 20 volts/cm for three hours, after which the gel is stained with ethidium bromide (1 ⁇ g/ml). The gel is then photographed. The photograph is shown in Figure 2.
  • the wells of a microtiter plate (Immulon II) are coated with 10 ⁇ g of egg white avidin (Precision Chemicals) in 100 ⁇ l of PBS and incubated overnight at 4°C. 2.
  • the wells of the microtiter plate are washed two times with PBS/0.5% Tween (PBST).
  • the wells are further coated by adding 100 ⁇ l of 2% BSA in PBS to each well for 30 min. at room temperature (RT).
  • the blocking solution is decanted and the wells are washed 4 times with PBST.
  • the wells of the microtiter plate are incubated with 0.01% H 2 O 2 for 30 minutes at RT.
  • reaction mixture 6 ⁇ l of each amplified sample (reaction mixture) is adjusted, with gentle mixing, to a volume of 100 ⁇ l with PBS. 50 ⁇ l of the adjusted reaction mixture is transferred from each reaction tube to a separate blocked well on the microtiter plate. The mixtures in the microtiter wells are incubated for one hour at room temperature.
  • the diluted reaction mixtures are decanted from the microtiter wells.
  • the wells of the plate are washed five times with PBST.
  • a horseradish peroxidase conjugated rabbit anti-fluorescein antibody (Biodesign) is diluted 1:10,000 in PBS. 100 ⁇ l of the conjugate is added to each well of the microtiter plate and incubated for one hour at room
  • microtiter wells The wells are washed five times with PBST. 100 ⁇ l of trimethyl benzidine (TMB) and hydrogen peroxide (1:1 vol.) substrate solution is added to each well of the

Abstract

L'invention concerne un procédé d'amplification de toute séquence d'acide nucléique spécifique désirée qui existe dans un acide nucléique ou un mélagne de celui-ci. Le procédé consiste à traiter un ARN monobrin ou les brins complémentaires séparés de la cible d'ADN avec un excédent molaire de paires oligonucléotides complémentaires dans lesquels les paires complémentaires d'oligonucléotides contiennent des séquences complétant la cible, dans des conditions d'hybridation dans lesquelles les brèches entre les oligonucléotides liés au même brin sont comblées au moyen d'au plus trois nucléotides, suivies de la ligature de ces oligonucléotides. Le procédé selon l'invention utilise des paires complémentaires d'oligonucléotides sélectionnées de façon que les membres d'une paire présentent des températures de fusion différentes. Les membres des oligonucléotides à tempéatures de fusion différentes permettent d'hybrider sélectivement les membres à la séquence cible.
PCT/US1993/007066 1992-07-21 1993-07-21 Amplification et detection d'acides nucleiques de remplissage de breches WO1994002648A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996006190A2 (fr) * 1994-08-19 1996-02-29 Perkin-Elmer Corporation Procede de ligature et d'amplification associees
WO1996040992A2 (fr) * 1995-06-07 1996-12-19 Abbott Laboratories Procede de masquage de sonde servant a limiter le signal d'arriere-plan dans une reaction d'amplification
EP0763133A1 (fr) * 1994-05-23 1997-03-19 Biotronics Corporation Procede de detection d'un acide nucleique cible
EP0785996A4 (fr) * 1993-07-13 1997-07-30
WO2006112780A1 (fr) * 2005-04-20 2006-10-26 Sedna Biotechnologies Ab Procede d'amplification

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WO1990001069A1 (fr) * 1988-07-20 1990-02-08 Segev Diagnostics, Inc. Procede d'amplification et de detection de sequences d'acide nucleique
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

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1990001069A1 (fr) * 1988-07-20 1990-02-08 Segev Diagnostics, Inc. Procede d'amplification et de detection de sequences d'acide nucleique
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

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0785996A4 (fr) * 1993-07-13 1997-07-30
EP0785996A1 (fr) * 1993-07-13 1997-07-30 Abbott Laboratories Sequences de nucleotides et procede d'amplification et de detection du virus de l'hepatite b
EP0763133A1 (fr) * 1994-05-23 1997-03-19 Biotronics Corporation Procede de detection d'un acide nucleique cible
EP0763133A4 (fr) * 1994-05-23 1999-02-10 Biotronics Corp Procede de detection d'un acide nucleique cible
WO1996006190A2 (fr) * 1994-08-19 1996-02-29 Perkin-Elmer Corporation Procede de ligature et d'amplification associees
WO1996006190A3 (fr) * 1994-08-19 1996-05-09 Perkin Elmer Corp Procede de ligature et d'amplification associees
US5912148A (en) * 1994-08-19 1999-06-15 Perkin-Elmer Corporation Applied Biosystems Coupled amplification and ligation method
US6130073A (en) * 1994-08-19 2000-10-10 Perkin-Elmer Corp., Applied Biosystems Division Coupled amplification and ligation method
WO1996040992A2 (fr) * 1995-06-07 1996-12-19 Abbott Laboratories Procede de masquage de sonde servant a limiter le signal d'arriere-plan dans une reaction d'amplification
WO1996040992A3 (fr) * 1995-06-07 1997-01-23 Abbott Lab Procede de masquage de sonde servant a limiter le signal d'arriere-plan dans une reaction d'amplification
US5814492A (en) * 1995-06-07 1998-09-29 Abbott Laboratories Probe masking method of reducing background in an amplification reaction
WO2006112780A1 (fr) * 2005-04-20 2006-10-26 Sedna Biotechnologies Ab Procede d'amplification

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