WO2001029248A2 - Method for amplifying and detecting nucleic acid - Google Patents
Method for amplifying and detecting nucleic acid Download PDFInfo
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- WO2001029248A2 WO2001029248A2 PCT/KR1999/000628 KR9900628W WO0129248A2 WO 2001029248 A2 WO2001029248 A2 WO 2001029248A2 KR 9900628 W KR9900628 W KR 9900628W WO 0129248 A2 WO0129248 A2 WO 0129248A2
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- primers
- nucleic acid
- dttp
- dctp
- solid support
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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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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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
- C12Q1/6869—Methods for sequencing
- C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
Definitions
- the present invention relates to a method of amplifying and detecting nucleic acid.
- PCR Polymerase Chain Reaction
- PCR works by hybridization of the separated complementary strands of the nucleic acid with an excess of oligonucleotide primers and extending the oligonucleotide primers to form complementary primer extension products. Because the extension product of each primer in turn serves as a template for synthesizing another copy of the desired nucleic acid sequence, the nucleic acid sequence of interest is amplified after successive rounds of primer extensions and strand separations.
- the asymmetric PCR amplification method that is a modified PCR method found in U.S. Patent 5,066,584 (Gyllensten, et al.) discloses a process for generating single- strand DNA by using an unequal ratio of two oligonucleotide primers.
- the products of single-strand DNA by use of the asymmetric PCR is particularly useful for post amplification applications such as sequencing and probe hybridization.
- the PCR method has a disadvantage of inconvenience of post amplification application when the PCR amplification is performed with many numbers of samples. This inconvenience mainly comes from the fact that the final PCR products present inside liquid reaction mixture along with target nucleic acids and other agents for amplification such as Taq DNA polymerase, dNTP and oligonucleotide primers.
- the PCR method also has disadvantages of generation of the spurious nonspecific amplification products and the difficulty of multi-number of target product amplification in single reaction mixture due to preferential amplification of some specific target sites. Summary of the invention
- the present invention provides a method for amplifyng at least one, existent or nonexistent, target nucleic acid which may be part of at least one larger nucleic acid, comprising the steps of:
- amplification reaction mixture which contains the target nucleic acid, at least one set of three oligonucliotide primers that consist of two equal or unequal amount of standard oligonucletide primers and one oligonucleotide primer bound to a solid support, mixture of four different nucleoside triphosphates and enzyme;
- the extension direction of the standard oligonucleotide primers having a lower concentration may be the same as that of the oligonucleotide primers bound to the solid support.
- the oligonucleotide primers bound to the solid support may be serially spotted on the solid support on the porpose of detecting mutation sites of target nucleic acid sequences.
- two standard oligonucleotide primers may be selected respectively from the group consisting of a unlabled oligonucleotide primer, a 5'end fluorescently labeled oligonucleotide primer, a 5'end Biotin labeled oligonucleotide primer, a 5'end radioisotope-labeled oligonucleotide primer and a 5'end Digoxigenin-labeled oligonucletide primer and the four different nucleoside triphosphates may be selected from the group consisting of the set of dATP, dTTP, dGTP and dCTP, the set of dTTP, dGTP, dCTP and fluorescently-labeled dATP, the set of dATP, dTTP, dCTP, and 7-Deaza-dGTP, the set of dTTP, dCTP, 7-Deaza-dGTP and
- the present invention also provides a method for amplifyng existent or nonexistent, plural target nucleic acids which may be part of plural larger nucleic acids, comprising the steps of:
- reaction mixture may further contain plural oligonucleotide primers bound to at least one solid support.
- the steps (a) through (g) may be performed by using a robotic workstation having a automatic multi-pipetting function for periodical addition of the oligonucleotide primers during the amplication process and a programmable temperature cycler which controls temperature levels, transitions between two temperatures and timing of temperature levels.
- Figs. 1A-1D illustrate a process of forming amplification products on a solid support by a oligonucleotide primer bound to a solid support
- Fig. 2 shows, in accordance with one aspect of the present invention, a process of amplifying a target nucleic acid with three different oligonucleotide primers
- Figs. 3A-3C illustrate, in accordance with one aspect of the present invention, a process of detecting mutation sites in a nucleic acid using a primer bound to a solid support;
- Fig. 4 shows, in accordance with another aspect of the present invention, a process of amplifying plural nucleic acids with plural sets of two oligonucleotide primers.
- the present invention provides a method for amplifying any target nucleic acid by preparing three different types of oligonucleotide primers, two of which are equal or preferably unequal amount of standard oligonucleotide primers and one of which is oligonucleotide primer bound to a solid support.
- the "standard oligonucleotide primer” means that the primer can be used for typical PCR.
- the oligonucleotide primer bound to the solid support contains the sequence which can be hybridized to the inside segment of the target nucleic acid.
- the extension products by the oligonucleiotide primer bound to the solid support enable convenient detection of the target nucleic acid and therefore the oligonucleotide primer bound to the solid support is the most important component of the present invention.
- Figs. 1A-1D illustrate a process of forming amplification products on a solid support by a oligonucleotide primer bound to a solid support .
- a target nucleic acid 110 two different standard oligonucleotide primers 120 and 130, and a primer 140 bound to a solid support 150 are prepared together with mixture of four different nucleoside triphosphates and enzyme (not shown).
- the target nucleic acid 110 are denaturated by heating or changing pH, so that two strands of the target nucleic acid are separated.
- the primers 120, 130 and 140 are hybridized to the separated target nucleic acid.
- the primers 120, 130 and 140 are extended by the enzyme to form extension products bound to the solid support 150 by the oligonucleotide primer 140 bound to the solid support 150.
- the method according to the present embodiment comprises repetitive cycles of the steps of separating the complementary strands of the target nucleic acid 110, hybridizing two standard oligonucleotide primers 120 and 130, and one oligonucleotide primer 140 bound to the solid support 150 to each strand of the target nucleic acid 110 and extending the hybridized primers 120, 130 and 140 to form complementary primer extension products which can be used as templates for synthesizing the target nucleic acid 110.
- the oligonucleotide primer 140 bound to the solid support 150 can initially be used to amplify the target nucleic acid 110, the extension efficiency of the oligonucleotide primer 140 bound to the solid support 150 is much less than that of the two standard oligonucleotide primers 120 and 130. However, as concentration of the standard oligonucleotide primers 120 and 130 is getting lower during the early cycles, the extension efficiency of the oligonucleotide primer 140 bound to the solid support 150 will be higher.
- the final amplification product will be the unbound nucleic acids extended by two unbound standard oligonucleotide primers 120 and 130, and the nucleic acids bound to the solid support 150 extended by the oligonucleotide primer 140 bound to the solid support 150.
- the concentrations of the standard oligonucleotide primer 130 is higher than that of the standard oligogonucleotide primer 120, the standard oligonucleotide primer 120 having lower concentration is getting exhausted during the early cycles, and the other standard oligonucleotide primer 130 having higher concentration and the oligonucleotide primer 140 bound to the solid support 150 remain in reaction mixture.
- the target nucleic acid 110 will be amplified by the cycling steps of denaturation of the target nucleic acid 110, hybridization of the oligonucleotide primers 130 and 140 to target nucleic acid , and extension of the primes 130 and 140.
- Fig. 2 shows, in accordance with one aspect of the present invention, a process of amplifying a target nucleic acid with three different oligonucleotide primers
- two standard oligonucleotide primers which consist of 8 to 50 nucleotides and the primer bound to the solid support which consists of 8 to 300 nucleotides are prepared along with the target nucleic acid which may be genome DNA, mRNA or plasmid DNA.
- the two standard oligonucleotide primers have an equal or preferably unequal molar concentration ratio, for example 50 to 1, 5 to 1 or 1 to 1.
- the size of amplification products is in the range of 40 to 40,000 nucleotides.
- step 22 the target nucleic acid is denatured by heating or changing pH to separate each strands of the target nucleic acid.
- the oligonucleotide primers are hybridized to the separated target nucleic acids in the location of the complementary sites.
- the concentrations of two stadandard oligonucleotide primers are different from each other, the extending direction of the oligonucleotide primer bound to the solid support is the same as that of the standard oligonucleotide primer having lower concentration and the sequences of these two oligonucleotide primers could be the same or different from each other.
- the binding site of the oligonucleotide primer bound to the solid support in the target nucleic acid is more downstream than that of the oligonucleotide primer having lower concentration, Therefore, the size of the amplified products between the oligonucleotide primer bound to the solid support and the standard oligonucleotide primer having higher concentration is equal to or smaller than that of the amplified products between the two standard oligonucleotide primers.
- step 24 the hybridized primers are extended using suitable enzyme, thermostable DNA polymerase such as Taq DNA polymerase, T4 DNA polymerase, Klenow fragment of E. coli DNA polymerase or reverse transcriptase.
- thermostable DNA polymerase such as Taq DNA polymerase, T4 DNA polymerase, Klenow fragment of E. coli DNA polymerase or reverse transcriptase.
- step 25 the steps 22 through 24 are repeated from 1 to 100 times.
- the important feature of this invention is the employment of equal or preferably unequal amount of two standard oligonucleotide primers and the addition of the oligonucleotide primer bound to the solid support.
- the efficiency of amplification with the two standard oligonucleotide primers is much higher than that of the oligonucleotide primer bound to the solid supports since the oligonucleotide primer bound to the solid support has a spatially limited hybridization condition with respect to the target nucleic acid.
- the two standard oligonucleotide primers accomplish the most amplification and then are exausted.
- the primer bound to the solid support is getting higher chance to hybridize to the target nucleic acids amplified from the standard oligonucleotide primers.
- the standard oligonucleotide primer having higher concentration forms the majority of extension products. As cycles go, the standard oligonucleotide primer having lower concentration is exhausted out. Accordingly, the primer bound to the solid support is getting higher chance to hybridize to the target nucleic acid amplifier from the standard oligonucleotide primers having higher concentration. As a result, the primer bound to the solid support and the standard oligonucleotide primer having higher concentration takes a chance of main amplification process.
- the employment of the primer bound to the solid support provides simple and convenient manipulation of the primer arrangement as well as isolation and detection of the amplification products bound to the solid support.
- the solid support containing the differentially amplified extension products is applied to fluorescently labeled probe for hybridization.
- fluorescently labeled single nucleotide such as fluorescently labeled dATP or fluorescently labeled dUTP can be also used for incorporation into the extension products. Therefore, detection of fluorescent signals on the spots contained in the extension products bound to the solid support is differentially achieved depending on the amount of the incorporation of fluorescently labeled single nucleotide into the extension products.
- Another way for detection is the use of the 5' end labeled oligonucleotide primers instead of the standard oligonucleotide primer having higher concentration.
- the efficiency of amplification for the primer bound to the solid support can be determined by the hybridization of the extension products from the 5 ' end labeled oligonucleotide primer having higher concentration.
- Biotin, Digoxigenin or radioisotope labeled nucleotide or primer can be used and detected by a suitable detection apparatus or process.
- the nucleic acids amplified from the oligonucleotide primer bound to the solid support is separated with other amplified nucleic acid by heating and changing pH and is detected with fluorescently labeled probe by hybridization and is also used as template for DNA sequencing.
- the solid support is of any solid material and has any shape that can hold oligonucleotide primers.
- the solid support may be of glass, magnetic silica, plastics, nylon, latex or metal and may have the shape of plate, bead, membrane or dipstick.
- DNA polymerase or reverse transcriptase is used for extension of the three oligonucleotide primers.
- the preferential DNA polymerase is thermostable enzyme such as Thermus Aquaticus DNA polymerase, Pfu DNA polymerase, vent DNA polymerase or
- the oligonucleotide primers hybridize to the target nucleic acids in a suitable temperature range, which is preferably 10 ⁇ 70 ° C .
- the extension of the hybridized oligonucleotide primers is performed in a suitable temperature range, which is preferably 20 ⁇ 40 ° C with thermostable enzyme such as T4 DNA polymerase or reverse transcriptase and 50 ⁇ 75 ° C with thermostable enzyme such as Tag DNA polymerase.
- the denaturation of the target nucleic acid is achieved by increasing temperature up to
- Another embodiment of the present invention provides a method for determining the position of known or unknown mutation by amplifying the target nucleic acid with two standard oligonucleotide primers and plural oligonucleotide primers bound to the solid support.
- the oligonucleotide primers bound to the solid support are complementary to multiple sites or sequential sites of the target nucleic acid.
- the positional arrangement such as serial spotting of plural oligonucleotide primers bound to the solid support allows simple detection of mutations in the area of the target nucleic acid.
- the oligonucleotide primers bound to the solid support hybridized to the mutation site can not efficiently be extended due to partial or complete mismatch between the amplified target nucleic acids and the oligonucleotide primers.
- spots containing the poorly-amplified nucleic acids bound to the solid support can be easily differentiated from spots containing the well-amplified nucleic acids bound to the solid support.
- Figs. 3A-3C illustrate, in accordance with one aspect of the present invention, a process of detecting mutation sites in the nucleic acid using the primer bound to the solid support.
- Fig. 3A shows an area of sequential binding sites 300 and locations of two standard oligonucleotide primers 320 and 330 at target nucleic acid 310a and 310b.
- Fig. 3B shows magnified examples of the oligonucleotide primers 340 bound to a solid support 350 whch are serially complementary to the area of sequential binding sites.
- Fig 3C shows oligonucleotide primers 340a-340v bound to the solid support 350 which are serially spotted on the solid support.
- the twenty-two arrayed primers 340a- 340v bound to the solid support 350 are employed for amplification along with the two standard oligonucleotide primers 320 and 330. If there are mutations inside of specific binding site 300, especially the 310a strand of the target nucleic acid, the efficiency of amplification with those primers is significantly low.
- Fig. 4 shows, in accordance with another aspect of the present invention, a process of amplifying plural target nucleic acids with plural sets of two standard oligonucleotide primers.
- step 41 amplification reaction mixture containing plural target nucleic acids, the mixture of four different triphosphates and enzyme is prepared.
- step 42 plural sets of two standard oligonucleotide primers which consist of 8 to 50 nucleotides are added.
- Two standard oligonucleotide primers are prepared in an equal or preferably unequal molar concentration ratio such as 50 to 1, 5 to 1 or 1 to 1.
- the size of amplification products is in the range of 40 to 40,000 nucleotides.
- step 43 the plural target nucleic acids are denatured by heating or changing pH to separate each strands of the target nucleic acids.
- step 44 the primers are hybridized to the respective target nucleic acids in the location of the complementary sites.
- step 45 the hybridized primers are extended using suitable enzyme, thermostable DNA polymerase such as Taq DNA polymerase, T4 DNA polymerase, Klenow fragment of E. coli DNA polymerase or reverse transcriptase.
- thermostable DNA polymerase such as Taq DNA polymerase, T4 DNA polymerase, Klenow fragment of E. coli DNA polymerase or reverse transcriptase.
- step 46 the steps 43 through 45 are repeated from 1 to 100 times.
- step 47 the steps 42 through 46 are repeated from 1 to 50 times.
- step 42 is contained in the repetitive cycling.
- the low concentration of plural sets of two different oligonucleotide primers such as less than 0.01 ⁇ m is applied. Therefore, the periodical addition of the oligonucleotide primers during amplication process is required.
- mixture of four different nucleoside triphosphates and enzyme may be added further in step 42 because four different nucleoside triphosphates and enzyme can be degenerated.
- reaction mixture may further contain plural oligonucleotide primers bound to at least one solid support and Biotin, Digoxigenin or radioisotope labeled nucleotides or primers may be used.
- the repetitive cycles of the steps are performed at robotic workstations which have an automatic multi-pipetting function and a programmable temperature cycling function.
- Example 1 the repetitive cycles of the steps are performed at robotic workstations which have an automatic multi-pipetting function and a programmable temperature cycling function.
- This example shows a method for amplification of a target nucleic acid from human genome.
- the amplification is performed in reaction mixture containing lOng of human genome DNA, 1.5 unit of Tag DNA polymerase, lOmM Tris-HCl, pH 8.3, 50mM KCl, 200 ⁇ M each dNTP, 1.5mM MgCl 2 , 30pmol 5' end fluorescently labeled primer, 3pmol primer and lOpmol primer immobilized to a 5mm ⁇ 5mmx0.5mm glass plate.
- the amplification reaction mixture is heated to 95 ° for 30 seconds for denaturation, cooled to 50 ° C for one minute for hybridization and heated to 72 ° C for one minute for extension, and these three steps are cycled 35 times.
- the glass plate containing the immobilized primers is isolated from the reaction mixture and washed using high stringency conditions to eliminate random background noise from the 5'end fluorescently labeled primers.
- the fluorescent signals of the hybridized amplification products on the glass plate can be detected by a fluorescent detector.
- This example shows a method for amplification of 20 different target nucleic acids from human genome.
- the amplification is performed in reaction mixture containing lOng of human genome DNA, 15 unit of Tag DNA polymerase, lOmM Tris-HCl, pH 8.3, 50mM KCl,
- the amplification reaction mixture is heated to 95 ° for 30 seconds for denaturation, cooled to 50 ° C for one minute for hybridization and heated to 72 ° C for one minute for extension, and these three steps are cycled 35 times.
- the glass plate containing the immobilized primers is isolated from the reaction mixture and washed using high stringency conditions to eliminate random background noise from the 5'end fluorescently labeled primer.
- the fluorescent signals of the hybridized amplification products on the glass plate can be detected by a fluorescent scanning detector.
- This example shows a method for amplification of a nucleic acid from human genome.
- the amplification is performed in reaction mixture containing lOng of human genome DNA, 0.1 unit of Tag DNA polymerase, lOmM Tris-HCl, pH 8.3, 50mM KCl, 200 ⁇ M each dNTP, 1.5mM MgCl 2 , lpmol 5' end fluorescently labeled primer, 0.05pmol primer and lOpmol primers immobilized to a 5mm ⁇ 5mm> ⁇ 0.5mm glass plate.
- the amplification reaction mixture is heated to 95 ° for 30 seconds for denaturation, cooled to 50 ° C for one minute for hybridization, heated to 72 ° C for one minute for extension, and added with lpmol 5' end fluorescently labeled primer,
- the glass plate containing the immobilized primer is isolated from the reaction mixture and washed using high stringency conditions to eliminate random background noise from the 5'end fluorescently labeled primer.
- the fluorescent signals of the hybridized amplification products on the glass plate can be detected by a fluorescent detector.
- Example 4 This example shows a method for amplification of 20 different nucleic acids from
- the amplification is performed in the reaction mixture containing lOng of human genome DNA, 0.5 unit of Tag DNA polymerase, lOmM Tris-HCl, pH 8.3, 50mM KCl,
- the amplification reaction mixture is heated to 95 ° for 30 seconds for denaturation. cooled to 50 ° C for one minute for hybridization, heated to 72 ° C for one minute for extension and added with 20different lpmol 5' end fluorescently labeled primers, 20 different 0.05pmol oligonucleotide primers and 0.05 unit Taq DNA polymerase after every 5 th extension step, and these four steps are cycled 45 times.
- the glass plate containing the immobilized primers is isolated from the reaction mixture and washed using high stringency conditions to eliminate random background noise from 5'end fluorescently labeled primer.
- the fluorescent signals of the hybridized amplification products on the glass plate can be detected by a fluorescent detector.
- This example shows a method for amplification of 10 different nucleic acids from human genome.
- the amplification is performed in reaction mixture containing lOng of human genome DNA, lOmM Tris-HCl, pH 8.3, 50mM KCl, 200 ⁇ M each dNTP, 1.5mM
- the amplification reaction mixture is heated to 95 ° for 30 seconds for denaturation, heated to 80 ° C for 30 sec to add ten different 0.5pmol 5' end fluorescently labeled primer, 10 different 0.5 pmol oligonucleotide primers and 0.1 unit of Taq DNA polymerase, cooled to 50 ° C for one minute for hybridization and heated to
- agarose or polyacrylamide gel electrophoresis is performed with the amplified product.
- amplication of target nucleic acid can be efficiently performed by using the oligonucleotide primer bound to the solid support together with unbounded standard oligonucleotide primers.
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Cited By (1)
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WO2007000408A1 (en) * | 2005-06-27 | 2007-01-04 | Siemens Aktiengesellschaft | Method for identifying nucleotide sequences, use of the method and test kit |
Citations (6)
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WO1990011369A1 (en) * | 1989-03-22 | 1990-10-04 | Cemu Bioteknik Ab | Solid phase diagnosis of medical conditions |
WO1995006753A1 (en) * | 1993-09-03 | 1995-03-09 | The United States of America, represented by The Secretary, Department of Health & Human Services | Method and compositions for primer specific solid-phase detection of pcr products |
WO1995015400A1 (en) * | 1993-12-03 | 1995-06-08 | The Johns Hopkins University | Genotyping by simultaneous analysis of multiple microsatellite loci |
WO1996036735A1 (en) * | 1995-05-19 | 1996-11-21 | American Type Culture Collection | A mycoplasma polymerase chain reaction testing system using a set of mixed and single sequence primers |
EP0787205A1 (en) * | 1994-10-28 | 1997-08-06 | Genset Sa | Solid-phase nucleic acid amplification method and reagent kit therefor |
WO1999047701A1 (en) * | 1998-03-18 | 1999-09-23 | november Aktiengesellschaft Gesellschaft für Molekulare Medizin | Method for detecting a nucleotide sequence |
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Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1990011369A1 (en) * | 1989-03-22 | 1990-10-04 | Cemu Bioteknik Ab | Solid phase diagnosis of medical conditions |
WO1995006753A1 (en) * | 1993-09-03 | 1995-03-09 | The United States of America, represented by The Secretary, Department of Health & Human Services | Method and compositions for primer specific solid-phase detection of pcr products |
WO1995015400A1 (en) * | 1993-12-03 | 1995-06-08 | The Johns Hopkins University | Genotyping by simultaneous analysis of multiple microsatellite loci |
EP0787205A1 (en) * | 1994-10-28 | 1997-08-06 | Genset Sa | Solid-phase nucleic acid amplification method and reagent kit therefor |
WO1996036735A1 (en) * | 1995-05-19 | 1996-11-21 | American Type Culture Collection | A mycoplasma polymerase chain reaction testing system using a set of mixed and single sequence primers |
WO1999047701A1 (en) * | 1998-03-18 | 1999-09-23 | november Aktiengesellschaft Gesellschaft für Molekulare Medizin | Method for detecting a nucleotide sequence |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2007000408A1 (en) * | 2005-06-27 | 2007-01-04 | Siemens Aktiengesellschaft | Method for identifying nucleotide sequences, use of the method and test kit |
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