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WO1999037820A1 - Methode d'amplification exponentielle d'un acide nucleique utilisant une seule amorce de polarite opposee - Google Patents

Methode d'amplification exponentielle d'un acide nucleique utilisant une seule amorce de polarite opposee Download PDF

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Publication number
WO1999037820A1
WO1999037820A1 PCT/US1999/000834 US9900834W WO9937820A1 WO 1999037820 A1 WO1999037820 A1 WO 1999037820A1 US 9900834 W US9900834 W US 9900834W WO 9937820 A1 WO9937820 A1 WO 9937820A1
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Prior art keywords
nucleic acid
primer
target
amplification
region
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PCT/US1999/000834
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English (en)
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Paul Haydock
Jack U'ren
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Saigene Corporation
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Priority to AU22285/99A priority Critical patent/AU2228599A/en
Publication of WO1999037820A1 publication Critical patent/WO1999037820A1/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
    • C12Q1/68—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]

Definitions

  • This invention relates to the amplification of nucleic acid using single nucleic acid primer having two 3' ends.
  • the present invention describes a novel nucleic acid amplification method utilizing a unique primer structure referred to as a single opposing polarity primer.
  • nucleic acid amplification technologies are now available. This method is distinct in that only a single primer addition is required for amplification.
  • This new primer structure can be synthesized on a standard nucleic acid synthesizer using commercially available reagents.
  • use of the single opposing polarity primer has the advantages that only a single primer need be added to the amplification mix, that since the single primer has two sequence specificities these are added to the reaction in perfect balance, that the unique dual 3' end of the primer avoids 5' exonucleolytic degradation, and that the amplification products all have perfectly single stranded tails that may aid in product detection by hybridization. 2
  • This invention provides for a method for amplification of a target nucleic acid sequence using 3' enzymatic extension and a single amplification primer.
  • the method comprises the steps of: (a) contacting in an aqueous solution the target nucleic acid having three defined regions said regions being a 5' P region, an intervening S region and a 3' Q region with an oligonucleotide primer comprising two 3' ends connected through a bond or linking composition wherein the first 3' end, 3P-1 is able to hybridize to the 3' Q region of the target nucleic acid sequence and the second 3' end 3P-2 is able to hybridize to the complement of the 5 'P end of the target oligonucleotide sequence; and, (b) further contacting the solution of step i with a DNA polymerase that carryouts 3 ' DNA extension wherein said contacting is (a) under conditions in which the primer is able to hybridize to either the complement of the 3'Q region or the 5
  • the copy is separated from the template target sequence, the primer hybridized to both the original target and the 3' extension copy and additional copies of the S region synthesized by 3' extension of the primers using DNA polymerase. Separation may be achieved by heat denaturation or by a helicase enzyme.
  • a preferred polymerase is thermostable as it resists denaturation under temperatures required to denature duplexed nucleic acid.
  • the target nucleic acid may be a double stranded DNA.
  • the amplification method can use a primer wherein the 3'P-l and 3'P-2 are linked by nucleotides.
  • the method can also include an amplification primer is covalently linked to a detectable reporter.
  • the method of this invention can involve target nucleic acid that is diagnostic of an animal or plant pathogen and where the target nucleic acid is an animal or plant gene.
  • nucleic acid amplification primers comprising nucleic acid sequences having two 3 ' ends where both ends complement different nucleic acid sequences of a target double stranded nucleic acid sequence where the target has three defined regions: a 5' P region, an intervening S region and a 3' Q region; 3 wherein the primer has a first 3' end, 3P-1 which is able to hybridize to the 3'Q sequence of the target nucleic acid sequence and a second 3' end, 3P-2 which is able to hybridize to the complement of a 5'P end of the target oligonucleotide sequence.
  • the primers can be directed to any type of target and be labelled as described above in the method provided herein.
  • aqueous solutions comprising the nucleic acid amplification primers described herein.
  • the solution can also comprise the amplification reaction components described above such as thermostable DNA polymerases, helicase enzymes and assorted target nucleic acids including genes of any sort and pathogens that need to be diagnosed in medical or agricultural applications.
  • Fig. 1 is a schematic representation of the amplification system.
  • Fig. 2 is a single primer of opposing polarity exponential amplification of the E. coli GroEL gene.
  • Fig. 3 is a single primer of opposing polarity exponential amplification of the HIV RT gene.
  • amplification refers to the duplication of a target nucleic acid while in its broadest context the term includes a single duplication, optimum use of the invention involves a series of repetitive steps that result in exponential duplication of a target nucleic acid.
  • complement refers to a strand of nucleic acid which is hybridized to, or which has the ability to hybridize to a reference strand of nucleic acid.
  • An exact complement indicates that the every base in the reference strand matches the bases in the hybridizing strand with which it can form hydrogen bonds.
  • An inexact complement means that at least one mismatched base is present between the reference strand and the hybridizing strand.
  • covalently linked refers to a bond between two distinct molecular species that is the result of shared electrons between constituent atoms of each species.
  • detectable reporter refers to a molecular species which can be identified based on either enzymatic activity or by chemical properties, and which may be used to identify a successful hybridization reaction.
  • an oligonucleotide may be radiolabeled which can be measured by autoradiography or scintillation counting.
  • Horseradish peroxidase is commonly used to identify hybridization when conjugated to a signal oligonucleotide either covalently or though a biotin residue.
  • diagnosis refers to a kit or a process which is used in identification of a specific nucleic acid sequence.
  • “Diagnostic” typically refers to medical and veterinary applications in which a disease causing species is being detected. In a broader sense, “diagnostic” may be referred to forensic or food applications in which specific nucleic acid sequences can refer to either contaminating food pathogens, adulteration with undesired food fillers, or for specific sequences used to identify an individual in forensic applications.
  • Another form of medical "diagnostic” may refer to a test for a specific DNA sequence associated with a genetic disease, or a predisposition to a disease state such as cancer.
  • hybridize refers to a process by which two single nucleic acid strands interact and form a stable or quasi-stable double stranded helical structure.
  • the two strands interact via hydrogen bonding between bases on opposing strands, with adenosine forming a hydrogen bond with either thymidine in DNA or uracil in RNA, and guanosine forming a hydrogen bond with cytosine.
  • nucleic acid refers to either double or single stranded DNA or RNA.
  • polarity refers to the directionality of the information encoded on a strand of nucleic acid. Nucleic acids are generally represented and read in the 5' to 3' direction. When representing a double-stranded nucleic acid, the upper strand is generally drawn as 5' to 3' while the lower complementary strand is drawn 3' to 5' .
  • opposite polarity refers to a switch in the natural polarity of a nucleic acid strand within a contiguous sequence. For example, a single DNA strand has polarity written as 5' to 3'. Therefore, the natural direction of reading this strand would be 5' to 3', or left to right. If a second segment of DNA could be read from the 5' to 3' direction from right to left (with the 3' end of the primer to the left), then even though the polarity is the same for both segments (5' to 3'), the polarities point outwards from a common 5' end of attachment in opposing directions. 5
  • single amplification primer refers to a single molecular species which can bind to a target nucleic acid template sequence and directs the synthesis of a new nucleic acid strand complementary to the template strand.
  • target refers to any nucleic acid species which may hybridize with the single opposing polarity primer and which can be copied by a polymerase activity.
  • 3' enzymatic extension refers to an activity carried out by a polymerase molecule in the addition of nucleotides to the 3 ' end of a nucleic acid molecule.
  • thermoostable DNA polymerase refers to an enzymatic activity that can be isolated from a number of bacterial or ' mammalian sources which exhibits the ability maintain enzymatic activity in response to repeated cycles of heating and cooling.
  • the present invention relates to a new method for the amplification of nucleic acid sequences utilizing a single primer of unique structure.
  • This amplification method is expected to have broad applicability in the areas of genetic research, diagnostics, and forensics. Methods for the production of the single amplification primer, reaction conditions, and diagnostic kits are described. Many techniques for the amplification of nucleic acid sequences have been described. The best known is the Polymerase Chain Reaction (or PCR). Others include the Ligase Chain Reaction, transcription based nucleic acid amplification such as NASBA, and single primer amplification systems such as SPA. PCR and LCR both require temperature cycling to cause nucleic acid amplification, while NASBA is an isothermal reaction.
  • nucleic acids have strand polarity. That is, sequences are directional defined by the chemical bond formed between adjacent bases within the nucleic acid strand. Thus, the 5' carbon of the sugar moiety of each nucleotide is linked to the 3' carbon of the sugar moiety in the adjacent nucleoside. Therefore, every natural nucleic acid strand has both a 5' and a 3' end.
  • the upper strand usually is drawn in the 5' to 3' direction.
  • the lower complementary strand would have the opposite polarity, or 3' to 5' .
  • All polymerases 6 require a free 3' hydroxyl group onto which nucleotides are added.
  • oligonucleotides are synthesized having a single 5' to 3' polarity. Therefore, for an amplification reaction such as PCR, two oligonucleotides must be synthesized, each complementary to the opposite strands.
  • a novel aspect of this invention is that this amplification method requires only a single primer having two 3' ends.
  • the single primer contains two sequence specificities defining the region of the target which is to be amplified. The two sequence specificities are arranged in opposite polarities and joined through their 5' ends as shown below:
  • this structure Since this structure has two 3' ends, it does not have a classical oligonucleotide structure which would normally have a single polarity sequence running 5' to 3'.
  • the bond between both sequence specificities can be directly through a phosphodiester linkage, or may be through other spacer arms of varying length.
  • the spacer arm itself may have functional groups useful in the analysis of the amplification products.
  • each sequence specificity may contain detectable groups such as biotin. Sequences may also be introduced that would allow cloning of amplification products, or production of RNA transcripts corresponding to the amplified product.
  • the use of the single primer in an amplification reaction is shown in Figure 1. Because the single primer contains both sequence specificities, only one addition of this primer is required for amplification.
  • the template strand is denatured so that the primer may hybridize to its complement.
  • the 3' end of the primer is then extended by a polymerase activity forming the complementary strand.
  • the two strands are once again denatured allowing binding of the second sequence specificity of the single primer.
  • the 3' end can then be extended once again by the polymerase activity to complete the second strand.
  • the product of one round of amplification will then have single strand tails.
  • this amplification system might not yield discrete products, but rather a smear of concatameric products. This is because the product of the amplification has functional priming sequences on it's ends which can bind to both template strands yielding amplification products joined through the opposing primer. Multiple cycles would then lead to concatameric products of increasing length. This potential problem may be exacerbated in the later amplification cycles when the product template concentration is much higher, and the primer concentration is lower which would encourage product primer binding to both template strands. The data presented below demonstrates that this does not happen, or if it does happen, it constitutes a very minor secondary reaction. In fact, very discrete amplification products were obtained.
  • the presence of the second sequence specificity with opposite polarity does not interfere with the hybridization of the first sequence specificity.
  • the use of the single opposing polarity primer is demonstrated using temperature cycling to effect strand denaturation.
  • any method to cause strand separation can be utilized including chemical methods and enzymatic activities such as that derived from helicase proteins.
  • RNA polymerase recognition sequences can likewise be incorporated into the primer for the generation of RNA transcripts form the amplification product.
  • the RNA polymerase binding site is generally 20 - 30 bases long.
  • RNA transcripts prepared this way are convenient research tools, or can be used to prepare controls for diagnostic kits.
  • Some additional features of the amplification system described in this application include the following. For diagnostic assays, only a single primer addition is required, which has built in both sequence specificities in a perfect balance. Also, some thermostable polymerases do have 5' - 3' nuclease activity which can result in the removal of 5' terminal groups, an activity which would be avoided in this system. In addition, products of the single primer amplification have single stranded tails on completed products which may be useful in hybridization detection of the reaction products.
  • the single opposing polarity primer may easily be prepared on a standard DNA synthesizer.
  • 3' nucleotide phosphoramidites are added to the 3' most base of the sequence being synthesized in a step-wise fashion.
  • 5' phosphoramidites are also available which can be added on the synthesizer in the opposite direction. Therefore, one would synthesize the first sequence specificity using the standard 3' nucleotide phosphoramidites. When the 5' end of that sequence is reached, the 5' phophoramidites are then added to complete the second sequence specificity.
  • a second method would be chemical linkage of two oligonucleotides with
  • Modifying reagents could be bifunctional or homofunctional.
  • the first oligonucleotide could be synthesized directly having a 5' sulfhydryl group.
  • the second oligonucleotide could be synthesized having a 5' amine group.
  • the 5' amine could be modified to a sulfhydryl specific 5' iodo- group using the bifunctional cross-linking agent Succinimidyl(4-iodoacetyl) Amino Benzoate (SIAB).
  • the sulfhydryl and iodo- modified oligonucleotides could be mixed and allowed to react. It would then be necessary to purify the opposing polarity primer from the reaction mix by HPLC, size exclusion chromatography, or gel electrophoresis. Homofunctional cross-linking reagents could likewise be used. For example, the first oligonucleotide could be reacted with a large excess of the reagent cyanuric chloride. Since the reaction products will include 9 oligonucleotide dimers, the reaction mix would be purified by HPLC. Singly modified oligonucleotide would then be mixed with the second oligonucleotide containing a 5 ' reactive group. The single opposing polarity primer would then be purified as above.
  • Bacterial samples are collected by centrifugation. Bacteria are protease digested and then lysed with SDS. The lysate is extracted 1 - 2X with buffer saturated phenol. Total nucleic acids are then precipitated with 2.5 vol. ethanol. The nucleic acid preparation can be stored at this point at -20 C. Alternatively, the nucleic acid may be collected by centrifugation and resuspended in buffer or water. The nucleic acid is stored at 4 C. ii. Eukaryotic DNA Common samples include blood and cell culture samples. In the case of blood, density centrifugation is first used to obtain a purified white cell population. Cells are suspended in a buffer containing 1 % Tween 20.
  • DNA is then prepared by protease digestion of the nuclei, lysis with SDS, phenol extraction, and preferably dialysis of the very high molecular weight DNA.
  • the DNA may be collected by ethanol precipitation.
  • DNA is stored under ethanol at -20 C, or suspended in water or buffer at 4 C.
  • iii. Eukaryotic RNA Cells are suspended in highly concentrated guanidine thiocyanate. Highly purified RNA is prepared by layering the lysate over a cesium chloride cushion and spinning the sample in an ultracentrifuge overnight at high speed. RNA will form a pellet under the cesium layer while DNA will remain soluble. The RNA pellet is 10 collected and resuspended in water or buffer and stored at -70 C.
  • RNA is selectively precipitated from the guanidine thiocyanate lysate with 0.5 vol. ethanol. The precipitate is collected by centrifugation and resuspended in water for storage at -70 C.
  • Bacterial cells carrying plasmid DNA are collected by centrifugation.
  • the cells are resuspended in buffer containing lysozyme. After digestion, the cells are lysed with an alkaline SDS solution which also denatures the bacterial genomic DNA and plasmid DNA.
  • the suspension is then neutralized causing renaturation of the plasmid DNA and precipitation of the genomic DNA.
  • the precipitate is removed by centrifugation, and the plasmid DNA in the supernatant is precipitated with ethanol.
  • the plasmid DNA is suspended in water or buffer and then stored at -70 C.
  • the dual opposing polarity primer is designed much like primers for other amplification systems.
  • the length of each recognition sequence in the primer is chosen to be, but not limited to, a range of sizes from 15 to 30 bases.
  • the preferred GC content is 50% but other compositions may be used as well.
  • Modified bases such as those containing biotin or chemically reactive groups such as free primary amines may be incorporated into the primer.
  • Sterile distilled water is added to the primer to make a final concentration of lOOuM.
  • the concentrated primer solution is stored at -20 C. ii. Reaction Conditions
  • a typical reaction will contain the single opposing polarity primer at a final concentration of 3uM, buffer salts, 20mM Tris-HCl (pH8.4), 50mM KC1, 2mM MgCl 2 , and 200uM dNTPs, the template, and the DNA polymerase. Amplification reactions are run using a standard DNA thermocycler.
  • the magnesium concentration is critical for efficient amplification.
  • the concentration must be above 1.5mM.
  • 2mM is chosen as a sufficient concentration, but for certain templates and primers, it may be necessary to titrate the magnesium concentration to the optimum concentration.
  • KC1 The amount of salt added to the reaction (KC1) maintains a sufficient ionic strength for 11 binding of the primer during amplification.
  • salt concentrations above lOOmM may adversely affect the polymerase activity.
  • concentration of the nucleotides must be relatively low (0.2mM). If the concentration is too high, misincorporation of nucleotides may result in sequence errors.
  • the primer concentration must be controlled since if the concentration is too high, then mis-priming or non-specific priming may result.
  • thermostable polymerase For the single opposing polarity primer amplification reaction, it is necessary to use a thermostable polymerase.
  • thermostable polymerases A variety of thermostable polymerases are now available commercially including but not limited to Taq, Pfu, and Pwo. The amount of added polymerase can influence the specificity of the amplification reaction.
  • Too much polymerase can lead to non-specific amplification, while too little will decrease the yield of product.
  • 1 unit of Taq DNA polymerase is sufficient for specific amplification with good yield of product.
  • the single opposing polarity primer When using the single opposing polarity primer to amplify a sequence from an RNA template, it is first necessary to copy the RNA strand into DNA utilizing a reverse transcriptase activity.
  • a 20ul reaction mix contains 20mM Tris-HCl (pH8.4), 50mM KC1, 2mM MgCl 2 , and ImM dNTPs, at least luM single opposing polarity primer, template RNA, and sufficient reverse transcriptase activity.
  • reverse transcriptases are commercially available. Commonly used is AMV reverse transcriptase or MMLV reverse transcriptase.
  • the reverse transcription reaction is incubated for 1 hour at 42 C. Then, the reaction mix is diluted with water and buffer to maintain the salt concentration and primer concentration, while diluting the dNTPs to 0.2mM. The reaction is then processed by thermal cycling.
  • the melting temperature in which the template is converted to single strands there are three segments to a typical thermal profile, the melting temperature in which the template is converted to single strands, the annealing temperature in which the primer will bind to the template, and the extension temperature, in which the polymerase has maximum activity for primer extension.
  • the best thermal profile is determined empirically and is based on the sequence of each segment of the single opposing polarity primer. For example, from the sequence, one can determine the Tm of each segment of the primer. Then, the annealing temperature can be chosen as about 5 degrees below the Tm. The number of cycles is chosen based on the amount of input template, and the level of sensitivity required. 12 iii. Helicase Mediated Amplification
  • An ideal use of the single opposing polarity primer is the design of an isothermal amplification system utilizing helicase activity to separate DNA strands instead of using heat.
  • Helicase proteins separate double stranded DNA at the expense of hydrolysis of ATP.
  • Helicase binds to the 3' end of single stranded DNA with high affinity and translocate down the strand.
  • a number of helicase proteins have been identified and studied. It has been found that helicases oligomerize when they bind to a nucleic acid. Some, such as the E coli DnaB protein and the SV40 large T antigen, form hexamers when bound.
  • a hexamer due to it's size would require a large binding site making these proteins less suitable for an amplification mediating protein.
  • Some activities such as the SV40 T antigen also possess' an RNA helicase activity.
  • Others such as the E. coli Rep protein, the E. coli UvrD protein, and the HeLa helicase form dimers which require much smaller binding sites. The mechanism of action of some of these proteins has been studied in detail.
  • the Rep helicase isolated from E. coli has been shown to be able to "jump" polarities, a situation that would be encountered using the opposing polarity primer.
  • thermostable helicase protein which can be isolated from a number of thermophylic bacteria. Possible sources are Thermus aquaticus and certain Thermophillus species. This would allow the amplification to be done at higher temperature which would increase the specificity of primer binding. iv. Detection of Amplification Products Amplification products can be detected by a number of methods. The simplest is by analyzing a portion of the amplification product by gel electrophoresis.
  • Nonspecific amplification products would appear as bands of weaker intensity at varying mobilities from the specific product.
  • Gel electrophoresis is particularly convenient when amplification conditions are being developed, since it is relatively easy to determine the specificity of the amplification by the yield of specific product and lack of spurious products.
  • Amplification products from the single opposing polarity primer amplification reaction have slightly slower mobilities than amplification products without the single 13 strand tails.
  • a convenient method is based on forming a sandwich between the amplification product, a capture oligonucleotide linked to a solid phase, and a specific signal oligonucleotide.
  • the signal oligonucleotide may contain a reporter group which will be recognized by a binding molecule conjugated to an enzymatic activity.
  • the signal oligonucleotide may have a biotin group which would be recognized by a streptavidin-horseradish peroxidase conjugate.
  • a signal oligonucleotide may not be needed if the amplification product is labeled with biotin during the amplification, or if the primer itself is labeled with biotin. In either event, the presence of a successful amplification can be determined by incubating the solid phase in contact with a colorimetric substrate and measuring the amount of color generated.
  • the hybridizations may be carried out at elevated temperatures, or in the presence of chaotropic salts.
  • Salt concentration and temperature combine to define the stringency of the hybridization.
  • low salt concentration together with high temperature would be considered high stringency conditions.
  • High stringency conditions would be used under conditions where an exact match between the capture probe and the amplification product is desired. For example, when differentiating between amplification products differing by a single base pair mismatch, high stringency is required.
  • Low or relaxed stringency conditions consists of high salt and lower temperatures. These conditions would allow hybridization analysis of identical genes between closely related species.
  • stringency may be controlled by either high concentrations of chaotrope (high stringency), or low concentrations of chaotrope (low stringency).
  • the single opposing polarity primer amplification products may offer an advantage when only detection of a single species is desired. This is due to the single strand tails present on the amplification products.
  • the amplification product may be labeled with biotin during the amplification reaction. Then, either the 14 complement of the sequence of either tail may be used to capture the amplification product. Since the tails are always single stranded, the lack of strand competition should result in a very sensitive detection step.
  • the single opposing polarity primer amplification reaction is best used in the design of diagnostic kits.
  • the primer could be supplied in such a kit in either of two forms, as a separate reagent to be added to a reaction mix for amplification of a specified target, or in an optimized reaction buffer ready to be added to a sample with only addition of the enzyme being otherwise required.
  • a kit for the detection of E. coli would contain: 1. 2X reaction mix containing a buffer component, the single primer, magnesium salts, and deoxynucleotide triphosphates. This reagent may be supplied pre-dispensed in appropriate tubes ready to use 2. Sterile water to dilute the sample 3. Thermostable polymerase which may be part of the 2X reaction mix 4. Detection reagents consisting of capture oligonucleotides in a preadjusted buffer, and a reagent plate containing the remainder of the sandwich hybridization reagents.
  • PCR has been used to detect E. coli using two primers directed towards the GroEL gene.
  • the primers have the following sequences
  • a single primer was synthesized having the sequence
  • the reaction conditions were in lOOul total volume 20mM Tris-HCl (pH8.4), 50mM KC1, 2mM MgCl 2 , and 200uM dNTPs.
  • each of the two primers was added to a final concentration of luM.
  • the amount of added primer was varied.
  • Each reaction was run using 1 unit of Taq polymerase (Life Technologies) and lug of DNA. Samples were overlayed with mineral oil and thermocycled using the following program: lmin. at 94°C, 1 min at 50°C, 1 min at 72°C for 35 cycles, a 5 min final extension at 72°C, and a 4°C soak. The results of the experiment are presented in Fig. 2.
  • a control tube is run without the addition of template to assess the presence- of background amplification.
  • the PCR products using two oligonucleotide primers yields a single band as expected when the template DNA is included in the reaction mix. Without template, no product is obtained.
  • the single primer reaction yields a single amplification product that migrates more slowly than the PCR product. This is due to the presence of the single strand tails on the single primer amplification products. The yield of product is dependent on the input amount of the single opposing polarity primer.
  • Detection of HIV sequences was accomplished using primers directed towards the reverse transcriptase gene. Primers were prepared for a standard PCR amplification, as well as a single primer for the method used in this application. The sequences are: 16
  • a single primer was synthesized having the sequence
  • the reaction conditions were in 50ul total volume 20mM Tris-HCl
  • the "S” refers to the single primer amplification
  • the “D” refers to the single opposing polarity primer (or "double primer") amplification.
  • the single opposing polarity primer amplification product has a slower mobility than the corresponding PCR product.
  • the double primer system is more sensitive than the single primer system.
  • the single primer system is less sensitive than the double primer.
  • the sensitivities of both systems are comparable.

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Abstract

L'invention concerne l'amplification d'un acide nucléique au moyen d'une seule amorce d'acide nucléique présentant deux extrémités 3'.
PCT/US1999/000834 1998-01-26 1999-01-13 Methode d'amplification exponentielle d'un acide nucleique utilisant une seule amorce de polarite opposee WO1999037820A1 (fr)

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AU22285/99A AU2228599A (en) 1998-01-26 1999-01-13 An exponential nucleic acid amplification method using a single primer of opposing polarity

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
WO2004027025A3 (fr) * 2002-09-20 2004-07-08 New England Biolabs Inc Amplification dependant de l'helicase des acides nucleiques
US7662594B2 (en) 2002-09-20 2010-02-16 New England Biolabs, Inc. Helicase-dependent amplification of RNA

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US4683202A (en) * 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US5043272A (en) * 1989-04-27 1991-08-27 Life Technologies, Incorporated Amplification of nucleic acid sequences using oligonucleotides of random sequence as primers
US5508178A (en) * 1989-01-19 1996-04-16 Rose; Samuel Nucleic acid amplification using single primer
US5610017A (en) * 1991-12-11 1997-03-11 Igen, Inc. Method for conducting a polymerase chain reaction using an improved electrochemiluminescent label
US5612199A (en) * 1991-10-11 1997-03-18 Behringwerke Ag Method for producing a polynucleotide for use in single primer amplification

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US4683202A (en) * 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
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WO2004027025A3 (fr) * 2002-09-20 2004-07-08 New England Biolabs Inc Amplification dependant de l'helicase des acides nucleiques
JP2006500028A (ja) * 2002-09-20 2006-01-05 ニュー・イングランド・バイオラブズ・インコーポレイティッド 核酸のヘリカーゼ依存性増幅
US7282328B2 (en) 2002-09-20 2007-10-16 New England Biolabs, Inc. Helicase dependent amplification of nucleic acids
AU2003272438B2 (en) * 2002-09-20 2009-04-02 New England Biolabs, Inc. Helicase dependent amplification of nucleic acids
US7662594B2 (en) 2002-09-20 2010-02-16 New England Biolabs, Inc. Helicase-dependent amplification of RNA
US7829284B2 (en) 2002-09-20 2010-11-09 New England Biolabs, Inc. Helicase-dependent amplification of nucleic acids

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