WO1990011374A1

WO1990011374A1 - Polymerase chain reaction products incorporating reporter moieties and affinity seperation

Info

Publication number: WO1990011374A1
Application number: PCT/US1990/001534
Authority: WO
Inventors: Frank Worden Hobbs, Jr.; Gerald Joseph Litt; Jeffrey Allan Miller
Original assignee: E.I. Du Pont De Nemours And Company
Priority date: 1989-03-27
Filing date: 1990-03-26
Publication date: 1990-10-04
Also published as: JPH04504203A; CA2012982A1; AU5359690A

Abstract

A nucleic acid assay which incorporates a deoxyribonucleotide triphosphate containing a reporter moiety into a nucleic acid amplification process, followed by the detection of the reporter moiety, is provided.

Description

TJTL-E

"Polymerase Chain Reaction Products Incorporating Reporter Moieties and Affinity Seperation"

FIELD OF INVENTION This invention relates to the detection of nucleic acid sequences and more specifically to a process of combining amplification of target nucleic acid sequences with detection of a reporter group specifically incorporated into the target nucleic acid sequence .

BACKGROUND OF THE INVENTION

The development of practical nucleic acid hybridization methods which can be used for detecting nucleic acid sequences of interest has been limited by several factors. These include lack of sensitivity, complexity of procedure, and the desire.to convert from radiometric to nonradiometric detection methods. A variety of methods have been investigated for the purpose of increasing the sensitivity nonradiometric procedures. In one general approach, improvements in the total assay procedure have been examined, with concomitant effects on the issues of complexity and nonradiometric detection. In another approach, methods which increase the amount of nucleic acid to be detected by such assays have been pursued. U.S. Patent 4,358,535, issued to Falko , describes a method of culturing cells to increase their number and thus the amount of nucleic acid of the organism suspected to be present, depositing the sample onto fixed support, and then contacting the sample with a labeled probe, followed by washing the support and detecting the label. One drawback to this method is that without culturing the organism first, the assay does not have adequate sensitivity. Adding a culture step, however, is time consuming and not always successful. Maniatis et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, pp.390-401 (1982), describe a method in which a nucleic acid of interest is amplified by cloning it into an appropriate host system. Then, when the host organism replicates in culture, the nucleic acid of interest is also replicated. This method also suffers from the requirement to perform a culture step and thus provides for a procedure that is time consuming and complicated.

An alternative approach to increasing the quantity of nucleic acids of organisms has been described in U.S. patents 4,683,202 and 4,683,195. These patents disclose "a process for amplification and detection of any target nucleic acid sequence contained in a nucleic acid or mixture thereof". This process employs an in vitro cycling mechanism which doubles the nucleic acid sequence to be amplified after each cycle is complete. This is carried out by separating the complementary strands of the nucleic acid sequence to be amplified, contacting these strands with excess oligonucleotide primers and extending the primers by enzymatic treatment to form primer extension products that are complementary to the nucleic acid annealed with each primer. The process is then repeated as many times as is necessary. An advantage of this method is that it can rapidly produce large quantities of a small portion of the sequence of the nucleic acid of an organism of interest. A disadvantage of this method is that the detection of the nucleic acids produced, using a direct assay method, is complicated in that the amplification process can produce nucleic acid sequences w c are no a u cop es o e original nucleic acid which was to be copied. These erroneous nucleic acid sequences can provide false positives in the assay which increase the background noise and thus decrease the sensitivity of the entire method.

Numerous DNA probe assays have been described in the past for the detection of nucleic acids of interest. Falkow's method (above) first renders the target nucleic acid single-stranded and then immobilizes it onto a solid support. A labeled probe which is complementary to the target nucleic acid is then brought into contact with the solid support. Any excess probe is washed away and the presence of the label in the resulting hybrid is determined. A disadvantage of this method is that it is time consuming and cumbersome. The assay steps, i.e., hybridization and washing steps are carried out in a sealed pouch which contains the membrane (solid support) as well as the buffer solution.

Hill et al., WO 86/05815, describe a variation of the above assay format employing nitrocellulose coated magnetic particles to which the target DNA is affixed, followed by direct hybridization with a biotinylated probe and detection using a streptavidin-conjugated reporter.

Dunn et al.. Cell. Vol. 12, 23-36 (1977), describe a different hybridization format which employs a two-step sandwich assay method employing polynucleotide probes in which the target nucleic acid is mixed with a solution containing a first or capture probe which has been affixed to a solid support. After a period of time, the support is washed and a second or reporter (labeled) probe, also complementary to the target nucleic acid but not to the capture probe, is added and allowed to hybridize with the capture probe - target nucleic acid complex. After washing to remove any unhybridized reporter probe, the presence of the reporter probe, hybridized to the target nucleic acid, is detected.

Ranki et al. U.S. patent 4,563,419, disclose EPA 0 154 505, W086/03782, and EPA 0 200 113. It is to be recognized that all of these employ an assay procedure in which the first or capture probe is immobilized onto a solid support prior to hybridization.

A further variation has been described in German Preliminary Published Application 3,546,312 Al. This method, like that described by Ranki et al., employs a capture probe and a reporter probe which hybridize to distinct portions of the target nucleic acid. The target nucleic acid is contacted in solution by the two probes. The first, or capture probe, contains a binding component, such as biotin, that is capable of binding with a receptor component, such as streptavidin, which has been affixed to a solid support. After formation of the capture probe - target nucleic acid - reporter probe complex, a streptavidin-modified solid support is added. Any unhybridized reporter probe is washed away followed by the detection of the label incorporated into the complex bound to the solid support. An advantage of this technique over that disclosed by Ranki et al. is that the hybridization, which takes place in solution, is favored kinetically. Some disadvantages are that the length of the target nucleic acid affects the overall efficiency of the reaction which decreases with increasing target nucleic acid length. Also, sandwich nucleic acid probe assays, whether heterogeneous two-step or one-step, or utilizing solution hybridization, are not as sensitive as the direct assay method.

DISCLOSURE OF THE INVENTION

The nucleic acid assay of this invention for the detection and/or measurement of a preselected nucleic acid sequence in a sample suspected of including a nucleic acid containing said preselected sequence comprises the steps of:

(A) rendering the target nucleic acid single- stranded;

(B) amplifying at least one specific nucleic acid sequence contained within the preselected nucleic acid sequence in the presence of at least one deoxyribonucleotide triphosphate containing a reporter moiety in an amount up to the total replacement of the corresponding dNTP, by (1) treating the strands with two oligonucleotide primers, for each different specific sequence being amplified, under conditions such that for each different sequence being amplified an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein said primers are selected so as to be sufficiently complementary to the different strands of each specific sequence to hybridize therewith such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer; (2) separating the primer extension products from the templates on which they were synthesized to produce single-stranded molecules; and (3) treating the single-stranded molecules generated from step (2) with the primers of step (1) under conditions that a primer extension product is synthesized using each of the single strands produced in step (2)- as a template; (4) repeating steps (2) and (3) to produce sufficient primer extension product for detection and/or measurement; (C) rendering the product of step (B) (4) single-stranded;

(D) contacting the product of step (C) with an oligonucleotide which is attached to a solid support and which is complementary to a portion of a primer extension product not including the nucleic acid sequences defined by both primers;

(E) removing any unhybridized material; and

(F) detecting and/or measuring the reporter moiety immobilized on the solid support.

DETAILED DESCRIPTION OF THE INVENTION

The nucleic acid assay of this invention comprises the following overall process for the detection of target nucleic acids of a preselected sequence: a) Using the polymerase chain reaction (PCR) nucleic acid amplification method described in U.S. 4,683,202, incorporated herein by reference, specific nucleic acid sequences are amplified by annealing the denatured target nucleic acid present in the sample w pr r r rm ng extension products. In this process, a deoxyribonucleotide triphosphate containing a reporter group (moiety) , dNTP-R, is used to replace some or all of at least one of the corresponding deoxyribonucleotide triphosphates (dNTP) employed. Each extension product formed is complementary to a portion of the preselected nucleic acid sequence contained within the target nucleic acid and becomes a template for further primer binding. This process is then repeated as necessary in order to produce the desired amount of primer extension product for detection and/or measurement. b) The resulting nucleic acid is rendered single-stranded by known methods, such as treatment with heat, chaotropic agents, or by raising or lowering the pH. The single-stranded nucleic acid so produced is then contacted with a capture probe which is attached to a solid support and allowed to hybridize with it. c) The amplified nucleic acid - capture probe complex is washed with appropriate buffers to remove unhybridized product from above and any unincorporated dNTP-R. The presence and quantity of the reporter group is detected and/or measured and is proportional to the amount of amplified target nucleic acid. The amount of amplified target nucleic acid present, in turn, is proportional to the unamplified target nucleic acid originally present in the sample. In an alternative detection method, a labeled antibody to the reporter group incorporated during the amplification process, is employed. It is brought into contact with the amplified product before or subsequent to capture of the amplified product. The label on the antibody can then be used to detect the presence of the amplified product.

The term "PCR" as used herein in referring to the process of amplifying target nucleic acid sequences employing primer oligonucleotides to produce by enzymatic means a greatly increased number of copies of a small portion of the target nucleic acid is described in U.S. patent 4,683,202.

The term capture probe as used herein refers to an oligonucleotide which is complementary to a portion of a preselected sequence of the target nucleic acid and which is attached to a solid support. The capture probe cannot be complementary to either primer or to those portions of a primer extension product whose nucleic acid sequences are defined by the primers. Reporter group-containing deoxyribonucleotide triphosphates are known materials. Reporter groups of interest include fluorescent compounds such as fluorescein. In the alternative detection method, the reporter group has to be a moiety capable of forming a stable complex with an antibody.

Useful reporter groups in this invention include: fluorescein, rhodamine, and other chromogenic or fluorogenic compounds. The PCR target amplification reaction requires approximately 20 to 30 repeat cycles in order to produce a sufficient quantity of the amplified target nucleic acid for further hybridization. Denaturation of the amplified nucleic acid can be accomplished by treatment with alkali, acid, chaotropic agents, or heat, although the preferred method is to place the amplified target nucleic acid in a boiling water bath for at least 10 minutes followed by a chilled water bath (4°C) for at least two minutes. The hybridization step can be accomplished by contacting the amplified single-stranded target nucleic acid containing the reporter group(s) in solution with the capture probe, which is attached to a solid support, in an appropriate buffer, for a period of from 1 to 30 minutes. The length of the capture probe is determined by the ease of its synthesis, by the desired reaction kinetics, and by the identity of the primers, and preferably is an oligonucleotide of approximately 20 to 30 bases. The capture probe can be attached to the solid support by known means through sugar groups, preferably through either the 5'-terminal or the 3'-terminal sugar groups; or by attachment through a modified nucleotide base group.

A variety of solid supports can be utilized. Among solid supports are included magnetic particles, such as the chromium dioxide particles disclosed by Lau et al. , U.S. Patent 4,661,408, incorporated herein by reference, microtiter plates, and membranes.

To remove non-hybridized amplified target nucleic acid and unincorporated reporter-group modified deoxyribonucleotide triphosphate, the immobilized target nucleic acid on the solid support can then be washed several times, for example, in the temperature range of 25°C - 37°C, for approximately 5 to 10 minutes per wash cycle.

A variety of known detection methods can be utilized in the assay of this invention depending on the type of the reporter groups incorporated into the amplified product. When the reporter group is a chromophor or fluorophor, the incorporated reporter group can be detected by known spectroscopic techniques. Alternatively, a labeled antibody to the reporter group, incorporated during the amplification process, is brought into contact with the amplified product before or subsequent to capture of the amplified product by hybridization to the capture probe. The label on the antibody can then be used to detect the presence of the amplified product subsequent to the washing step.

The Example below exemplifies the invention.

EXAMPLE Detection of HIV I

A. Amplification of Target Nucleic Acid bv PCR

The procedure as described in U.S. Patent 4,683,202 and in a product bulletin for GeneAmp DNA Amplification Reagent Kit (#N801-0043) can be followed utilizing the following specific conditions and reagents. A 103-nucleotide base sequence located within the GAG pl7 region of HIV I, incorporated into a plas id (the plasmid incorporating most of the HIV I genome is designated pBH10-R3) , can be amplified using primers A and B as shown below:

5'-TGGGCAAGCAGGGAGCTAGG

Primer A 5'-TCTGAAGGGATGGTTGTAGC Primer B

Aliquots of serial dilutions (lxl0⁺⁷, lxl0⁺⁶, lxl0⁺⁵, lxlO÷⁴, 1x10+³, lχl0+², lxl0⁺¹, and zero copies) of plasmid pBH10-R3 can be amplified using PCR. Each aliquot can be combined with a buffer 200 μM in each of dATP, dTTP, dCTP, and dGTP and 10 μM in succinyl-fluorescein dTTP, 1.0 μM in each of Primer A and Primer B, and containing 1 μg of human placental DNA/reaction and 2.5 units of a DNA polymerase enzyme in a total reaction volume of 100 μl. Each reaction mixture can then be temperature cycled as described in the product bulletin thirty (30) times.

This process is expected to result in the estimated increase in the number of target molecules by lxl0⁺⁵ to lxl0⁺⁶.

B. Hybridization

1) Denaturation: 10 μl of amplified target DNA from each of the above aliquots can be combined with 30 μl of H2O in a 1.5-ml Eppendorf tube and placed in a boiling water bath for 10 minutes. The tubes can then be transferred to a chilled water bath (4°C) for two minutes and then centrifuged in a microcentrifuge for 10 seconds.

2) Hybridization:

The denatured samples can be split into two 20-μl portions (in Eppendorf tubes) in order that duplicate samples could be run. 100 μl of hybridization mix, pre-equilibrated at 37°C for 10 minutes, can then be added. Hybridization buffer can be prepared by combining: 3 ml of 20X SSC, pH 7.0, 0.1 ml of Triton X-100, an alkylaryl polyether alcohol having 9-10 ethoxy units, 1.0 ml of deionized formamide, 6.375 ml of H2O, and 25 μl of 1.0 N HC1.

12 μl of capture oligonucleotide bound to chromium dioxide particles (12 μg) , prepared as described in U.S. 4,661,408, can then be added to the samples, incubated for 10 minutes at 37°C, centrifuged in a microfuge for 5 seconds, and placed in a Corning magnetic rack for two minutes at 25°C. The pellets can then be washed three times at 25°C by adding 200 μl of wash buffer containing IX SSC, pH 7.0, and 0.17% Triton X-100, mixing, placing the samples in a magnetic rack for 2 minutes, and removing the wash buffer.

3. Detection

Detection can be accomplished by adding 200 μl of 10 mM Tris, pH 7.0, to each sample and placing them in boiling water bath for 10 minutes. The tubes can then be transferred to a chilled water bath (4°C) for two minutes and centrifuged in a microcentrifuge for 10 seconds. 200 μl portions can then be removed from each tube and the amplified nucleic acid product detected by direct fluorescence visualization.

Claims

1. A nucleic acid assay for the detection and/or measurement of a preselected nucleic acid sequence in a sample suspected of including a nucleic acid containing said preselected sequence comprises the steps of:

(A) rendering the target nucleic acid single-stranded;

(B) amplifying at least one specific nucleic acid sequence contained within the preselected nucleic acid sequence in the presence of at least one deoxyribonucleotide triphosphate containing a reporter moiety in an amount up to the total replacement of the corresponding dNTP, by (1) treating the strands with two oligonucleotide primers, for each different specific sequence being amplified, under conditions such that for each different sequence being amplified an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein said primers are selected so as to be sufficiently complementary to the different strands of each specific sequence to hybridize therewith such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer; (2) separating the primer extension products from the templates on which they were synthesized to produce single-stranded molecules; and

(3) treating the single-stranded molecules generated from step (2) with the primers of step (1) under conditions that a primer extension product is synthesized using each of the single strands produced in step (2) as a template;

(4) repeating steps (2) and (3) to produce sufficient primer extension product for detection and/or measurement: (C) rendering the product of step (B) (4) single-stranded; (D) contacting the product of step (C) with an oligonucleotide which is attached to a solid support and which is complementary to a portion of a primer extension product not including the nucleic acid sequences defined by both primers;

(E) removing any unhybridized material; and

(F) detecting and or measuring the reporter moiety immobilized on the solid support.

2. The assay of claim 1 wherein said preselected nucleic acid sequence is HIV I DNA.

3. The assay of claim 1 wherein said reporter moiety is a fluorescent moiety.

4. The assay of claim 1 wherein said reporter moiety is an antigenic moiety.