CA2012983A1 - Process for nucleic acid detection by binary amplification - Google Patents
Process for nucleic acid detection by binary amplificationInfo
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- CA2012983A1 CA2012983A1 CA002012983A CA2012983A CA2012983A1 CA 2012983 A1 CA2012983 A1 CA 2012983A1 CA 002012983 A CA002012983 A CA 002012983A CA 2012983 A CA2012983 A CA 2012983A CA 2012983 A1 CA2012983 A1 CA 2012983A1
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- nucleic acid
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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
- C12Q1/702—Specific hybridization probes for retroviruses
- C12Q1/703—Viruses associated with AIDS
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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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Abstract
TITLE
PROCESS FOR NUCLEIC ACID DETECTION
BY BINARY AMPLIFICATION
ABSTRACT
A nucleic acid assay based on the enzymatic combination of the products of two amplification reactions carried out with two sets of two primers specific for two different regions within the target nucleic acid is provided.
PROCESS FOR NUCLEIC ACID DETECTION
BY BINARY AMPLIFICATION
ABSTRACT
A nucleic acid assay based on the enzymatic combination of the products of two amplification reactions carried out with two sets of two primers specific for two different regions within the target nucleic acid is provided.
Description
- ~0~29~33 ; N~-0234 PROCESS FOR NUCLEIC ACID DE~ECTION
BY BINARY AMPLIFIC~TION
FI~LD OF I~VEtlTIO~l This in~.~ention relates to the detection of nucleic acid sequences and more specifically to a process of combining the products f rom the amplification of two portions of a target nucleic acid sequence.
BACKGRO~ OF THE INV~TIOI~
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 ~or 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 Falkow, describes a method of culturing cells to increase their number and thus the amount of nucleic acid of the organism suspected to be present, depositinq 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 Z~3129~3 always successful. Maniatis et al., Mole~ular Clonina: A L2boratorY 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 ou~ 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 which are not faithful copies of the original nucleic acid which was to be copied. These 3 . 20129~3 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., ~811, 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 A1. 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 i5 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 reactlon 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.
20~29~33 A disadvantage of all of these techniques relates to the need to employ a specific hybridization step in order to obtain the necessary specificity and thus proper identification of the target nucleic acid sequence of interest.
DI~CLOSURE OF THE INVEN~IQN
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 two specific nucleic acid sequences contained within the preselected nucleic acid sequence, said specific nucleic acid sequences being positioned such that when either sequence is amplified under amplification conditions, the extension product of either sequence cannot serve as a template for the synthesis of the other sequence,by ~1) treating the strands with two sets of two oligonucleotide primers, one set 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 sets of primers are selected so as to be sufficiently complementary to the different strands of each specific sequence to hybridize therewith such that the extension 6 2~ 983 products synthesized from one primer from each of the two sets of primers, when separated from their respective complements, can serve as templates for the synthesis of the extension products of the other primer from each of the two sets of primers; wherein one of the primers of each set of primers contains a LoxP sequence at its S'-end;
BY BINARY AMPLIFIC~TION
FI~LD OF I~VEtlTIO~l This in~.~ention relates to the detection of nucleic acid sequences and more specifically to a process of combining the products f rom the amplification of two portions of a target nucleic acid sequence.
BACKGRO~ OF THE INV~TIOI~
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 ~or 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 Falkow, describes a method of culturing cells to increase their number and thus the amount of nucleic acid of the organism suspected to be present, depositinq 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 Z~3129~3 always successful. Maniatis et al., Mole~ular Clonina: A L2boratorY 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 ou~ 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 which are not faithful copies of the original nucleic acid which was to be copied. These 3 . 20129~3 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., ~811, 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 A1. 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 i5 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 reactlon 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.
20~29~33 A disadvantage of all of these techniques relates to the need to employ a specific hybridization step in order to obtain the necessary specificity and thus proper identification of the target nucleic acid sequence of interest.
DI~CLOSURE OF THE INVEN~IQN
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 two specific nucleic acid sequences contained within the preselected nucleic acid sequence, said specific nucleic acid sequences being positioned such that when either sequence is amplified under amplification conditions, the extension product of either sequence cannot serve as a template for the synthesis of the other sequence,by ~1) treating the strands with two sets of two oligonucleotide primers, one set 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 sets of primers are selected so as to be sufficiently complementary to the different strands of each specific sequence to hybridize therewith such that the extension 6 2~ 983 products synthesized from one primer from each of the two sets of primers, when separated from their respective complements, can serve as templates for the synthesis of the extension products of the other primer from each of the two sets of primers; wherein one of the primers of each set of primers contains a LoxP sequence at its S'-end;
(2) separating the primer extension products from the templates on which they were synthesized to produce single-stranded molecules;
(3) treating the single-stranded molecules generated from step ~2) with the two sets of primers of step (1) under conditions that primer extension products are synthesized using each of the single strands produced in step (2) as templates; and (4) repeating steps (2) and (3) to produce sufficient primer extension products for detection and/or measurement;
(C) treating the products of the two separate amplification reactions by the recombinase enzyme Cre; and (D) detecting and/or measuring that product of step (C) resulting from the two separate amplification reactions.
20~29~
12~J:LT:D 12E;SCRI~ l OF THE INVENTIO
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, two specific nucleic acid sequences, within the preselected sequence, are amplified by first annealing the denatured target nucleic acid present in the sample with two sets of oligonucleotide primers complementary to the specific nucleic acid sequences.
These primers are designed such that one primer of each set of primers contains a LoxP sequence attached to its 5'-end. The LoxP sequence is defined by a 34 -nucleotide sequence 5'...ATAACTTCGTATAGCATACATTATACGAAGTTAT...3' . [See Sternberg et al.,J. Mol. Biol., Volume 197, 197-212 ~1986).] The specific nucleic acid sequences are positioned such that when either sequence is amplified under amplification conditions, the extension product of either sequence cannot serve as a template for the synthesis of the other sequence. During amplification, each extension product formed from each set of primers is complementary to one of the two specific nucleic acid sequences within the preselected nucleic acid sequence and is a template for further primer extension. This process is then repeated as necessary in order to produce the desired amount of primer extenslon products for detection and/or measurement.
b) Adding the recombinase enzyme Cre to the products of the two separate amplification reactions, allowing the enzyme to combine the amplification ZQ1~9~3 product from the first amplification with the amplification product from the second amplification.
The resulting product is of a length of the combination of the two specific nucleic acid sequences plus 3q additional nucleotides.
c) Detecting the product of the above step by, for examplel gel electrophoresis. Such detection can differentiate between the desired combination product on the one hand and the combination products formed from the coupling of each amplification product with itself.
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 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 ~4C) for at least two minutes.
The Example below exemplifies the invention.
20~ 298~3 EY~MPLE
Detection of HIV I
A. Amplification of Target Nucleic Acid by 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 foIlowed utilizing the following specific conditions and reagents. Two sequences of the HIV I genome can be selected to be amplified. The first is a 103-nucleotide base sequence located within the GAG pl7 region of HIV I, incorporated into a plasmid (the plasmid incorporating most of the HIV I genome is designated pBH10-R3), and can be amplified using primers A and B as shown below:
5'..ATAACTTCGTATAGCATACATTATACGAAGTTATTGGGCAAGCAGGGAGCT
AGG..3' Primer A
5'..ATAACTTCGTATAGCATACATTATACGAAGTTATTCTGAAGGGATGGTTGTA
CG..3' Primer B
The second is a 160-base region also located within the GAG pl7 region of HIV I, incorporated into a plasmid (the plasmic incorporating most of the HIV I
genome is designated pBH10-R3), and can be amplified using Primers C and D as shown below:
5'.... ....ATAACTTCGTATAGCATACATTATACGAAGTTATTTCCCTCAGACCCTTTTAG
TC..3' erimer C
5'..ATAACTTCGTATAGCATACATTATACGAAGTTATTGGCGTACTCACCAGTCGC
CT..3' Primer D
201Z98;~
Aliquots of serial dilutions ~lx10+7, 1X10+6,1X10~S~ 1X1014, lx10~3, lx10+2, lxlO+l, and zero copies) of plasmid p~H10-R3 can be amplified using PCR. Each aliquot can be combined with a buffer 200 ~M in each of dA~P, dTTP, dCTP, and dGTP, 1.0 ~M in each of Pri~ers A, B, C, and D, and containing 1 ~g of human placental DNA/reaction and 2.5 units of a DNA
polymerase, in a total reaction volume of 100 ~1.
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 lx10+5 to lx10+6.
The products of the amplification reactions can be placed onto a 6% acrylamide gel run under standard conditions. After electrophoresis, the gel can be soaked in a 10 ~g/ml solution of ethidium bromide in 10 mM Tris, pH 7.0, for 15 minutes. The gel can then be rinsed in 10 mM Tris, pH 7.0, and the resulting product bands can be detected and/or measured by irradiating the gel at 302 nm and visualizing the fluorescent bands produced.
(C) treating the products of the two separate amplification reactions by the recombinase enzyme Cre; and (D) detecting and/or measuring that product of step (C) resulting from the two separate amplification reactions.
20~29~
12~J:LT:D 12E;SCRI~ l OF THE INVENTIO
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, two specific nucleic acid sequences, within the preselected sequence, are amplified by first annealing the denatured target nucleic acid present in the sample with two sets of oligonucleotide primers complementary to the specific nucleic acid sequences.
These primers are designed such that one primer of each set of primers contains a LoxP sequence attached to its 5'-end. The LoxP sequence is defined by a 34 -nucleotide sequence 5'...ATAACTTCGTATAGCATACATTATACGAAGTTAT...3' . [See Sternberg et al.,J. Mol. Biol., Volume 197, 197-212 ~1986).] The specific nucleic acid sequences are positioned such that when either sequence is amplified under amplification conditions, the extension product of either sequence cannot serve as a template for the synthesis of the other sequence. During amplification, each extension product formed from each set of primers is complementary to one of the two specific nucleic acid sequences within the preselected nucleic acid sequence and is a template for further primer extension. This process is then repeated as necessary in order to produce the desired amount of primer extenslon products for detection and/or measurement.
b) Adding the recombinase enzyme Cre to the products of the two separate amplification reactions, allowing the enzyme to combine the amplification ZQ1~9~3 product from the first amplification with the amplification product from the second amplification.
The resulting product is of a length of the combination of the two specific nucleic acid sequences plus 3q additional nucleotides.
c) Detecting the product of the above step by, for examplel gel electrophoresis. Such detection can differentiate between the desired combination product on the one hand and the combination products formed from the coupling of each amplification product with itself.
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 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 ~4C) for at least two minutes.
The Example below exemplifies the invention.
20~ 298~3 EY~MPLE
Detection of HIV I
A. Amplification of Target Nucleic Acid by 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 foIlowed utilizing the following specific conditions and reagents. Two sequences of the HIV I genome can be selected to be amplified. The first is a 103-nucleotide base sequence located within the GAG pl7 region of HIV I, incorporated into a plasmid (the plasmid incorporating most of the HIV I genome is designated pBH10-R3), and can be amplified using primers A and B as shown below:
5'..ATAACTTCGTATAGCATACATTATACGAAGTTATTGGGCAAGCAGGGAGCT
AGG..3' Primer A
5'..ATAACTTCGTATAGCATACATTATACGAAGTTATTCTGAAGGGATGGTTGTA
CG..3' Primer B
The second is a 160-base region also located within the GAG pl7 region of HIV I, incorporated into a plasmid (the plasmic incorporating most of the HIV I
genome is designated pBH10-R3), and can be amplified using Primers C and D as shown below:
5'.... ....ATAACTTCGTATAGCATACATTATACGAAGTTATTTCCCTCAGACCCTTTTAG
TC..3' erimer C
5'..ATAACTTCGTATAGCATACATTATACGAAGTTATTGGCGTACTCACCAGTCGC
CT..3' Primer D
201Z98;~
Aliquots of serial dilutions ~lx10+7, 1X10+6,1X10~S~ 1X1014, lx10~3, lx10+2, lxlO+l, and zero copies) of plasmid p~H10-R3 can be amplified using PCR. Each aliquot can be combined with a buffer 200 ~M in each of dA~P, dTTP, dCTP, and dGTP, 1.0 ~M in each of Pri~ers A, B, C, and D, and containing 1 ~g of human placental DNA/reaction and 2.5 units of a DNA
polymerase, in a total reaction volume of 100 ~1.
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 lx10+5 to lx10+6.
The products of the amplification reactions can be placed onto a 6% acrylamide gel run under standard conditions. After electrophoresis, the gel can be soaked in a 10 ~g/ml solution of ethidium bromide in 10 mM Tris, pH 7.0, for 15 minutes. The gel can then be rinsed in 10 mM Tris, pH 7.0, and the resulting product bands can be detected and/or measured by irradiating the gel at 302 nm and visualizing the fluorescent bands produced.
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 two specific nucleic acid sequences contained within the preselected nucleic acid sequence, said specific nucleic acid sequences being positioned such that when either sequence is amplified under amplification conditions, the extension product of either sequence cannot serve as a template for the synthesis of the other sequence, by:
(1) treating the strands with two sets of two oligonucleotide primers, one set 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 sets of primers are selected so as to be sufficiently complementary to the different strands of each specific sequence to hybridize therewith such that the extension products synthesized from one primer from each of the two sets of primers, when separated from their respective complements, can serve as templates for the synthesis of the extension products of the other primer from each of the two sets of primers;
wherein one of the primers of each set of primers contains a LoxP sequence at its 5'-end;
(2) separating the primer extension products from the templates on which they were synthesized to produce single-stranded molecules;
(3) treating the single-stranded molecules generated from step (2) with the two sets of primers of step (1) under conditions that primer extension products are synthesized using each of the single strands produced in step (2) as templates; and (4) repeating steps (2) and (3) to produce sufficient primer extension products for detection and/or measurement;
(C) treating the products of the two separate amplification reactions by the recombinase enzyme Cre; and (D) detecting and/or measuring that product of step (C) resulting from the two separate amplification reactions.
(A) rendering the target nucleic acid single-stranded;
(B) amplifying two specific nucleic acid sequences contained within the preselected nucleic acid sequence, said specific nucleic acid sequences being positioned such that when either sequence is amplified under amplification conditions, the extension product of either sequence cannot serve as a template for the synthesis of the other sequence, by:
(1) treating the strands with two sets of two oligonucleotide primers, one set 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 sets of primers are selected so as to be sufficiently complementary to the different strands of each specific sequence to hybridize therewith such that the extension products synthesized from one primer from each of the two sets of primers, when separated from their respective complements, can serve as templates for the synthesis of the extension products of the other primer from each of the two sets of primers;
wherein one of the primers of each set of primers contains a LoxP sequence at its 5'-end;
(2) separating the primer extension products from the templates on which they were synthesized to produce single-stranded molecules;
(3) treating the single-stranded molecules generated from step (2) with the two sets of primers of step (1) under conditions that primer extension products are synthesized using each of the single strands produced in step (2) as templates; and (4) repeating steps (2) and (3) to produce sufficient primer extension products for detection and/or measurement;
(C) treating the products of the two separate amplification reactions by the recombinase enzyme Cre; and (D) detecting and/or measuring that product of step (C) resulting from the two separate amplification reactions.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US32899989A | 1989-03-27 | 1989-03-27 | |
| US328,999 | 1989-03-27 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2012983A1 true CA2012983A1 (en) | 1990-09-27 |
Family
ID=23283411
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002012983A Abandoned CA2012983A1 (en) | 1989-03-27 | 1990-03-23 | Process for nucleic acid detection by binary amplification |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0465528A1 (en) |
| JP (1) | JPH04504201A (en) |
| AU (1) | AU5345790A (en) |
| CA (1) | CA2012983A1 (en) |
| WO (1) | WO1990011375A1 (en) |
Families Citing this family (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5614389A (en) * | 1992-08-04 | 1997-03-25 | Replicon, Inc. | Methods for the isothermal amplification of nucleic acid molecules |
| US5834202A (en) * | 1992-08-04 | 1998-11-10 | Replicon, Inc. | Methods for the isothermal amplification of nucleic acid molecules |
| US6261808B1 (en) | 1992-08-04 | 2001-07-17 | Replicon, Inc. | Amplification of nucleic acid molecules via circular replicons |
| US5733733A (en) * | 1992-08-04 | 1998-03-31 | Replicon, Inc. | Methods for the isothermal amplification of nucleic acid molecules |
| WO1994003624A1 (en) * | 1992-08-04 | 1994-02-17 | Auerbach Jeffrey I | Methods for the isothermal amplification of nucleic acid molecules |
| SG63589A1 (en) * | 1993-05-14 | 1999-03-30 | Johnson & Johnson Clin Diag | Diagnostic compositions elements methods and test kits for amplification and detection of two or more dna's using primers having matched melting temperatures |
| US6720140B1 (en) | 1995-06-07 | 2004-04-13 | Invitrogen Corporation | Recombinational cloning using engineered recombination sites |
| US6964861B1 (en) | 1998-11-13 | 2005-11-15 | Invitrogen Corporation | Enhanced in vitro recombinational cloning of using ribosomal proteins |
| JP4020429B2 (en) * | 1995-06-07 | 2007-12-12 | インヴィトロジェン コーポレーション | Recombination cloning using engineered recombination sites |
| US5851808A (en) | 1997-02-28 | 1998-12-22 | Baylor College Of Medicine | Rapid subcloning using site-specific recombination |
| WO1999021977A1 (en) * | 1997-10-24 | 1999-05-06 | Life Technologies, Inc. | Recombinational cloning using nucleic acids having recombination sites |
| EP2119788A1 (en) | 1999-03-02 | 2009-11-18 | Life Technologies Corporation | Compositions and methods for use in recombinational cloning of nucleic acids |
| US7198924B2 (en) | 2000-12-11 | 2007-04-03 | Invitrogen Corporation | Methods and compositions for synthesis of nucleic acid molecules using multiple recognition sites |
| NZ532577A (en) * | 2001-10-03 | 2004-07-30 | Iseao Technologies Ltd | Methods for detection of target molecules and molecular interactions between nucleic acid tags attached to binding entities specific for the target molecules of interest |
| EP2287341B1 (en) | 2003-12-01 | 2013-02-13 | Life Technologies Corporation | Nucleic acid molecules containing recombination sites and methods of using the same |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4683202A (en) * | 1985-03-28 | 1987-07-28 | Cetus Corporation | Process for amplifying nucleic acid sequences |
| GB8612087D0 (en) * | 1986-05-19 | 1986-06-25 | Ici Plc | Hybridisation probes |
-
1990
- 1990-03-23 CA CA002012983A patent/CA2012983A1/en not_active Abandoned
- 1990-03-26 JP JP2505279A patent/JPH04504201A/en active Pending
- 1990-03-26 WO PCT/US1990/001535 patent/WO1990011375A1/en not_active Application Discontinuation
- 1990-03-26 AU AU53457/90A patent/AU5345790A/en not_active Abandoned
- 1990-03-26 EP EP90905265A patent/EP0465528A1/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| WO1990011375A1 (en) | 1990-10-04 |
| JPH04504201A (en) | 1992-07-30 |
| AU5345790A (en) | 1990-10-22 |
| EP0465528A1 (en) | 1992-01-15 |
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