Amplifying and detecting target nucleic acids using a post...

Details Amplifying and detecting target nucleic acids using a post... Amplifying and detecting target nucleic acids using a post...

1997-04-25

2001-08-28

Arthur, Lisa B. (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200

Reexamination Certificate

active

06280930

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a method for amplifying and detecting target nucleic acids. In particular, it relates to improved methods of detecting amplified nucleic acid products. The present invention can be used in various medical and research studies, forensic investigations, and diagnostic procedures, such as for the detection of genetic disorders or infectious diseases.
BACKGROUND OF THE INVENTION
Technology to detect minute quantities of nucleic acids has advanced rapidly over the last two decades including the development of highly sophisticated hybridization assays using probes in amplification techniques such as polymerase chain reaction (PCR). Researchers have readily recognized the value of such technology to detect diseases and genetic features in human and animal test specimens. The use of primers and probes in the amplification and detection of nucleic acids is based upon the concept of complementarity, that is, the bonding of two strands of a nucleic acid by hydrogen bonds between complementary nucleotides (which are known as nucleotide pairs).
Much research has been carried out to find ways to amplify and detect small quantities of DNA. Various procedures are known and have been used to amplify or greatly multiply the number of nucleic acids in a specimen for detection. Such amplification techniques include PCR, ligase chain reaction (LCR), and branched DNA.
PCR is the most well known of these amplification methods. Details of PCR are well described in the art, including, for example, U.S. Pat. No. 4,638,195 (Mullis et al.), U.S. Pat. No. 4,683,202 (Mullis), and U.S. Pat. No. 4,965,188 (Mullis et al.). Without going into extensive detail, PCR involves hybridizing primers to the strands of a target nucleic acid in the presence of polymerization agent (such as a DNA polymerase) and deoxyribonucleoside triphosphates under the appropriate conditions. The result is the generation of primer extension products along the templates, the products having added thereto nucleotides that are complementary to the templates.
Once the primer extension products are denatured and one copy of the templates has been prepared, the cycle of priming, extending and denaturation can be carried out as many times as desired to provide an exponential increase in the amount of nucleic acid that has the same sequence as the target nucleic acid. In effect, the target nucleic acid is duplicated (or “amplified”) many times so that it is more easily detected. Once the target nucleic acid has been sufficiently amplified, various detection procedures can be used to detect, qualitatively and/or quantitatively, the presence of the target.
Once the target nucleic acid has been sufficiently amplified, various detection procedures can be used to detect its presence. A standard detection method used to detect PCR products has been ethidium bromide stained agarose gels. Use of ethidium bromide stained gels, however, has several disadvantages including, for example, relatively poor sensitivity and specificity.
Improved methods of detecting PCR products that eliminate the use of radiolabels and electrophoresis have been developed. These nonisotopic oligonucleotide capture detection methods rely on specific hybridization to probes and enzymatic signal generation. Such nonisotopic oligonucleotide capture detection methods, also known as reverse dot blot detection, are described in U.S. Pat. No. 5,229,297 (Schnepilsky et al.), U.S. Pat. No. 5,328,825 (Warren et al.), and U.S. Pat. No. 5,422,271 (Chen et al.). Such a method is also described in Findlay et al., Clinical Chemistry, 39:1927-1933 (1993).
These nonisotopic detection methods have higher sensitivity and specificity than ethidium bromide staining detection and avoid the use of radioactivity. The methods operate by either carrying out amplification with biotinylated primer(s) or using a biotinylated probe to detect the amplified nucleic acids. Biotinylated products or probes are subsequently reacted with an avidin or streptavidin conjugated enzyme such as horseradish peroxidase (HRP). A dye precursor (or light generating signal reagent) can then be brought into contact with the enzyme and be converted into a dye (luminescence) thereby generating a detectable signal.
Nonisotopic oligonucleotide capture detection methods are not, however, without their own drawbacks. If nonisotopic oligonucleotide capture detection is carried out utilizing standard PCR denaturation conditions (95° C.) to denature concentrated or minimally diluted amplified nucleic acid products, the enzymes utilized to carry out the amplification reaction, such as thermostable polymerases or DNA ligases, will still be present and active. The presence of such active enzymes during detection results in competition between and binding of the probe to the denatured amplification product. Such competition can reduce the amount of amplified nucleic acid products bound to probe and therefor, the detection signal.
One solution to this problem has been to add high levels of ethylenediamine tetraacetic acid (EDTA), a chelator of Mg
++
, to the PCR amplification mixture after amplification has been carried out but prior to detection.
EDTA is able to inhibit many enzymes requiring Mg
++
for activity including DNA polymerases and DNA ligases. Use of EDTA, however, adds an additional step to the PCR amplification and detection process. In addition, use of EDTA requires opening up the reaction vessel to add the EDTA. As those skilled in the art are aware, opening the reaction vessel is to be avoided because of contamination concerns.
Thus, blocking the amplification process during detection through the addition of EDTA or other such enzyme inhibitors is not desired. Rather, it is desirable to have a method of inactivating the amplification enzymes prior to detection without the increased risk of contamination.
SUMMARY OF THE INVENTION
It is an object of the present invention to overcome the problems noted above by using a post amplification incubation step prior to detection to inactivate the amplification enzymes.
In one embodiment, the present invention relates to a method for amplifying and detecting a target nucleic acid in an enclosed reaction vessel comprising:
(i) contacting a sample suspected of containing the target nucleic acid with at least two oligonucleotides and a thermostable amplification enzyme, wherein the oligonucleotides are substantially complementary to a portion of the target nucleic acid, under conditions such that the target nucleic acid is amplifiable;
(ii) amplifying the target nucleic acid;
(iii) denaturing amplified target nucleic acids to form single stranded nucleic acids;
(iv) incubating the sample for between 1 second and 30 minutes at between 95° C. and 120° C., as a post amplification incubation step to inactivate the thermostable amplification enzyme; and
(v) detecting the presence or absence of the amplified target nucleic acids,
wherein the reaction vessel remains closed.
In a further embodiment, the present invention relates to a method for amplifying and detecting a target nucleic acid in a reaction vessel comprising:
(i) contacting a sample suspected of containing the target nucleic acid with four different nucleoside triphosphates, a thermostable DNA polymerase, and at least two primers, wherein the primers are substantially complementary to the target nucleic acid, under conditions such that the target nucleic acid is amplifiable;
(ii) amplifying the target nucleic acid;
(iii) denaturing amplified target nucleic acids to form single stranded nucleic acids;
(iv) incubating the sample for between 1 second and 30 minutes at between 95° C. and 120° C., as a post amplification incubation step to inactivate the polymerization agent; and
(v) detecting the presence or absence of the amplified target nucleic acids, wherein the reaction vessel remains closed during amplification and detection.
In another embodiment, the present invention relates to a method for amplifying and detecting a target nucleic acid in a reaction

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