Exogenous nucleic acid detection

Details Exogenous nucleic acid detection Exogenous nucleic acid detection

1999-09-27

2001-08-07

Fredman, Jeffrey (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S007800, C435S091200, C435S091500, C436S173000, C436S501000, C536S026110, C536S027400, C536S028500

Reexamination Certificate

active

06270974

ABSTRACT:

DESCRIPTION
1. Field of the Invention
The invention relates to nucleic acid detection. More specifically, the invention relates to the detection of a predetermined exogenous nucleic acid target sequence in a nucleic acid target/probe hybrid, and the various applications of such detection.
2. Background of the Invention
Methods to detect nucleic acids provide a foundation upon which the large and rapidly growing field of molecular biology is built. There is widespread application of such general methods to the detection of specific, exogenous nucleic acids. There is constant need for alternative methods and products. The reasons for selecting one method over another are varied, and include a desire to avoid radioactive materials, the lack of a license to use a technique, the cost or availability of reagents or equipment, the desire to minimize the time spent or the number of steps, the accuracy or sensitivity needed for a certain application, the ease of analysis, or the ability to automate the process.
The detection of nucleic acids, including specific exogenous nucleic acids, is often a portion of a process rather than an end in itself. There are many applications of the detection of nucleic acids in the art, and new applications are always being developed. The ability to detect and quantify exogenous nucleic acids is useful in detecting microorganisms and viruses and biological molecules (e.g. non-native promoter or terminator sequences or foreign genes) in a biological sample, and thus affects many fields, including human and veterinary medicine, food processing and environmental testing. Additionally, the detection and/or quantification of specific biomolecules from biological samples (e.g. tissue, sputum, urine, blood, semen, saliva) has applications in medicine and forensic science.
Hybridization methods to detect nucleic acids are dependent upon knowledge of the nucleic acid sequence. Many known nucleic acid detection techniques depend upon specific nucleic acid hybridization in which an oligonucleotide probe is hybridized or annealed to nucleic acid in the sample or on a blot, and the hybridized probes are detected.
A traditional type of process for the detection of hybridized nucleic acid uses labeled nucleic acid probes to hybridize to a nucleic acid sample. For example, in a Southern blot technique, a nucleic acid sample is separated in an agarose gel based on size and affixed to a membrane, denatured, and exposed to a labeled nucleic acid probe under hybridizing conditions. If the labeled nucleic acid probe forms a hybrid with the nucleic acid on the blot, the label is bound to the membrane. Probes used in Southern blots have been labeled with radioactivity, fluorescent dyes, digoxygenin, horseradish peroxidase, alkaline phosphatase and acridinium esters.
Another type of process for the detection of hybridized nucleic acid takes advantage of the polymerase chain reaction (PCR). The PCR process is well known in the art (U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159). To briefly summarize PCR, nucleic acid primers, complementary to opposite strands of a nucleic acid amplification target sequence, are permitted to anneal to the denatured sample. A DNA polymerase (typically heat stable) extends the DNA duplex from the hybridized primer. The process is repeated to amplify the nucleic acid target. If the nucleic acid primers do not hybridize to the sample, then there is no corresponding amplified PCR product. In this case, the PCR primer acts as a hybridization probe. PCR-based methods are of limited use for the detection of nucleic acid of unknown sequence.
In a PCR method, the amplified nucleic acid product may be detected in a number of ways, e.g. incorporation of a labeled nucleotide into the amplified strand by using labeled primers. Primers used in PCR have been labeled with radioactivity, fluorescent dyes, digoxygenin, horseradish peroxidase, alkaline phosphatase, acridinium esters, biotin and jack bean urease. PCR products made with unlabeled primers may be detected in other ways, such as electrophoretic gel separation followed by dye-based visualization.
Enzymes having template-specific polymerase activity for which some 3′→5′ depolymerization activity has been reported include
E. coli
DNA Polymerase (Deutscher and Kornberg,
J. Biol. Chem.,
244(11):3019-28 (1969)), T7 DNA Polymerase (Wong et al.,
Biochemistry
30:526-37 (1991); Tabor and Richardson,
J. Biol. Chem.
265: 8322-28 (1990)),
E. coli
RNA polymerase (Rozovskaya et al.,
Biochem. J.
224:645-50 (1994)), AMV and RLV reverse transcriptases (Srivastava and Modak,
J. Biol. Chem.
255: 2000-4 (1980)), and HIV reverse transcriptase (Zinnen et al.,
J. Biol. Chem.
269:24195-202 (1994)). A template-dependent polymerase for which 3′ to 5′ exonuclease activity has been reported on a mismatched end of a DNA hybrid is phage 29 DNA polymerase (de Vega, M. et al.
EMBO J.,
15:1182-1192, 1996)
There is a need for highly sensitive, diagnostic applications that are capable of determining the number of virus molecules present in a body (“viral load”). For example, the presence of viral particles in the circulation system or in specific tissues is a means of monitoring the severity of viral infection. Several methods are currently used in the art for determining viral load. U.S. Pat. No. 5,667,964 discloses a method for the determination of the number of HIV-1 infected patient cells using reactive oxygen-intermediate generators. U.S. Pat. No. 5,389,512 discloses a method for determining the relative amount of a viral nucleic acid segment in a sample using PCR.
G. Garinis et al.,
J. Clin. Lab. Anal.
13:122-5 (1999) compare the determination of viral load results using an enzyme-linked immunosorbent assay (ELISA), a recombinant immunoblot assay (RIBA), and a reverse transcriptase polymerase chain reaction method (RT-PCR) in the detection of hepatitis C virus (HCV) infection in haemodialysis patients. The quantitative hepatitis HCV RT-PCR assay had a detection level of about 2,000 viral copies/mL serum. Holguin et al.,
Eur. J. Clin. Microbiol. Infect. Dis.
18:256-9 (1999) compare plasma HIV-1 RNA levels using several commercially available assays, namely the second-generation HIV-1 branched DNA assay, the Nuclisens assay, the Amplicor® Monitor reverse transcriptase polymerase chain reaction assay, and the Ultradirect Monitor. Differing values were noted in comparing results among these various assays. Boriskin et al.,
Arch. Dis. Child.
80:132-6 (1999) used a nested polymerase chain reaction to measure HIV-1 proviral DNA and CMV genomic DNA in peripheral blood leukocytes of children infected with HIV-1. There remains a need for a reliable means to detect and quantify viral load. There is a demand for methods to determine viral load when the quantities of viral particles are very low.
There is a need for alternative methods for detection of nucleic acid hybrids. There is a demand for highly sensitive methods that are useful for determining the presence or absence of specific nucleic acid sequences that are non-native or “exogenous” to an organism's nucleic acid. For example, there is a need to determine the presence of non-native nucleic acid present in a cell, both when the non-native nucleic acid is incorporated into the native nucleic acid and when it is not incorporated. For example, there is a need for methods to determine viral load that are able to reliably detect as few as 10 copies of a virus present in a body, tissue, fluid, or other biological sample. There is great demand for methods to determine the presence of a mutant virus, e.g. a drug-resistant mutant, in a biological sample containing a viral population. There is great demand for methods to determine the presence or absence of non-native sequences unique to a particular species in a sample, for example the identification of bacterial contamination present in a primarily non-bacterial biological sample. There is also great demand for methods that are more highly sensitive than the known methods, methods

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