Selective amplification of target polynucleotide sequences

Details Selective amplification of target polynucleotide sequences Selective amplification of target polynucleotide sequences

1995-04-24

2002-06-25

Fredman, Jeffrey (Department: 1655)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S006120, C435S091100, C536S024300

Reexamination Certificate

active

06410276

ABSTRACT:

FIELD OF THE INVENTION
This invention is related to diagnostic assays in which the presence of a particular gene is being detected, whether for detection of the gene itself or detection of the organism containing the gene, and is particularly directed to techniques in which the number of copies of the gene being detected is enzymatically increased prior to the detection process. It is further related to any process requiring the generation of many copies of a specific polynucleotide sequence.
BACKGROUND OF THE INVENTION
A number of diagnostic assays have been developed which rely on detection of the presence of a particular DNA or RNA sequence as an indication of the presence of an analyte, e.g., a bacterium, virus, or genetic defect, in a sample. In some cases the diagnostic gene is present in sufficient quantities to be detected directly, whether by hybridization, reaction with a specific antibody, or by some other method. However, if the gene of interest is present in a small amount or the background caused by similar sequences present in the sample is sufficiently high, reliable and sensitive detection of the targetted gene is difficult. An ambiguous result is not satisfactory in a diagnostic test.
Various techniques for increasing the sensitivity and specificity of such diagnostic procedures have been developed. Amplification of the target by cell culture, an efficient but time consuming technique, has been for long the only reliable method. Other techniques increase sensitivity of the detection system using sensitive reporter groups attatched to the probe which will combine with the target. Examples of sensitive reporter groups would include radioactive and fluorescent molecules. Enzymes, such as peroxidase or alkaline phosphatase coupled to the probe, also improve sensitivity through their catalytic action on substrate chromophores. Increased sensitivity may also be obtained by an amplification of the reporter groups. Such amplification has been achieved through avidin-biotin interactions, networking with nucleic acids, or the direct enzymatic replication of an RNA reporter group. This latter technique generates up to 1,000,000 copies of the RNA in about 12 minutes. Another technique amplifies the target nucleic acid sequence rather than the reporter groups used in the detection system.
One method for amplification of target nucleic acid is known as the polymerase chain reaction or PCR technique and has been developed for detecting the genes responsible for genetic defects. This method uses specific oligonucleotide primers in repeated cycles of target DNA denaturation, primer annealing, and extension with a DNA polymerase. Extension products generated from one primer serve as additional target sequences for the other primer. The degree of amplification of a target sequence is controlled by the number of cycles that are performed and is theoretically calculated by the simple formula 2
n
where n is the number of cycles. Given that the average efficiency per cycle ranges from about 65% to 85%, 25 cycles are needed to yield from 0.3 to 4.8 million copies of the target sequence.
Although the polymerase chain reaction is a very sensitive and promising method, there are some limitations and disadvantages inherent in this technique. For example, each cycle of polymerase chain reaction provides at best only a 2-fold amplification, and thus a high number of cycles (between 20 and 30) is required to achieve substantial amplification. Furthermore, the high-temperature denaturation that occurs in each PCR cycle typically inactivates the enzyme used and thus requires repeated addition of expensive enzyme.
Accordingly, techniques that increase the rate of gene amplification (thereby requiring less enzyme and fewer cycles) would be highly advantageous to all diagnostic techniques that involve the detection of a specific target nucleotide sequence and any other procedure needing an increased number of specifically amplified polynucleotides (RNA or DNA).
RELEVANT LITERATURE
The PCR method is described in a number of publications, including Saiki et al., “Enzymatic amplification of beta-globin genomic sequences and restriction site analysis of sickle cell anemia”,
Science
(1985) 230:1350-1354; Saiki et al., “Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes”,
Nature
(1986) 324:163-166; and Scharf et al., “Direct cloning and sequence analysis of enzymatically amplified genomic sequences”,
Science
(1986) 233:1076-1078. Also see European Patent Application publication no. 0 200 362 A2 (Application No. 86302298-4), published Dec. 10, 1986, which claims priority to three U.S. patent applications: Ser. No. 716,975, filed Mar. 28, 1985; Ser. No. 791,308, filed Oct. 10, 1985; and Ser. No. 828,144, filed Feb. 7, 1986.
SUMMARY OF THE INVENTION
The present invention provides a method for rapidly multiplying (through an enzymatic cycle) the number of copies of a target polynucleotide sequence by alternating two procedures that make copies from a target template. In a first series of steps, an intermediate double-stranded polynucleotide is produced comprising a promoter followed by the target sequence. The double-stranded intermediate is then used in a second process to prepare multiple RNA copies using an RNA polymerase that binds to the promoter region of the double-stranded intermediate. Each of the RNA copies can then be used as a target sequence to initiate another amplification cycle by preparing a second (or further) collection of double-stranded promoter-containing intermediates using reverse transcriptase. The process of the invention therefore provides a technique that multiplies in vitro the number of copies of a target polynucleotide sequence present in a sample much faster than prior processes. The method is applied in a specific embodiment to a diagnostic assay for
Toxoplasma gondii.


REFERENCES:
patent: 4683194 (1987-07-01), Saiki et al.
patent: 4683195 (1987-07-01), Mullis et al.
patent: 4683202 (1987-07-01), Mullis
patent: 5130238 (1992-07-01), Malek et al.
patent: 5437990 (1995-08-01), Burg et al.
patent: 0200362 (1986-03-01), None
patent: 0201184 (1986-03-01), None
patent: 0229701 (1987-01-01), None
Krupp et al, (Feb. 1987), “Slmplified in vitro synthesis of mutated RNA molecules”, FEBS Lett. 212(2):271-275.*
Milligan et al, (Nov. 1987), “Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates”, Nucleic Acid Res. 15(21):8783-8798.*
Krupp et al, (1988), “RNA synthesis: strategies for the use of bacteriophage RNA polymerases”, Gene 72:75-89.*
Axelrod, V.D., et al., “Transciption from Bacteriophage T7 and SP6 RNA Polymeraase Promoters in the Presence of 3′ -Deoxyribonucleoside 5′—Triphosphate Chain Terminators”,Biochemistry, vol. 24(8):5716 (Oct. 1985).
Genetic Engineering News, “PCR's Competitors Are Alive and Well and Moving Rapidly Towards Commercialization”, pp. 1, 8, 9 (6/92).
Guatelli, et al., “Isothermal, in vitro Amplification of Nucleic Acids by a Mulitenzyme Reaction Modeled After Retroviral Replication”,Proc. Nat'l. Acad. Sci.87:1874-78 (1990).
Melton, et al., “Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes form plasmids containing a bacteriophage SP6 promoter”,Nuc. Acids. Res., vol. 12(18):7035-2056 (1984).
Mullis, Karry B.; slide of May 1986; Talk at the University of California Berkeley.

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