Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1996-03-01
1999-06-15
Marschel, Ardin H.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 6, 435 912, 536 231, 536 243, 536 2433, C12Q 168, C12P 1934, C07H 2102
Patent
active
059121459
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to assay procedures involving nucleic acid analogues by which quantitative or semi-quantitative determinations of such analogues or nucleic acid sequences to which they hybridise can be made.
Nucleic acid amplification techniques are now in widespread use. These include the "PCR" (polymerase chain reaction) procedure described in EP-A-0200362 and EP-A-0201184, which is the technique in most widespread use, but also the "LCR" (ligase chain reaction) described in EP-A-0320308, the so-called "NASBA" or "3SR" technique which is described in "Proc. Natl. Acad. Sci. USA" Vol. 87 pp 1874-1878 March 1990 and "Nature" Vol. 350, No. 6313. pp 91-92 Mar. 7, 1991 and the "SDA" method described in "Nucleic Acid Research", Vol. 20 pp 1691-1696.
A number of strategies now exist to obtain quantitative information from amplification systems such as the polymerase chain reaction. In the simplest procedure the amount of amplified DNA produced by the test sample is compared directly with a set of standard reactions prepared with known amounts of the same DNA. A linear relationship has been shown to exist between the amount of DNA present in the sample and the amount of amplification product eventually formed in the PCR assay. However, the exponential nature of the PCR process means that minor differences in amplification efficiency between the sample tube and the separate tubes containing the known amounts of DNA will lead to large and unpredictable differences in the final product yield (Gilliland et al (1990) "Proc. Natl Acad Sci. USA" 87, 2725-2729). Such direct comparisons between the amount of PCR product from the sample and standards tubes are therefore fundamentally flawed. A number of alternative procedures have been proposed to overcome this problem. These include the use of limiting dilution of the sample (Thang et al (1991) "AIDS", 5,678-681) or the co-amplification of internal or external standards (Kellog et al (1990) "Anal. Biochem," 189, 202-208). The limiting dilution method is a very simple strategy that employs a Poisson distribution analysis of positive reactions to calculate the amount of the target sequence in the sample. The limiting dilution method has a fundamental limitation because the linear relationship that exists between the initial amount of template DNA in the sample and the amount of amplification product obtained is only maintained for amounts of starting DNA within a limited range. Hence the method is very imprecise when samples containing highly variable amounts of target DNA are examined. The alternative methods of co-amplification can provide a more reliable approach, in that they rely on the measurement of the ratio of target DNA and a co-amplified standard within the same tube. However, they still suffer from a number of difficulties. The co-amplification of internal standards, such as ubiquitously expressed genes that are also present in the sample DNA is limited in its usefulness by the difference in amplification efficiency that exists between target and reference sequences. This will affect the relative amounts of the products obtained in an uncontrollable manner. The alternative method of co-amplification of an external template possessing a similar length and the same primer recognition sequences as the target DNA is the most successful strategy so far for a quantitative PCR assay (Gilliland et al "Proc.
Natl. Acad. Sci. USA" 87, 2725-2729) but it still has three major disadvantages. The first problem is that the product from the target DNA and the co-amplified reference standard must be separated, usually by gel electrophoresis, and the separated products must be quantified to determine the ratio of the products. Usually, 4 reactions or more are carried out at different dilutions of the external competitor template and the products from all these reactions must be scanned to provide a standard curve. The external competitive PCR approach is therefore restricted to measurement methods that are capable of separating different PCR fragments and this intr
REFERENCES:
Gilliland et al. "Analysis of cytokine mRNA and DNA : Detection and quantitation by competitive polymerase chain reaction" Proc. Natl. Acad. Sci. USA, vol. 87, pp. 2725-2729 Apr. 1990.
Marschel Ardin H.
PNA Diagnostics A/S
Riley Jezia
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