Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-04-09
1999-02-16
Arthur, Lisa B.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 9121, 935 17, 935 78, 536 2433, 536 243, C12Q 168, C12P 1934, C07H 2104
Patent
active
058719148
DESCRIPTION:
BRIEF SUMMARY
This application was filed under 35 USC 371 from PCT Application PCT/US94/06034.
FIELD OF THE INVENTION
The present invention concerns a method and kit for the detection of specific nucleic acid sequence in a sample.
BACKGROUND OF THE INVENTION
Detection of the presence of a specific DNA or RNA sequence in a sample is required for a variety of experimental, diagnostic and therapeutic purposes, e.g. detection of a specific mutation in a sample of amniotic fluid, parenterage testing, testing for incorporation of a viral DNA into a cell's genomic DNA, etc. The task of direct detection of a specific DNA or RNA sequence, which is routinely performed by the use of an appropriately labelled probe, is often hindered by the fact that the specific DNA or RNA is present in a sample only in minute amounts.
Examples of methods which enable the amplification of DNA sequences present in a sample in only minute quantities are: LCR (ligase chain reaction), 3SR (self-sustained sequence replication) or PCR (polymerase chain-reaction). In PCR a sample is contacted with a primer DNA complimentary to a 3' end sequence of the specific DNA, a DNA polymerase and with single DNA nucleotides. Following a number of replication cycles, the sample is enriched with the specific assayed DNA. A typical cycle of PCR comprises three distinct stages: a first stage in which the double-stranded DNA is melted to two single strands; a second stage of annealing of the primer to the single-stranded DNA; and a third stage of polymerization where the annealed primers are extended by the DNA polymerase, to produce a double-stranded DNA. The cycle of melting, annealing and DNA synthesis is repeated many times, the products of one cycle serving as templates for the next ad thus, each successive cycle enriches the sample with the specific DNA.
PCR suffers from several shortcomings, the most serious of which being its lack of specificity. The effective hybridization temperature, i.e. the temperature in which the two strands of DNA hybridize, determines the specificity of the reaction. A low effective hybridization temperature results in a higher percentage of non-specific binding. In PCR this temperature, which is defined by the temperature of the annealing stage, is relatively low and this brings about non-specific binding of the probe to the target sequences resulting in amplification of undesired sequences which brings about a relatively high background reading.
This non-specificity also requires an additional and time-consuming detection procedure such as electrophoretic separation of the amplification products on an agarose gel, in order to separate between the various amplification products, and does not enable detection of the presence of the assayed DNA by a mere detection of amplification.
PCR also suffers from a severe problem of contamination which is due to amplification of sequences that did not originate from the test sample being sequences unintentionally introduced to the sample.
Another disadvantage of PCR is that it is a complex procedure. Typically, each of the stages of melting, annealing and polymerization is carried out at a different temperature, e.g. melting at 94.degree. C., annealing at 50.degree. C. and polymerization at 72.degree. C. Since the samples have to be constantly cycled through several temperatures a special apparatus is required rendering the procedure laborious and time consuming.
Another shortcoming of PCR is in the time required therefor. A typical cycle lasts several minutes, and usually 25-30 cycles are required to produce sufficient copies of amplified DNA. Thus, a typical PCR even in a completely automated system lasts at least 2 to 3 hours.
Finally, PCR is basically suited for the detection of DNA sequences. Where detection of RNA sequences is desired, RNA has to be converted first to DNA (by reverse transcription). This conversion to DNA requires additional time, effort and enzymes, and also introduces many errors due to the inherent inaccuracy of reverse transcription.
It should be noted that althoug
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Arthur Lisa B.
Intelligene Ltd.
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