Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1997-02-14
1999-07-20
Horlick, Kenneth R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 911, 435 912, 536 2433, C12Q 168, C12P 1934, C07H 2104
Patent
active
059255200
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to methods of nucleotide sequencing, and more particularly to methods for rapidly determining the identity of several single bases at given locations simultaneously within one or more target nucleotide base sequences within a sample comprising one or more polynucleotide chains.
BACKGROUND OF THE INVENTION
The ability to determine the identity of a nucleotide within a characterised sequence of DNA has many applications in the fields of medical and forensic science. For instance, changes in one or more individual, ie. single, bases in genomic DNA have been shown to be associated with a number of human hereditary diseases including muscular dystrophy and cystic fibrosis. The identification of such mutations at the prenatal and postnatal stages can be a valuable diagnostic tool. Similarly, the identity of single bases at several polymorphic sites in human DNA can provide an accurate method for matching forensic samples with genetic material taken from known subjects.
Methods for the detection of characterised sequences or variations are known in which the region of DNA containing the variation is first amplified by the Polymerase Chain Reaction (PCR) and the sample is then tested using immobilised oligonucleotide probes which correspond to the possible variations in the region (Saiki et al. 1989; Proc Natl Acad Sci U.S.A. 86: 6230-6234). Such methods are cumbersome because a probe is required for each possible variation, and a separate reaction must be carried out for each probe.
Methods are also known for detecting a single base variation in which first a segment of DNA is amplified by PCR using two primers, one of which has been conjugated to biotin. The resulting biotin-DNA is immobilised and used as a template for a single detection-step primer which anneals to the DNA immediately upstream of the site of the variation. The variation is then investigated using a pair of radiolabelled nucleoside triphosphates corresponding to two possible base variations. These are added to the immobilised DNA/primer mixture in the presence of a suitable polymerase.
The identity of the base variation can then be ascertained by using a scintillation counter to measure the radioactivity incorporated into the eluted detection primer. Alternatively a digoxigenin label can be used which can be detected by spectrophotometery. This method has the disadvantage that a separate incorporation experiment must be carried out for each possible variation in each variable region. By using two distinguishable radiolabels, the number of experiments can be reduced slightly. However, each variable region must still be analysed separately which makes it laborious when analysing several polymorphic sites, for instance when compiling stringent forensic data or screening for several different inherited diseases. The present inventors have now provided a method that addresses some, and in preferred forms all, of these problems.
According to a first aspect of the present invention there is provided a method for determining the identity of at least two discrete single nucleotide bases each adjacent to a predetermined target nucleotide base sequence in a target sample comprising one or more types of polynucleotide chain, the method comprising mixing the target sample with (i) nucleotide primers which are complementary to the predetermined base sequences such that they anneal thereto at positions adjacent to the bases to be identified, (ii) at least two types of chain terminator each type labelled with a characteristic fluorescent group, and (iii) a nucleotide chain extending enzyme such that terminators complementary to the bases to be identified are incorporated into the nucleotide primers; separating the types of extended nucleotide primer on basis of size and/or charge and identifying the terminators incorporated into each type of nucleotide primer by reference to its fluorescent characteristics. Using the preferred embodiments the present invention provides a method for rapidly determining several discrete bases sim
REFERENCES:
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Sullivan Kevin
Tully Gillian
Horlick Kenneth R.
The Secretary of State for the Home Department
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