Glycosylase mediated detection of nucleotide sequences at candid

Details Glycosylase mediated detection of nucleotide sequences at candid Glycosylase mediated detection of nucleotide sequences at candid

1998-01-08

1999-09-14

Campbell, Eggerton A.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 912, C12Q 168, C12P 1934

Patent

active

059521767

DESCRIPTION:

BRIEF SUMMARY
This application is the national phase under 35 U.S.C. .sctn.371 of prior PCT International Application No. PCT/IE95/00067 which has an International filing date of Dec. 21, 1995 which designated the United States of America, the entire contents of which are hereby incorporated by reference.


TECHNICAL FIELD

This invention relates to a method for rapidly detecting the presence or absence of a particular nucleic acid sequence at a candidate locus in a target nucleic acid sample. In particular, the invention relates to a method for detecting specific mutations in a DNA sample.


BACKGROUND ART

Detection of specific sequences at candidate loci in target nucleic acid samples is highly important for several reasons relating to diagnosis of inherited disorders and of infectious diseases. Detection of multiple different mutations is necessary for screening for the presence of specific gene tic disorders. Detection of specific sequences in amplified DNA samples significantly enhances current DNA diagnostic methods for the detection of organisms of interest and especially of infectious organisms. Current techniques for the detection of sequences at candidate loci are either cumbersome, lacking in specificity, difficult to optimise and use or poorly adaptable to high sample throughput.
There are several methods known for the detection of a particular nucleic acid sequence at a candidate locus in a target nucleic acid sample. Details of these methods are as follows.
1) Restriction enzyme analysis
Detection of a particular DNA sequence by restriction enzyme analysis. Restriction enzymes cleave DNA at specific sequences. For example, the enzyme EcoRI cleaves double stranded DNA at the sequence GAATTC. Thus, by checking a candidate locus in a particular DNA sample for cleavage with EcoRI, one may deter mine whether the sequence GAATTC is present or absent at the candidate locus. The appearance or disappearance of a restriction site from a candidate locus indicates that one or more of the bases in the sequence at the candidate locus has been altered. Thus, the creation or loss of a restriction site at a candidate locus can involve the alteration of any base or bases of the respective restriction site. Therefore, the appearance or disappearance of a restriction site at a candidate locus may not inform the investigator of the exact sequence change. However, there are a number of problems with the use of restriction enzymes for the detection of specific sequences at candidate loci. These include: regularly does not occur at a restriction site; sequences, different restriction enzymes are regularly required for the detection of the presence or absence of a particular sequence at different candidate loci; and presence or absence of a particular sequence at different candidate loci, a high throughput is difficult to achieve with this approach and automation is difficult.
2) DNA sequencing
DNA sequencing allows detection of a particular sequence at any candidate locus. The main methods of DNA sequencing are the Sanger method (Sanger, F. and Coulson, A. R. (1975) J. Mol. Biol. 94, 444-448), also known as the dideoxy method or chain termination method, and the Maxam Gilbert method (Maxam, A. M. and Gilbert, W. (1977) Proc. Natl. Acad. Sci. USA 74, 560-564), also known as the chemical method.
While DNA sequencing is the ultimate way of determining if a particular sequence is presence or absence at a particular candidate locus, it suffers from a number of drawbacks as follows: presence or absence of a particular sequence at different candidate loci; or absence of a particular sequence at a given candidate locus; obtain good quality DNA sequences; problematic; detect the presence or absence of a specific sequence at a candidate locus.
3) Uracil interference
A method has been described whereby uracil is incorporated into an amplified DNA molecule randomly and at a low level. This is achieved by amplifying the DNA in the presence of the normal DNA precursor nucleotides and dUTP. The ratio of dTTP to dUTP is chosen so that in the am

REFERENCES:
Sanger et al., J. Mol. Biol. (1975) 94, 441-448.
Maxam et al., Proc. Natl. Acad. Sci. USA, vol. 74, No. 2, pp. 560-564, Feb. 1977.

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