Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1998-07-29
2000-07-04
Jones, W. Gary
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 912, C12Q 168, C12P 1934
Patent
active
060837012
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
Simple sequence repeats or microsatellites have found widespread usage in the linkage analysis of genetic traits. These sequences are tandem repeats of simple motifs that occur abundantly at random locations throughout the genomes of most eukaryotes. Microsatellites are ideal substrates for PCR due to the fact that they are generally flanked by unique sequence and the overall amplicon size is usually in the region of 100 bases. These sequences display polymorphic variation in the number of repeat units between the flanking sequences. The repeat length polymorphism is not only stable enough to facilitate genetic analysis but is also highly informative for the purpose of linkage analysis. Microsatellites thus serve as ideal markers for the construction of high resolution genetic maps and for the genomic localisation of regions of biological and medical interest.
Current methods for typing the number of repeats at such loci are time consuming and tedious. The number of repeats is determined by PCR product sizing in various electrophoretic separation systems. The PCR products generated upon amplification of genomic DNA may be labelled in a variety of ways. The currently preferred method employs the attachment of a fluorescent moiety to the PCR fragment in order to facilitate detection. A major limitation is the number of samples that can be analysed on a single gel. The current art--using multiple emission wavelength fluors--only allows up to about 10 PCR products to be analysed per gel lane.
In the current art, electrophoretic sizing of PCR products is complicated not only by mobility distortions introduced by the attached fluorescent moieties but also by the exact sequences chosen for the size markers (different sequence compositions for the same fragment length may result in small differences in mobility for some electrophoretic separation systems). Also, addition of non-template directed bases by the polymerase during PCR may lead to multiple fragments appearing in the electropherogram--complicating the subsequent analysis of repeat length considerably.
SUMMARY OF THE INVENTION
The present invention uses a hybridisation approach to obtaining information about microsatellite sequences. The system is immune to the electrophoretic mobility differences induced by such fluorescent moieties and is also insensitive to the addition of an extra base to the end of PCR fragments. Once set up, the system permits a rapid throughput and allows for the parallel analysis of a large number of different microsatellite repeat lengths.
In one aspect the invention provides a method of analysing a nucleic acid target comprising a tandem repeat sequence and two flanking sequences, which method comprises providing at least two nucleic acid probes in an array, each probe immobilised on a surface of a support and comprising a tandem repeat sequence and two flanking sequences complementary to the tandem repeat sequence and the two flanking sequences of the target, each probe immobilised at a spaced location on the surface of the support, the tandem repeat sequence length of one probe being different from that of another probe, labelling the target, applying the labelled target to the surface, washing the surface to remove labelled target that has not formed a perfectly matched hybrid with a probe, and using the label to observe a perfectly matched hybrid where the tandem repeat sequence of the target is the same length as the tandem repeat sequence of a probe.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
FIG. 1 presents a schematic depiction of a method of practice of the invention.
DETAILED DESCRIPTION OF THE INVENTION
The nucleic acid target to be analysed comprises a tandem repeat sequence and two flanking sequences. The tandem repeat sequence contains a variable number (VNTR) of repeats of a simple motif. Generally analysis involves determining the number of repeats of that motif, or at least ascertaining whether the number of repeats in the target is the same as the number of repe
REFERENCES:
Yaar, R. et al., "In situ detection of tandem DNA repeat length,"Genetic Analysis: Biomolecular Engineering, 13:113-118 (1996).
Amersham Pharmacia Biotech UK Limited
Jones W. Gary
Souaya Jehanne
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