Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-01-03
2004-03-16
Whisenant, Ethan (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S024300
Reexamination Certificate
active
06706480
ABSTRACT:
The present invention relates to screening for sequence copy number and in particular to screening for changes in copy number of a plurality of nucleic acid sequences substantially simultaneously.
The loss or reduction in the normal number of copies of a genetic sequence (deletion) or the increase in copy number (amplification) are of widespread general importance. Such genetic alterations are known to underlie phenotype characteristics both somatic and germline including disorders as diverse as idiopathic mental retardation and neoplasia. The demonstration of the site and nature of such genetic alteration is critical in the identification of the genes responsible and to the development of appropriate and effective treatments and therapies.
In principle, it is possible to screen for genetic deletions by using Southern blot hybridisation. However, this method requires heterozygosity, and whilst the loss of heterozygosity at polymorphic loci can be used to demonstrate the absence of one allele in the soma or in the germline, its effectiveness is limited by the requirement for heterozygosity and the number of repeated tests needed to screen even a small fraction of the genome.
If sufficient precautions are taken to assure quantitative yields, the polymerase chain reaction (PCR) can be used to screen for copy number, and specialised systems have been developed to assure quantitative PCR by following the accumulation of products as amplification proceeds. However, such systems do not provide for the satisfactory analysis of a plurality of alterations substantially simultaneously.
According to the present invention there is provided a method of screening for copy number of target nucleic acid sequences in a sample of genetic material, the method comprising introducing to the sample a plurality of different genetic probes suitable to hybridise with respective target sequences and all flanked by the same or substantially the same primer binding sites, subjecting the sample to conditions favouring hybridisation of the probes to their respective sequences, and amplification of sample-bound probes using a pair of primers, wherein analysis of the respective amounts of amplified probe provides for quantitative determination of the copy number of the respective nucleic acid sequences in the sample.
Preferably each probe is distinguishable from the other(s), for example by having distinguishing mobility characteristics through a separating gel. Preferably the plurality of different probes comprises a predetermined set of different probes each chosen to be specific for a respective target nucleic acid sequence. The set may comprise probes suitable to screen a plurality of different nucleic acid sequences simultaneously or substantially simultaneously such that determination of the quantity of each probe product produced, preferably using the polymerase chain reaction (PCR), enables quantitative determination of the copy number of the respective sequences in the sample. The method may be used to screen sequences of different genes or different sequences within a gene, such as different exons in a eukaryotic gene. Preferably the method is used to detect genetic alterations such as genetic deletions (reduction in sequence copy number) and genetic amplification (increase in sequence copy number).
Preferably the genetic material is immobilised prior to hybridisation, such that hybridised flanking primers are likewise immobilised. Preferably an excess of probes is used.
Probes labelled for ready identification, such as with fluorescent labels are preferably used. More than one set of probes may be used, either simultaneously or sequentially. The flanking primer pairs may be the same or different for each set of probes.
Preferably the method comprises means to obviate or mitigate hybridisation between primer binding sequences. Competing oligonucleotides may be introduced to the sample preferably during the hybridisation stage to releasably bind to the primer binding sites flanking each probe whereby to mitigate primer binding site interactions.
Preferably unbound probes and primers are thoroughly washed away from the bound probes following hybridisation stage and prior to analysis.
The method may be used to screen DNA, RNA and/or cDNA with appropriate probe sets. The method may be used to screen somatic and/or germline sequences. The method may be used to screen for polymorphic alterations.
The invention further provides a set of probes substantially as described above in any of the preceding seven paragraphs.
The invention may still further comprise a kit for such a method described above in any of the preceding eight paragraphs, which kit comprises a probe set generally as defined above, amplification primers and means to enable amplification and analysis of amplification product(s).
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University of Nottingham
Watts, Hoffmann, Fisher & Heinke Co. LPA
Whisenant Ethan
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