Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-10-29
2001-08-07
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S024300, C536S025320, C536S026600
Reexamination Certificate
active
06270967
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to novel methods for the detection of diagnostic base sequences in sample nucleic acid. In particular the invention relates to the use of tailed primers in such methods.
2. Description of Related Art
The invention is an improvement on currently established procedures for the detection of nucleic acid sequences. The detection of nucleic acid sequences is a desirable and necessary procedure in the following exemplary areas; detection and diagnosis of alleles responsible for genetic diseases in humans and other species; detection and diagnosis of DNA sequences associated or linked to genes that may or may not be involved in disease in humans and other species; detection and diagnosis of neoplasms and the effects of therapy of neoplasms; detection of and distinction between different pathogens (eg. viruses, bacteria and fungi); determining the purity of animal strains and pedigrees; distinguishing and identifying different humans and animal samples in forensic medicine.
The polymerase chain reaction (PCR) as disclosed for example in U.S. Pat. Nos. 4,683,202 and 4,683,195 has been used to amplify specific DNA sequences. However, PCR does not, by itself, provide a method to detect single base mutations. It has been necessary to combine the PCR with other techniques, for example allele specific oligonucleotide probing of PCR amplification products.
SUMMARY OF THE INVENTION
We have now devised a novel assay system for the detection of diagnostic base sequences which uses tailed diagnostic primers having a tag region and a detector region. Under appropriate conditions any diagnostic primer extension product acts as a template for extension of a further primer. In which case a sequence complementary to the tag region and the detector region will arise in the further primer extension product. A tag primer is provided which can hybridise to the complement of the tag region in the further primer extension product and be extended. A diagnostic base sequence is identified by reference to the sequence complementary to the detector region in the tag primer extension product.
Therefore in a first aspect of the present invention we provide a method for the detection of a diagnostic base sequence in nucleic acid comprised in a sample, which method comprises contacting the sample under hybridising conditions and in the presence of appropriate nucleoside triphosphates and an agent for polymerisation thereof, with a diagnostic primer for the diagnostic base sequence, the diagnostic primer having a tail sequence comprising a tag region and a detector region, such that an extension product of the diagnostic primer is synthesised when the corresponding diagnostic base sequence is present in the sample, no extension product being synthesised when the corresponding diagnostic base sequence is not present in the sample and any extension product of the diagnostic primer acts as template for extension of a further primer which hybridises to a locus at a distance from the diagnostic base sequence, and contacting the sample with a tag primer which selectively hybridises to the complement of the tag sequence in an extension product of the further primer and is extended, and detecting the presence or absence of the diagnostic base sequence by reference to the detector region in the further primer extension product.
The detector region in the further primer extension product may be detected in a number of ways. For example the sample may be contacted with detector species capable of emitting a detectable signal upon interaction with the detector region in the further primer extension product whereby the presence or absence of the diagnostic base sequence is detected by reference to the detectable signal. It will be appreciated that the detector species cannot become associated with the corresponding detector region until target dependent hybridisation and further primer extension has occurred. This system is well suited for homogeneous assays and real time or end point analysis. A detector species is any species capable of selective association with the detector region in a further primer extension product and release of a detectable signal. It will be appreciated that by “selective association” we mean that the detector species identifies and binds to the detector region in the further primer extension product to the exclusion of other nucleic acid seqences in the sample. Such detector species may include antibodies and hybridisation probe(s). A particular detector species is a detector probe such as a labelled hybridisation probe. Label is conveniently released by the action of for example an exonuclease associated with the polymerase mediated extension of a tag primer. In the specific description hereinafter we describe a number of alternative systems. These include detection of the change in shape of a probe upon hybridisation, the use of two or more probes having interactive labels such as for example the use of fluorescence resonance energy transfer, the use of scintillation proximity assays (SPA), the measurement of a change in fluorescence polarisation upon hybridisation of a fluorescently labelled probe. Further systems will be apparent to the scientist of ordinary skill. These include the use of a solid phase capture probe for the detector region in the further primer extension product. It will be appreciated that both direct and indirect labelling methods may be used to detect the immobilised further primer extension product. By way of example a further labelled probe for a region other than the detector region may be used. Alternatively, intercalation may be used to detect the detector region/probe DNA duplex. Also, labelled dNTPs may be incorporated into the further primer extension product.
The sequence of the detector probe need not be the same but is conveniently identical to the sequence of the detector region in the tail. It will be appreciated that minor changes may be made to the sequence of the detector probe without affecting its performance to any significant extent.
Alternatively the complement of the detector region is detected by reference to its size contribution to the overall amplification product of the tag and further primers. A convenient size difference may be used, even as little as one base pair difference can be detected on a gel. Generally however size differences of at least 5, conveniently at least 10, at least 15 or at least 20 base pairs are used. This aspect of the invention is of particular use where two or more alleles of a genetic locus are to be detected in a single assay mixture.
The tag primer is capable of hybridisation to the complement of the tag sequence in the further primer extension product. It will be understood that the diagnostic primer extension product is separated from the further primer extension product prior to hybridisation of the tag primer. The sequence of the tag primer is conveniently identical to the sequence of the tag region in the tail. The tag primer preferably comprises a sequence capable of hybridisation to all tag sequences. All tag sequences are preferably identical. Again it will be appreciated that minor changes may be made to the sequence of the tag primer without affecting its performance to any significant extent. The use of a common tag primer and common tail sequences has significant cost advantages for a typical assay.
It will be understood that the diagnostic primer tail is non-complementary to any relevant genomic sequence or adjacent region so as not to compromise the assay.
In known diagnostic PCR procedures mispriming may occur at each amplification cycle, especially where the primer is used to detect for example single base mismatches or to detect a particular sequence against a background of related sequences. Such mispriming may only occur as a very low percentage of total priming events per amplification cycle but will increase significantly as a function of the overall number of cycles. The present invention comprises a two stage procedure wh
Brownie Jannine
Little Stephen
Whitcombe David Mark
Horlick Kenneth R.
Rothwell Figg Ernst & Manbeck
Tung Joyce
Zeneca Limited
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