Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-06-10
2004-07-06
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200
Reexamination Certificate
active
06759202
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a probe composition and method for use in detecting selected sequences in a target polynucleotide.
BACKGROUND OF THE INVENTION
A variety of DNA hybridization techniques are available for detecting the presence of one or more selected polynucleotides sequences in a sample containing a large number of sequence regions. In a simple method, which relies on fragment capture and labeling, a fragment containing a selected sequence is captured by hybridization to an immobilized probe. The captured fragment can be labeled by hybridization to a second probe which contains a detectable reporter label.
Another widely used method is Southern blotting. In this method, a mixture of DNA fragments in a sample are fractionated by gel electrophoresis, then fixed on a nitrocellulose filter. By reacting the filter with one or more labeled probes under hybridization conditions, the presence of bands containing the probe sequence can be identified. The method is especially useful for identifying fragments in a restriction-enzyme DNA digest which contain a given probe sequence, and for analyzing restriction-fragment length polymorphisms (RFLPs).
Another approach to detecting the presence of a given sequence or sequences in a polynucleotide sample involves selective amplification of the sequencers) by polymerase chain reaction (Mullis, Saiki). In this method, primers complementary to opposite end portions of the selected sequence(s) are used to promote, in conjunction with thermal cycling, successive rounds of primer-initiated replication. The amplified sequence may be readily identified by a variety of techniques. This approach is particularly useful for detecting the presence of low-copy sequences in a polynucleotide-containing sample, e.g., for detecting pathogen sequences in a body-fluid sample.
More recently, methods of identifying known target sequences by probe ligation methods have been reported (Wu, Whiteley, Landegren, Winn-Deen). In one approach, known as oligonucleotide ligation assay (OLA), two probes or probe elements which span a target region of interest are hybridized with the target region. Where the probe elements match (basepair with) adjacent target bases at the confronting ends of the probe elements, the two elements can be joined by ligation, e.g., by treatment with ligase. The ligated probe element is then assayed, evidencing the presence of the target sequence.
In a modification of this approach, the ligated probe elements act as a template for a pair of complementary probe elements. With continued cycles of denaturation, reannealing and ligation in the presence of the two complementary pairs of probe elements, the target sequence is amplified geometrically, allowing very small amounts of target sequence to be detected and/or amplified. This approach is also referred to as Ligase Chain Reaction (LCR).
There is a growing need, e.g., in the field of genetic screening, for methods useful in detecting the presence or absence of each of a large number of sequences in a target polynucleotide. For example, as many as 200 different mutations have been associated with cystic fibrosis. In screening for genetic predisposition to this disease, it is optimal to test all of the possible different gene sequence mutations in the subject's genomic DNA, in order to make a positive identification of a “cystic fibrosis”. Ideally, one would like to test for the presence or absence of all of the possible mutation sites in a single assay.
These prior-art methods described above are not readily adaptable for use in detecting multiple selected sequences in a convenient, automated single-assay format. It is therefore desirable to provide a rapid, single-assay format for detecting the presence or absence of multiple selected sequences in a polynucleotide sample.
SUMMARY OF THE INVENTION
The present invention includes, in a first general embodiment, a method of detecting the presence or absence of a plurality of selected target sequences in a target polynucleotide. In practicing the method, a plurality of different-sequence probe pairs are added to a target polynucleotide, where each probe pair includes two polynucleotide probe elements which are complementary in sequence to adjacent portions of a selected one of the target sequences in the target polynucleotide. In each probe pair, one of the probe elements contains a polymer chain which imparts a distinctive electrophoretic mobility in a sieving matrix, to the associated probe pair, when the elements in the pair are ligated. In one embodiment, the polymer chains are nonpolynucleotide chains. The other element in the pair contains a detectable reporter label.
After the probe pairs have been allowed to hybridize with the target polynucleotide, the hybridized polynucleotides are treated under conditions effective to ligate the end subunits of target-bound probe elements when their end subunits are base-paired with adjacent target bases. The ligated probe pairs are then released from the target polynucleotide and separated by electrophoresis in a sieving matrix.
In one embodiment, the polynucleotide portions to all of the probe pairs, in ligated form, are substantially the same in length. In this embodiment, separability of the ligated probe pairs relies predominantly on the non-polynucleotide polymers attached to each probe.
In a second embodiment, the ligated probe(s) are amplified by repeated cycles of probe binding and ligation. The ligated probe(s) may be amplified linearly by repeated binding and ligation of unligated probe to the target sequence. Alternatively, the ligated probe(s) mays be amplified exponentially, by repeated cycles of probe binding and ligation in the presence of a second pair of first and second probe oligonucleotides which, together, make up a sequence that is complementary to the selected ligated probe.
In another embodiment, the second probe element in each probe includes two alternative-sequence oligonucleotides which (i) are complementary to alternative sequences in the same portion of an associated target sequence and (ii) are derivatized with different detectable reporters. This method allows the mutation state of the target sequence to be determined according to (a) a signature electrophoretic migration rate of each probe, which identifies the target sequence associated with that probe, and (b) a signature reporter label, which identifies the mutation state of that target sequence.
In another embodiment, one of the elements (e.g., the first-mentioned element) in each probe includes two alternative-sequence oligonucleotides which (i) are complementary to alternative sequences in the same portion of an associated target sequence and (ii) are derivatized with different polymer chains which impart a distinctive mobility to each associateed probe pair, when the elements in the pair ar ligated. This method allows the mutation state of the target sequence to be determined according to (a) a signature reporter label which identifies the target sequence associated with the associated probe, and (b) a signature mobility, which identifies the mutation state of the associated target sequence.
The polymer chain used in the method may be a substantially uncharged, water-soluble chain, such as a chain composed of polyethylene oxide (PEO) units or a polypeptide chain, where the chains attached to different-sequence binding polymers have different numbers of polymer units. Also included are polymers composed of units of multiple subunits linked by charged or uncharged linking groups.
In another embodiment, hybridization of the probes to the target polynucleotide is carried out with the target polynucleotide immobilized on a solid support. Following hybridization of the probes to the immobilized target polynucleotide, the target polynucleotide is washed to remove probe pairs not bound to the target polynucleotide in a sequence-specific manner. The target polynucleotide is then denatured to release probes bound in a sequence-specific manner.
In a second general embodiment, the inv
Fung Steven
Grossman Paul David
Menchen Steven Michael
Winn-Deen Emily Susan
Woo Sam Lee
Applera Corporation
Dehlinger Peter J.
Finnegan Henderson Farabow Garrett & Dunner LLP
Horlick Kenneth R.
Powers Vincent M.
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