Oligonucleotides for discriminating related nucleic acid sequences
OwnerIvan Brukner, Damian Labuda +1inventors
InventorsIvan Brukner, Damian Labuda, Maja Krajinovic
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Abstract
An in vitro selection method is described which identifies oligonucleotide probes that discriminate amongst closely related nucleic acid sequences and which involves iterative hybridizations, including subtractive hybridization. Using the method, oligonucleotides are identified which can discriminate among human papilloma virus (HPV) subtypes. Corresponding methods and kits for the detection of nucleic acids are described, which methods and kits may be used in analytical, diagnostic, research and related applications.
Claims62
A method for identifying an oligonucleotide for discriminating a first nucleic acid from a second nucleic acid, said method comprising:
- (a) hybridizing said first nucleic acid with a pool of oligonucleotides in a hybridization mixture, said oligonucleotides comprising a random nucleotide sequence flanked by primer recognition sequences;
- (b) removing oligonucleotides which are not bound to said first nucleic acid from said hybridization mixture;
- (c) dissociating bound oligonucleotides from said first nucleic acid;
- (d) amplifying said bound oligonucleotides using primers capable of binding to said primer recognition sequences to obtain amplified oligonucleotide duplexes comprising a first strand corresponding to said bound oligonucleotides and a second strand corresponding to the complement of said bound oligonucleotides;
- (e) treating said duplexes to remove or degrade said second strand to obtain single-stranded amplified oligonucleotides;
- (f) repeating (a) to (e), wherein said pool of oligonucleotides of (a) is the amplified oligonucleotides obtained in (e) thereby to obtain further amplified oligonucleotides;
- and
- (g) repeating (a) to (e), wherein said hybridization step (a) is performed in the further presence of said second nucleic acid;
- wherein an oligonucleotide comprising the random nucleotide sequence of said further amplified oligonucleotides can be used for discriminating said first nucleic acid from said second nucleic acid.
An oligonucleotide capable of discriminating a first nucleic acid from a second nucleic acid, wherein said oligonucleotide is not exactly complementary to said first nucleic acid.
An oligonucleotide comprising a nucleotide sequence selected from SEQ ID NOs:
- 1-43, 100-104 and 116.
A collection of two or more oligonucleotides, wherein said oligonucleotides comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs:
- 1-43, 100-104 and 116.
Dependencies are inferred from the claim text.