Method of detecting and discriminating between nucleic acid sequences
First Claim
1. An allele specific primer having a 3'"'"' portion and a 5'"'"' portionwherein said 3'"'"' portion is complementary to a target sequence adjacent an allele specific nucleotide, andwherein said 5'"'"' portion is complementary to a preselected and randomly generated nucleic acid sequence which is different from said target sequence and which said preselected nucleic acid sequence is immobilized at a preselected location in an array of immobilized, preselected and randomly generated nucleic acid sequences on a solid support, wherein the length, base composition and percentage of each base in each of said immobilized preselected nucleic acid sequences are substantially the same to permit hybridization between said 5'"'"' portion and said preselected nucleic acid sequence to be carried out at the same temperature for all said immobilized preselected nucleic acid sequences.
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Abstract
The present invention is directed to a method for detecting the presence or absence of any specific target nucleic acid sequence contained in a sample. The target sequence can be present in the sample in a relatively pure form or as a component of a member of a mixture of different nucleic acids. The method of the invention utilizes a novel primer design. The sequence of the novel primer is composed of two portions, the 3'"'"' portion is a primer specific for the desired nucleic acid sequence and the 5'"'"' portion is complementary to preselected nucleic acid sequence. Extension of the 3'"'"' portion of the primer with a labeled deoxynucleosides triphosphate yields a labeled extension product if, but only if, the template includes the target sequence. The labeled extension product is detected by hybridization of the 5'"'"' portion to the preselected sequence. The preselected sequence is preferably bound to a solid support as one member of a grid having a group of sequences.
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Citations
11 Claims
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1. An allele specific primer having a 3'"'"' portion and a 5'"'"' portion
wherein said 3'"'"' portion is complementary to a target sequence adjacent an allele specific nucleotide, and wherein said 5'"'"' portion is complementary to a preselected and randomly generated nucleic acid sequence which is different from said target sequence and which said preselected nucleic acid sequence is immobilized at a preselected location in an array of immobilized, preselected and randomly generated nucleic acid sequences on a solid support, wherein the length, base composition and percentage of each base in each of said immobilized preselected nucleic acid sequences are substantially the same to permit hybridization between said 5'"'"' portion and said preselected nucleic acid sequence to be carried out at the same temperature for all said immobilized preselected nucleic acid sequences.
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4. A method for identifying an allele specific nucleotide in a target nucleic acid sequence in a nucleic acid sample, said method comprising
(a) combining under hybridizing conditions to form a duplex (i) a nucleic acid sample which may include a target nucleic acid sequence, (ii) an allele specific primer having a 3'"'"' portion and a 5'"'"' portion wherein said 3'"'"' portion is complementary to a target nucleic acid sequence adjacent an allele specific nucleotide, and wherein said 5'"'"' portion is complementary to a preselected and randomly generated nucleic acid sequence which is different from said target nucleic acid sequence and which said preselected nucleic acid sequence is immobilized at a preselected location in an array of immobilized, preselected and randomly generated nucleic acid sequences on a solid support, wherein the lengths base composition and percentage of each base in each of said immobilized preselected nucleic acid sequences are substantially the same to permit hybridization between said 5'"'"' portion and said preselected nucleic acid sequence to be carried out at the same temperature for all said immobilized preselected nucleic acid sequences, (b) adding a DNA polymerase and deoxynucleoside triphosphates, one of which is an allele specific nucleotide containing a label, to the duplex of step (a), if present, under conditions to form an allele specific labeled primer extension product which includes the 3'"'"' and 5'"'"' portions of the allele specific primer of (a)(ii), (c) screening the allele specific labeled primer extension product of (b) by combining the 5'"'"' portion with the array of immobilized, preselected and randomly generated nucleic acid sequences on the solid support whereby the label identifies the allele specific nucleotide and the location of the immobilized preselected nucleic acid sequence is associated with the locus of the target nucleic acid sequence in the nucleic acid sample.
- 8. A solid support for capturing a plurality of nucleic acids, each of said nucleic acids having a 3'"'"' portion complementary to a target sequence and a 5'"'"' portion, said solid support comprising a plurality of immobilized, preselected and randomly generated nucleic acid sequences, wherein the length, base composition and percentage of each base in each of said immobilized preselected nucleic acid sequences are substantially the same to permit hybridization between said 5'"'"' portion and its complementary preselected nucleic acid sequence to be carried out at the same temperature for all said immobilized preselected nucleic acid sequences.
Specification