US 6,190,868 B1 · Method for identifying a nucleic acid sequence

Abstract

The present invention discloses a methodology which is directed to providing positive confirmation that nucleic acids, possessing putatively identified sequence predicted to generate observed GeneCalling™ signals, are actually present within the sample from which the signal was originally derived. The putatively identified nucleic acid fragment within the sample possesses 3′- and 5′-ends with known terminal subsequences, said method comprising; contacting said nucleic acid fragments in said sample in amplifying conditions with (i) a nucleic acid polymerase; (ii) “regular” primer oligonucleotides having sequences comprising hybridizable portions of said known terminal subsequences; and (iii) a “poisoning” oligonucleotide primer, said poisoning primer having a sequence comprising a first subsequence that is a portion of the sequence of one of said known terminal subsequences and a second subsequence that is a hybridizable portion of said putatively unidentified sequence which is adjacent to said one known terminal subsequence, wherein nucleic acids amplified with said poisoning primer are distinguishable upon detection from nucleic acids amplified with said nucleic acids amplified only with said regular primers; separating the products of the contacting step; and the detecting sequence is confirmed if the nucleic acids amplified with said poisoning primer are detected.

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Claims14

Dependent claims

A method for identifying one or more nucleic acids in a sample comprising a plurality of nucleic acids having different nucleotide sequences, said method comprising:

(a) providing a sample contacted by one or more recognition means, each of said recognition means recognizing a different target nucleotide sequence or different set of target nucleotide subsequences;
(b) generating one or more signals from said sample by amplifying one or more sequences in said sample with at least one amplification means, each generated signal arising from a nucleic acid in said sample and comprising a representation of (i) the size of the sequence comprising said target nucleotide subsequences in said nucleic acid, and (ii) the identities of said target subsequences in said nucleic acid;
(c) searching a nucleotide sequence database for sequences that have a corresponding size and identified target subsequence as one or more of the nucleic acids generating said signal, said database comprising a plurality of known nucleotide sequences of nucleic acids that may be present in the sample, a sequence from said database matching the generated signal when the sequence from said database has both (i) the same size of the sequence comprising said target nucleotide subsequences as is represented by the generated signal, and (ii) the same target subsequences as are represented by the generated signal, or target subsequences that are members of the same sets of target subsequences represented by the generated signal, thereby identifying a putative matching sequence;
(d) contacting under amplifying conditions said sample with said amplification means and one or more poisoning primers to form a reamplified signal corresponding to said putative matching sequence, wherein said poisoning primers are capable of hybridizing to and amplifying only nucleic acids having said putative matching sequence, said poisoning primers being unlabeled or differentially labeled from said amplification means and having a sequence comprising a first subsequence that is a portion of said putative matching sequence and a second subsequence;
and
(e) comparing the reamplified signal of said putative matching sequence to the signal of said putative matching sequence in step (b), wherein a decrease in the reamplified signal relative to the amplified signal verifies the identity of said matching sequences thereby identifying one or more nucleic acids in said sample.

3 more elements

A method of confirming a putatively identified sequence of a nucleic acid fragment in a sample of nucleic acid fragments, wherein each nucleic acid fragment in said sample has ends with known terminal subsequences, said method comprising;

(a) providing an amplified nucleic acid sample comprising an amplified signal associated with said putatively identified sequence, said amplified nucleic acid sample generated by contacting said nucleic acid fragments in said sample in amplifying conditions with a nucleic acid polymerase and at least one amplification means having sequences comprising hybridizable portions of said known terminal subsequence;
(b) contacting said amplified nucleic acid sample in amplifying conditions with a nucleic acid polymerase, said amplification means and poisoning primers to generate a reamplified signal associated with said putatively identified sequence, said poisoning primers having a sequence comprising a first subsequence that is a portion of the sequence of one of said known terminal subsequences and a second subsequence that is a hybridizable portion of said putatively identified sequence that is adjacent to said one known terminal subsequence, wherein nucleic acids amplified with said poisoning primer are distinguishable upon detection from nucleic acids amplified only with said amplification means;
and
(c) comparing said reamplified signal with said amplified signal, wherein a decrease in the amplified signal relative to said reamplified signal confirms the identity of said putatively identified sequence.

1 more element

Dependencies are inferred from the claim text.

Description15,204 words

Summary4,807 words

#The field of the invention is DNA sequence classification, identification or determination, and quantification; more particularly it is the quantitative classification, comparison of expression, or identification of preferably all DNA sequences or genes in a sample without performing any associated sequencing.

#As molecular biological and genetics research have advanced, it has become increasingly clear that the temporal and spatial expression of genes plays a vital role in processes occurring in both health and in disease. Moreover, the field of biology has progressed from an understanding of how single genetic defects cause the traditionally recognized hereditary disorders (e.g., the thalassemias), to a realization of the importance of the interaction of multiple genetic defects in concert with various environmental factors in the etiology of the majority of the more complex disorders, such as neoplasia.

#For example, in the case of neoplasia, recent experimental evidence has demonstrated the key causative roles of multiple defects in several pivotal genes causing their altered expression. Other complex diseases have been shown to have a similar etiology. Therefore, the more complete and reliable a correlation which can be established between gene expression and disease states, the better diseases will be able to be recognized, diagnosed and treated. This important correlation may be established by the quantitative determination and classification of DNA expression in tissue samples.

Brief description of the drawings6 figures

#The present invention may be better understood and its advantages appreciated by those individuals skilled in the relevant arts by referring to the accompanying drawings wherein:

#FIG. 1 is an illustration of the DNA primers utilized for a PCR amplification-mediated embodiment of the GeneCalling™ methodology.

#FIG. 2 is a flow chart illustrating the various steps utilized in the oligo-poisoning methodology of the present invention as applied to the conformation of the results obtained from GeneCalling.™

Detailed description10,113 words

#In order that the present invention disclosed herein is better understood and appreciated, the following detailed description is set forth.

#Without limitation, the oligo-poisoning methodology disclosed herein has been primarily applied in quantitative expression analysis (GeneCalling™), as disclosed in WO97115690 entitled “METHOD AND APPARATUS FOR IDENTIFYING, QUANTIFYING, AND CONFIRMING DNA SEQUENCES IN A SAMPLE WITHOUT SEQUENCING,” and which is incorporated herein by reference in its entirety. However, individuals possessing ordinary skill within the relevant arts will immediately appreciate how to adapt this methodology to the other protocols for generating appropriate nucleic acids samples of the described structure. Prior to proceeding with the detailed description of the oligo-poisoning methodology, a review of GeneCalling™ methodology will be set forth.

#1. Quantitative Expression Analysis (GeneCalling™)

Facts

Application

09/381,779Filed 23 Sep 1999 · us provisional application

Priority

7 Aug 1997

Granted

20 Feb 2001

Term ended

23 Sep 2019

Estimated · how

20 years from earliest known nonprovisional domestic filing. Maintenance-fee lapses, terminal disclaimers and statutory extensions have not been checked. The continuity chain may be incomplete; this is not an enforceability determination. Patent term adjustment is unavailable; no adjustment was added.

Classification6 codes

C12N 15/1093
Isolating an individual clone by screening libraries: general methods of preparing gene libraries, not provided for in other subgroups
C12Q 1/6809
Involving nucleic acids: methods for determination or identification of nucleic acids involving differential detection
C12Q 1/6813
Involving nucleic acids: hybridisation assays
C12Q 1/6855
Using modified primers or templates: ligating adaptors

Method for identifying a nucleic acid sequence

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Method for identifying a nucleic acid sequence

Life of this patent

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Abstract

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Claims14

Description15,204 words

Drawings

Classification6 codes

Life of this patent

Description15,204 words