Binary DNA Probe for Fluorescent Analysis of Nucleic Acids

Abstract

The invention is directed to binary oligonucleotide probes for nucleic analysis, which probes can be made of DNA or RNA that recognize nucleic acid analytes (both DNA and RNA) with unprecedented high selectivity under mild conditions and are highly sensitive to single nucleotide mismatches (SNP single nucleotide polymorphisms) without PCR amplification. In one group, the binary probes indicate that they have hybridized to a particular nucleic analyte by binding to a molecular beacon that gives off a fluorescent signal. A second group of binary probes bind to a dye such as malachite green, where upon hybridization to analyte the fluorescence of the dye increases dramatically and is easily detected and measured. The new binary probes require only about five minutes at room temperature to generate a detectable signal.

1 . A method for identifying an SNP in an analyte nucleotide fragment using fluorescent oligonucleotide tandems, comprising:
 1. identifying a known SNP in a chromosome in the genome of an animal,   2. obtaining an single stranded analyte fragment from human or an experimental animal which fragment comprises a part of the chromosome having the known SNP identified in step 1,   3. identifying a five nucleotide long target analyte sequence in the analyte fragment, in which the position of the known SNP is at the center (the number 3 nucleotide),   4. designing a fluorescent oligonucleotide tandem probe of claim  35  that binds to the target analyte,   5. designing four oligonucleotides 5 nucleotides long of known sequence in which the nucleotides in all positions except one are complementary to respective nucleotides in the target sequence,   6. mixing the analyte with the first and second oligonucleotide probes under conditions that permit hybridization,   7. determining the amount of fluorescence in the mixture of step 6, to obtain a baseline fluorescence,   8. adding one of the four penta-oligonucleotides probes under conditions that permit hybridization,   9. measuring the fluorescence of the mixture in step 8,   10. determining that a penta-oligonucleotide has bound to the target sequence and is fully complementary to the target sequence if the fluorescence determined in step 9 is significantly greater than the baseline fluorescence,   11. if the penta-oligonucleotide of step 10 is determined to be fully complementary to the target sequence, then identifying the nucleotide at the center position of the target sequence as that corresponding to the nucleotide at the center position of the pentanucleotide added in step 6, and   12. determining whether the nucleotide at the center position of the target sequence represents an SNP by comparing it with the known SNP, and   13. if the penta-oligonucleotide is not fully complementary to the target sequence, then washing out the penta-oligonucleotide and adding another penta-oligonucleotide before repeating steps 8-12.   
     
     
         2 . A binary DNA probe having on the first strand a molecular beacon binding arm comprising the nucleotide sequence 5′ CATAGGTC and an analyte binding arm comprising the sequence set forth in SEQ ID No. 17, and on the second strand a molecular beacon binding arm comprising the nucleotide sequence 5′ TTAACTTC and an analyte binding arm comprising the sequence set forth in SEQ ID No. 18, which probe binds to a fragment of the  Francisella tularensis  16S rRNA. 
     
     
         3 . A binary oligonucleotide probe hybridization assay to detect a single stranded nucleotide analyte having a known sequence in a sample containing a heterogeneous mixture of nucleic, having the following steps:
 a) providing a first binary oligonucleotide probe comprising two oligonucleotide strands, wherein (1) a first oligonucleotide strand comprises at its 5′-terminus a reporter binding arm that is complementary to and is capable of selectively hybridizing with a reporter, and at its 3′-terminus, a first analyte binding arm that is complementary to and is capable of selectively hybridizing with a first region of the oligonucleotide analyte, and (2) a second oligonucleotide strand comprises: at its 3′-terminus a reporter binding arm that is complementary to and is capable of selectively hybridizing with a nucleotide sequence in the reporter, and at its 5′ terminus, a second analyte binding arm that is complementary to and is capable of selectively hybridizing with a second region of the oligonucleotide analyte, wherein the nucleotides in the analyte binding arms of the first probe are complementary to the known nucleotide sequence in the first analyte,   b) providing a first molecular beacon that fluoresces at a first wavelength and that selectively hybridizes to the molecular beacon binding arms on the first probe,   c) creating a mixture comprising the first binary probe and the first molecular beacon,   d) determining a first background level of fluorescence of the first molecular beacon for the mixture of step c,   e) adding the sample to the mixture of step c,   f) maintaining said mixture of step e for a sufficient period of time and under predetermined reaction conditions to allow the analyte to hybridize to the analyte binding arms on the first probe, and for the first molecular beacon to hybridize to the molecular beacon binding arms on the first probe, and   g) determining that the analyte is present in the sample if the level of fluorescence of the first molecular beacon increases above the first background level.   
     
     
         4 . The hybridization assay of  claim 3 , further comprising:
 1. in step a) providing a second binary oligonucleotide probe to detect a second different nucleic acid analyte in the sample, wherein the analyte binding arms on the second probe are complementary to and hybridize with a known nucleotide sequence in a second different analyte,   2. in step b) providing a second molecular beacon that fluoresces at a second wavelength and that selectively hybridizes to the molecular beacon binding arms on the second probe,   3. in step c) creating a mixture further comprising the second binary probe and the second molecular beacon,   4. in step d) determining a second background level of fluorescence of the second molecular beacon for the mixture of step c,   5. in step f) maintaining said mixture of step e for a sufficient period of time and under predetermined reaction conditions to allow the second analyte to hybridize to the analyte binding arms on the second probe, and for the second molecular beacon to hybridize to the molecular beacon binding arms on the second probe, and   6. in step g) determining that the second analyte is present in the sample if the level of fluorescence of the second molecular beacon increases above the second background level.