Sensing strategies and methods for nucleic acid detection using biosensors

Abstract

Embodiments of the present invention relate generally to strategies and methods of amplifying short target sequences and removing flanking sequences from target nucleic acids to remove background signal when detecting hybridizations events using sensitive detection biosensors, such as biosensors based on nanowires, carbon nanotubes, nanopores etc, that may be capable of detecting molecules at small molar concentrations (fM and less), or even at the single molecule level. Furthermore, by cropping and therefore standardizing the size of the target sequences to be detected, when detecting many target sequences in an array, the signals across each biosensor can be compared and the hybridization conditions standardized easily.

1 - 13 . (canceled) 
     
     
         14 . An oligonucleotide primer for amplifying a target nucleotide sequence, comprising: a 5′ anchor region, wherein the 5′ anchor region is configured to hybridize to a nucleotide sequence outside of the target nucleotide sequence and is at least partially complimentary to said nucleotide sequence outside of the target nucleotide sequence; a 3′ extension region, wherein the 3′ extension region is configured to hybridize to a portion of the target nucleotide sequence; and a bubble region located between the 5′ anchor region and the 3′ extension region, wherein at least one of the 5′ anchor region and the bubble region comprise a nuclease cleavage sequence. 
     
     
         15 . The oligonucleotide primer of  claim 14 , wherein the 5′ anchor region is about 15 to 30 bp and about 70 to 100% complimentary to said nucleotide sequence outside of the target nucleotide sequence; the 3′ extension region is about 5 to 30 bp and about 80 to 100% complimentary to said portion of the target nucleotide sequence, and wherein a melting temperature of the 3′ extension region hybridized to the portion of the target nucleotide sequence is optimized such that the 3′ extension region can only hybridize to the portion of the target nucleotide sequence under selective conditions when the 5′ anchor region is hybridized to said nucleotide sequence outside of the target nucleotide sequence; and the bubble region is about 50% or less complimentary to the target nucleotide sequence, and comprises a nuclease cleavage sequence. 
     
     
         16 . The oligonucleotide primer of  claim 14 , wherein the oligonucleotide primer is configured to facilitate PCR multiplexing in a single reaction. 
     
     
         17 . The oligonucleotide primer of  claim 14 , wherein the oligonucleotide primer is configured to facilitate consistent amplification of short amplicons. 
     
     
         18 . A biosensor for detecting an analyte in a sample, wherein the analyte comprises a target nucleotide sequence, comprising: a signal sensing unit, the signal sensing unit comprising a probe immobilized on a conducting medium, wherein the probe is prepared by amplifying the target nucleotide sequence and optionally at least one additional nucleotide sequence with one or more primers comprising a 5′ anchor region, a 3′ extension region, and a bubble region therebetween, wherein at least part of the 5′ anchor region is removed after amplification; a signal detecting unit operably coupled to the conducting medium of the signal sensing unit; and a signal processing unit operably coupled to the signal detecting unit. 
     
     
         19 . The biosensor of  claim 18 , wherein the signal processing unit further comprises one of more of a signal sensing unit, a signal recording unit, a data processing unit, and a data reporting unit. 
     
     
         20 . (canceled) 
     
     
         21 . A method of detecting an analyte comprising a target nucleotide sequence, the method comprising the steps of: obtaining the analyte comprising the target nucleotide sequence; contacting the analyte with an oligonucleotide, wherein the oligonucleotide hybridizes to the target nucleotide sequence, thereby generating a duplex of the target nucleotide sequence and the oligonucleotide; removing single stranded nucleotide sequences that are not present in the duplex; obtaining the target nucleotide sequence from the duplex; contacting the obtained target nucleotide sequence with a probe, which is immobilized on a conducting medium, under hybridizing conditions, wherein the probe is at least partially complimentary to the target nucleotide sequence, such that the probe hybridizes to the target nucleotide sequence, thereby generating a duplex of the target nucleotide sequence and the probe; and detecting a signal related to the presence, level and/or sequence variation of the target nucleotide sequence. 
     
     
         22 . The method of  claim 21 , wherein the probe is generated by amplifying the target nucleotide sequence and optionally at least one additional nucleotide sequence with one or more primers comprising a 5′ anchor region, a 3′ extension region, and a bubble region therebetween, wherein at least part of the 5′ anchor region is removed after amplification. 
     
     
         23 . The method of  claim 22 , wherein at least one of the 5′ anchor region and the bubble region comprise a nuclease cleavage sequence. 
     
     
         24 . The method of  claim 21 , wherein the analyte is obtained by one of the following methods: amplifying the analyte comprising the target nucleotide sequence with one or more primers comprising nucleotide sequences corresponding to part of the target nucleotide sequence, obtaining nucleotide sequences from an organism and sheering the nucleotide sequences into a size suitable for contacting with the probe, and obtaining nucleotide sequences from an organism and cleaving the nucleotide sequences using one or more nucleases into a size suitable for contacting with the probe. 
     
     
         25 . The method of  claim 21 , wherein removing single stranded nucleotide sequences comprises cleaving the single stranded nucleotide sequences using one or more nucleases. 
     
     
         26 . The method of  claim 25 , wherein said one or more nucleases comprise sequence-specific nucleases and/or sequence-nonspecific nucleases. 
     
     
         27 . The method of  claim 25 , wherein said one or more nucleases are selected from the group consisting of S1 nuclease, P1 nuclease, micrococcal nuclease, mung bean nuclease, exonuclease, II, III, IV, V, VI, VII and VIII, and Klenow fragment. 
     
     
         28 . The method of  claim 21 , wherein said conducting medium is selected from the group consisting of nanowire, nanotube, nanogap, nanopore, nanobead, and any other nanostructure that is able to conduct electricity. 
     
     
         29 . The method of  claim 28 , wherein the signal is generated when hybridization of the target nucleotide sequence to the probe causes a change in the electrical charge of the probe. 
     
     
         30 . A method of amplifying a target nucleotide sequence, using one or more primers comprising a 5′ anchor region, a 3′ extension region, and a bubble region therebetween, the 5′ anchor region being configured to hybridize to a nucleotide sequence outside of the target nucleotide sequence and being at least partially complimentary to said nucleotide sequence outside of the target nucleotide sequence; the 3′ extension region being configured to hybridize to a portion of the target nucleotide sequence; and the bubble region being located between the 5′ anchor region and the 3′ extension region and being not complementary or only partly complementary to the target nucleotide sequence.