Single molecule amplification and detection of DNA length

Abstract

Methods and systems for performing single molecule amplification for detection, quantification and statistical analysis of nucleic acids are provided. Methods and systems are provided for determining and quantifying lengths of nucleic acids of interest.

1 . A method of determining whether a nucleic acid of interest in a sample comprises at least a given length, the method comprising: 
 contacting the nucleic acid of interest in a reaction mixture with two or more different probes, wherein the probes each comprise a detectable marker;    flowing the nucleic acid of interest into a detection region; and,    detecting one or more detectable marker signals from the probes;    wherein coincident detection of signals from two or more of the different probes indicates the nucleic acid of interest is not fragmented between the probes, thereby determining the nucleic acid of interest in the sample has at least a given length.    
     
     
         2 . The method of  claim 1 , wherein the reaction mixture comprises a single copy of the nucleic acid of interest.  
     
     
         3 . The method of  claim 1 , wherein detecting a signal from only one of the different probes indicates the nucleic acid of interest is fragmented.  
     
     
         4 . The method of  claim 1 , wherein determining the given length comprises determining the integrity of the nucleic acid of interest.  
     
     
         5 . The method of  claim 1 , wherein the two or more probes each comprise detectable markers with different signals.  
     
     
         6 . The method of  claim 1 , wherein at least one of the probes comprise a fluorescent resonant energy transfer (FRET) detectable marker or a molecular beacon (MB) marker.  
     
     
         7 . The method of  claim 1 , further comprising correlating one or more lengths of one or more nucleic acids of interest to a disease state by identifying a ratio or quantitative threshold of lengths associated with the disease state.  
     
     
         8 . The method of  claim 1 , further comprising: 
 contacting the nucleic acid of interest with a first primer pair;    contacting the nucleic acid of interest with a second primer pair comprising at least one primer complimentary to a sequence of the nucleic acid of interest or its compliment outside of a sequence defined by the first primer pair; and,    amplifying the nucleic acid of interest in the reaction mixture contained in a microchannel or microchamber with primer extensions initiated at primers to produce first amplicons defined by the first primer pair or second amplicons defined by the second primer pair;    wherein at least a first probe is complimentary to a sequence of the first amplicons and at least a second probe is complimentary to a sequence of the second amplicons;    whereby a sensitivity of said detecting is increased.    
     
     
         9 . The method of  claim 8 , wherein one primer pair comprises control primers defining amplicons of 100 base pairs or less in length and another primer pair comprises test primers defining longer amplicons ranging in length from about 100 base pairs to about 3000 base pairs.  
     
     
         10 . The method of  claim 8 , wherein a region of the nucleic acid of interest defined by the first primer pair does not overlap with a region of the nucleic acid of interest defined by the second primer pair.  
     
     
         11 . The method of  claim 8 , wherein at least one of the probes is complimentary to an amplicon sequence defined by one primer pair but not complimentary to an amplicon sequence defined by another primer pair.  
     
     
         12 . The method of  claim 8 , further comprising: 
 contacting the nucleic acid of interest with one or more additional primer pairs comprising at least one primer complimentary to a sequence of the nucleic acid of interest or its compliment outside a sequence defined by the first primer pair or second primer pair;    whereby one or more additional amplicons are produced, and whereby coincident detection of signals from a probe specific for the one or more additional amplicons and the first or second probes indicates the nucleic acid of interest is not fragmented between sequences complimentary to probes providing the signals.    
     
     
         13 . The method of  claim 8 , wherein said amplifying comprises: a polymerase chain reaction (PCR), reverse-transcriptase PCR (RT-PCR), ligase chain reaction (LCR), a Q-β replicase or RNA/transcription mediated techniques.  
     
     
         14 . The method of  claim 1 , further comprising: 
 aliquotting the sample into at least 25 reaction mixtures comprising 2 or fewer copies of the nucleic acid of interest each;    individually subjecting the sample aliquots to the contacting and the detecting; and,    individually counting a number of aliquots resulting in detection of a signal from one probe or individually counting a number of aliquots resulting in detection of signals from two or more probes.    
     
     
         15 . The method of  claim 14 , wherein the at least 25 reaction mixtures comprise one or more reaction mixtures comprising single copies of the nucleic acid of interest.  
     
     
         16 . The method of  claim 14 , wherein the at least 25 reaction mixtures comprise one or more reaction mixtures comprising zero copies of the nucleic acid of interest.  
     
     
         17 . The method of  claim 14 , further comprising evaluating the number of one probe signals and two probe signals to determine a proportion of nucleic acids of interest having different lengths.  
     
     
         18 . The method of  claim 17 , further comprising correlating a disease state with the proportion.  
     
     
         19 . The method of  claim 1 , further comprising quantifying the nucleic acid of interest.  
     
     
         20 . The method of  claim 19 , wherein said quantifying comprises counting a number of signals from one or more of the different probes.  
     
     
         21 . The method of  claim 19 , wherein said quantifying comprises detecting a volume, width, height, length, area, shape, or ratio, of the one or more signals.  
     
     
         22 . The method of  claim 19 , wherein said quantifying comprises comparison of a probe detectable marker signal to an internal standard signal.  
     
     
         23 . The method of  claim 19 , wherein said quantifying comprises comparison of signals from two or more reaction mixtures comprising different degrees of amplification.  
     
     
         24 . The method of  claim 23 , wherein the two or more reaction mixtures comprise different amplification due to: flowing through a thermocycler at different flow rates, flowing different distances into a thermocycler, remaining in a thermocycler for different amounts of time, or experiencing different numbers of amplification cycles.  
     
     
         25 . The method of  claim 1 , further comprising diluting the sample to obtain one or more reaction mixtures containing a single copy of the nucleic acid of interest.  
     
     
         26 . The method of  claim 25 , wherein the nucleic acid of interest is diluted to a concentration of about 1 molecule per nanoliter or less.  
     
     
         27 . The method of  claim 1 , wherein the sample comprises a sample selected from the group consisting of: whole blood, serum, plasma, stool, urine, vaginal secretions, ejaculatory fluid, a cervical swab, synovial fluid, a biopsy, cerebrospinal fluid, amniotic fluid, sputum, saliva, lymph, tears, sweat, and urine.  
     
     
         28 . A method of differentiating lengths of a nucleic acid of interest in a sample, the method comprising: 
 a) contacting the nucleic acid of interest with a first primer pair;    b) contacting the nucleic acid of interest with a second primer pair comprising at least one primer complimentary to a sequence of the nucleic acid of interest or its compliment outside of a sequence defined by the first primer pair;    c) amplifying the nucleic acid of interest in a reaction mixture comprising a single copy of the nucleic acid of interest to produce first amplicons defined by the first primer pair or second amplicons defined by the second primer pair;    d) contacting the reaction mixture with a first probe complimentary to a sequence of the first amplicons or with a second probe complimentary to a sequence of the second amplicons, which probes comprise signals from detectable markers; and,    e) detecting one or more of the signals;    wherein detection of a signal from only one of the probes indicates a fragmented nucleic acid of interest or detecting signals from both probes indicates a nucleic acid that has a given length, thereby differentiating the length of the nucleic acid of interest.    
     
     
         29 . The method of  claim 28 , wherein the reaction mixture is contained in a microchannel or microchamber.  
     
     
         30 . A method of differentiating lengths of nucleic acids of interest in a sample, the method comprising: 
 a) contacting a nucleic acid of interest with a first primer pair;    b) contacting the nucleic acid of interest with a second primer pair comprising at least one primer complimentary to a sequence of the nucleic acid of interest or its compliment outside of a sequence defined by the first primer pair;    c) amplifying the nucleic acid of interest in a reaction mixture contained in a microchannel or microchamber with primer extensions initiated at the primers to produce first amplicons defined by the first primer pair or second amplicons defined by the second primer pair;    d) contacting the reaction mixture with a first probe complimentary to a sequence of the first amplicons or with a second probe complimentary to a sequence of the second amplicons, which probes comprise signals from detectable markers; and,    e) detecting one or more signals;    wherein detection of a signal from only one of the probes indicates a fragmented nucleic acid of interest or detecting signals from both probes indicates a nucleic acid that has a given length, thereby differentiating the length of the nucleic acid of interest.    
     
     
         31 . The method of  claim 1 , wherein the reaction mixture comprises a single copy of the nucleic acid of interest.  
     
     
         32 . A method of quantifying a nucleic acid of interest in a sample, the method comprising: 
 amplifying the nucleic acid of interest through a plurality of amplification cycles;    detecting signals associated with amplicons produced for two or more of the amplification cycles;    preparing a sample curve of a signal parameter versus a number of amplification cycles; and,    comparing one or more identifiable points from the sample curve to a standard curve of identifiable points versus concentration, thereby quantifying the nucleic acid of interest.    
     
     
         33 . The method of  claim 32 , wherein the identifiable points comprise: points of inflection, points having a certain slope, points having a certain absolute signal amplitude, or points having a certain fraction of a maximum signal amplitude.  
     
     
         34 . A method of quantifying a nucleic acid of interest in a sample, the method comprising: 
 amplifying the nucleic acid of interest through a plurality of amplification cycles in a reaction mixture defining two or more different amplicons of the nucleic acid of interest;    detecting from homogenous reaction mixtures different signals associated with each of the different amplicons after at least two of the plurality of amplification cycles;    preparing sample curves of the different signals versus numbers of amplification cycles; and,    comparing one or more identifiable points from the sample curves to one or more standard curves of identifiable points versus nucleic acid concentration, thereby quantifying one or more sequences of the nucleic acid of interest associated with one or more of the amplicons.    
     
     
         35 . The method of  claim 34 , wherein said detecting comprises detecting one or more signals from a low copy or single copy reaction mixture.  
     
     
         36 . The method of  claim 35 , wherein coincident detection of two or more of the different signals indicates a nucleic acid of a given length, or the detection of only one of the different signals indicates a fragmented nucleic acid.  
     
     
         37 . The method of  claim 34 , wherein the identifiable points comprise: points of inflection, points having a certain slope, points having a certain absolute signal amplitude, or points having a certain fraction of a maximum signal amplitude.  
     
     
         38 . The method of  claim 34 , wherein the number of amplification cycles is controlled by: flowing an amplification reaction through a thermocycler at different flow rates, flowing an amplification reaction different distances into a thermocycler, an amplification reaction remaining in a thermocycler for different amounts of time, or an amplification reaction experiencing different numbers of amplification cycles.  
     
     
         39 . A method of quantifying a nucleic acid of interest in a sample, the method comprising: 
 amplifying a plurality of nucleic acid of interest standard materials through a number of amplification cycles;    detecting signals associated with standard amplicons produced for standard materials having different known concentrations of the nucleic acid;    amplifying the sample nucleic acid of interest the number of amplification cycles;    detecting a signal associated with sample amplicons produced for the sample nucleic acid of interest; and,    comparing one or more standard amplicon signals to the sample amplicon signal to determine a concentration value for the nucleic acid of interest in the sample, thereby quantifying the nucleic acid of interest.    
     
     
         40 . The method of  claim 39 , wherein said comparing comprises comparison of signal parameters selected from the group consisting of: a shape of a signal peak, points of inflection on a signal peak, slopes of signal peaks, signal peak amplitude, signal peak areas, and signal peak widths at half height.  
     
     
         41 . The method of  claim 39 , further comprising: 
 repeating said amplifying, detecting, and comparing steps one or more times, but with different numbers of amplification cycles, thereby determining additional concentration values for the sample nucleic acid of interest; and,    Statistically evaluating the concentration values, thereby providing a more precise or more accurate concentration value result for the nucleic acid of interest in the sample.    
     
     
         42 . A system for differentiating the lengths of nucleic acids of interest in a sample, the system comprising: 
 a microfluidic device comprising an amplification microchannel or microchamber containing a reaction mixture under conditions that provide one or more amplicons of the nucleic acid of interest;    a detector integral with or proximal to the microfluidic device, which detector is configured to detect the amplicons as one or more signals from a homogenous mixture; and,    a software system that interprets one or more coincidently detected signals to indicate lengths of one or more individual nucleic acid molecules of interest, thereby differentiating the lengths of the nucleic acids of interest.    
     
     
         43 . The system of  claim 42 , wherein the sample comprises: a nucleic acid with single nucleotide polymorphism (SNP), a cancer associated nucleic acid, a nucleic acid from an infective agent, whole blood, serum, plasma, stool, urine, a vaginal secretion, cervical swab, ejaculatory fluid, synovial fluid, a biopsy, cerebrospinal fluid, amniotic fluid, or a forensic nucleic acid.  
     
     
         44 . The system of  claim 42 , wherein the reaction mixture comprises: the nucleic acid of interest, a first primer pair, a second primer pair comprising at least one primer complimentary to a sequence of the nucleic acid of interest outside a sequence defined by the first primer pair, and a polymerase that can synthesize amplicons defined by the primer pairs.  
     
     
         45 . The system of  claim 44 , wherein one primer pair comprises control primers defining amplicons of 100 base pairs or less in length and another primer pair comprises test primers defining longer amplicons ranging in length from about 100 base pairs to about 3000 base pairs.  
     
     
         46 . The system of  claim 44 , wherein a region of the nucleic acid of interest defined by the first primer pair does not overlap with a region of the nucleic acid of interest defined by the second primer pair.  
     
     
         47 . The system of  claim 42 , wherein the amplification microchannel or microchamber comprises: electrodes to apply a heating current to the microchannel, a resistive heating element, a Joule-Thompson device, or a Peltier device.  
     
     
         48 . The system of  claim 42 , wherein the amplification microchannel or microchamber is configured to thermocycle the reaction mixture to produce amplicons of the nucleic acid of interest in a volume sufficiently small to substantially separate amplification products of a single nucleic acid of interest molecule from other nucleic acid of interest molecules in the sample or from additional nucleic acids in the sample.  
     
     
         49 . The system of  claim 42 , wherein the amplicons are detected without resolution in a size selective media or affinity media.  
     
     
         50 . The system of  claim 42 , wherein the system software interprets a volume, width, height, length, area, shape, or ratio, of the signals detected by the detector to indicate: a number of copies of the nucleic acid of interest in the sample, a number of the nucleic acids of interest having a given length, or a proportion of nucleic acids of interest having different lengths.  
     
     
         51 . The system of  claim 42 , further comprising one or more nucleic acid probes comprising one or more detectable markers and a sequence complimentary to one or more of the amplicons, wherein the detectable markers provide a signal detectable by the detector.  
     
     
         52 . The system of  claim 51 , wherein the detector comprises: a fluorometer, a charge coupled device, a laser, an enzyme, or an enzyme substrate, a photo multiplier tube, a spectrophotometer, scanning detector, microscope, or a galvo-scanner.  
     
     
         53 . The system of  claim 52 , wherein the fluorometer can simultaneously detect emissions at two or more frequencies.  
     
     
         54 . The system of  claim 51 , wherein the detector can independently detect signals from two or more detectable markers with different signals.  
     
     
         55 . The system of  claim 51 , wherein at least one of the probes is complimentary to an amplicon sequence defined by one primer pair but not complimentary to an amplicon sequence defined by another primer pair.  
     
     
         56 . The system of  claim 51 , wherein at least one of the probes is complimentary to a first amplicon sequence and to a second amplicon sequence.  
     
     
         57 . The system of  claim 51 , wherein two or more probes each comprise different signals.  
     
     
         58 . The system of  claim 57 , wherein the different signals comprise different fluorescent emissions.  
     
     
         59 . The system of  claim 51 , wherein at least one of the probes comprise a fluorescent resonant energy transfer (FRET) detectable marker or a molecular beacon (MB) marker.  
     
     
         60 . The system of  claim 59 , wherein the FRET detectable marker comprises a quencher removable from the FRET probe by nuclease activity.  
     
     
         61 . The system of  claim 42 , wherein the system is a high throughput system.  
     
     
         62 . The system of  claim 42 , further comprising a dilution module.  
     
     
         63 . The system of  claim 62 , wherein the dilution module is configured to dilute the sample to a concentration which provides one or more single copy reaction mixtures for nucleic acids of interest in the amplification microchannel or microchamber.  
     
     
         64 . The system of  claim 62 , wherein the dilution module comprises serial multiwell plate dilutions or a dilution channel in a microfluidic device.  
     
     
         65 . The system of  claim 62 , further comprising system instructions that direct the dilution module to aliquot the sample into a plurality of aliquots, including a plurality of zero copy aliquots comprising no copies of the nucleic acid of interest, and one or more single copy aliquots comprising a single copy of the nucleic acid of interest.  
     
     
         66 . The system of  claim 42 , further comprising a computer in communication with the detector.  
     
     
         67 . The system of  claim 42 , wherein the microfluidic device further comprises multiple amplification channels.  
     
     
         68 . The system of  claim 42 , further comprising a sample storage module that stores the sample before preparation of the reaction mixture, or a sample retrieval module that retrieves the sample from the storage module before preparation of the reaction mixture.  
     
     
         69 . The system of  claim 42 , further comprising a capture oligonucleotide bound to a solid support to capture nucleotides of interest before or during preparation of the reaction mixture or to capture amplicons for detection.