System for differentiating the lengths of nucleic acids of interest in a sample
Systems for differentiating the lengths of nucleic acids of interest in a sample are provided. The system includes a microfluidic device, a detector, and a software system. The microfluidic device includes an amplification microchannel or microchamber containing a reaction mixture under conditions that provide one or more amplicons of the nucleic acid of interest. The detector is integral with or proximal to the microfluidic device and is configured to detect the amplicons as one or more signals from a homogenous mixture. The software system interprets one or more coincidentally 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.
1. 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 acids of interest in the sample;
a detector integral with or proximal to the microfluidic device, which detector is configured to detect the one or more amplicons as one or more signals from a homogenous mixture of reaction mixture constituents flowing through the microfluidic device; and
a software system that interprets two or more coincidentally detected signals from the homogenous mixture to indicate lengths of one or more individual nucleic acid molecules of interest, thereby differentiating the lengths of the nucleic acids of interest.
2. The system of claim 1 , 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.
3. The system of claim 1 , wherein the reaction mixture comprises: the nucleic acid of interest, a first primer pair, a second primer pair comprising at least one primer complementary 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, wherein the sequence or amplicon is defined by a primer pair when only the sequence disposed between the two primers of the primer pair is amplified by the polymerase.
4. The system of claim 3 , 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.
5. The system of claim 3 , 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.
6. The system of claim 1 , 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.
7. The system of claim 1 , 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.
8. The system of claim 1 , wherein the amplicons are detected without resolution in a size selective media or affinity media.
9. The system of claim 1 , 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.
10. The system of claim 1 , further comprising one or more nucleic acid probes comprising one or more detectable markers and a sequence complementary to one or more of the amplicons, wherein the detectable markers provide a signal detectable by the detector.
11. The system of claim 10 , 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.
12. The system of claim 11 , wherein the fluorometer can simultaneously detect emissions at two or more frequencies.
13. The system of claim 10 , wherein the detector can independently detect signals from two or more detectable markers with different signals.
14. The system of claim 10 , wherein at least one of the probes is complementary to an amplicon sequence defined by one primer pair but not complementary to an amplicon sequence defined by another primer pair.
15. The system of claim 10 , wherein at least one of the probes is complementary to a first amplicon sequence and to a second amplicon sequence.
16. The system of claim 10 , wherein two or more probes each comprise different signals.
17. The system of claim 16 , wherein the different signals comprise different fluorescent emissions.
18. The system of claim 10 , wherein at least one of the probes comprise a fluorescent resonant energy transfer (FRET) detectable marker or a molecular beacon (MB) marker.
19. The system of claim 18 , wherein the FRET detectable marker comprises a quencher removable from the FRET probe by nuclease activity.
20. The system of claim 1 , wherein the system is a high throughput system.
21. The system of claim 1 , further comprising a computer in communication with the detector.
22. The system of claim 1 , wherein the microfluidic device further comprises multiple amplification channels.