SYSTEM FOR DIFFERENTIATING THE LENGTHS OF NUCLEIC ACIDS OF INTEREST IN A SAMPLE

• US 20100129896A1
• Filed: 01/08/2010
• Published: 05/27/2010
• Est. Priority Date: 12/20/2002
• Status: Active Grant

• Current Assignee: Caliper Life Sciences Incorporated (Revvity, Inc.)
• Original Assignee: Caliper Life Sciences Incorporated (Revvity, Inc.)
• Inventors: KNAPP, MICHAEL R., Chow, Andrea W., Spaid, Michael, Kopf-Sill, Anne R., Baker, Jill M.
• Granted Patent: US 8,275,554 B2
• Time in Patent Office: Days
• US Class Current: 435/285.100
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2527/143 Concentration of primer or probe
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2547/107 Use of permeable barriers, e.g. waxes
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

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.

Claims

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 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 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.

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 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.

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 complimentary 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 complimentary to an amplicon sequence defined by one primer pair but not complimentary to an amplicon sequence defined by another primer pair.

15. The system of claim 10, wherein at least one of the probes is complimentary 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 dilution module.

22. The system of claim 21, 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.

23. The system of claim 21, wherein the dilution module comprises serial multiwell plate dilutions or a dilution channel in a microfluidic device.

24. The system of claim 21, 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.

25. The system of claim 1, further comprising a computer in communication with the detector.

26. The system of claim 1, wherein the microfluidic device further comprises multiple amplification channels.

27. The system of claim 1, 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.

28. The system of claim 1, 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.