Devices and methods for monitoring genomic DNA of organisms

• US 20060257893A1
• Filed: 02/17/2006
• Published: 11/16/2006
• Est. Priority Date: 02/18/2005
• Status: Active Grant

• Current Assignee: Canon USA, Inc. (Canon Inc.)
• Original Assignee: Canon USA, Inc. (Canon Inc.)
• Inventors: Takahashi, Toru, Inoue, Hiroshi
• Granted Patent: US 7,604,938 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• 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 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0645 Electrodes
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/087 Multiple sequential chambers
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502738 characterised by integrated valves microvalves F16K99/0001
• CPC Class Codes: B01L 3/502784 specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• CPC Class Codes: B01L 7/525 with physical movement of samples between temperature zones
• CPC Class Codes: B01L 7/54 using spatial temperature gradients
• CPC Class Codes: C12Q 1/6839 Triple helix formation or other higher order conformations in hybridisation assays
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2535/139 : Random amplification polymorphism detection [RAPD]
• CPC Class Codes: C12Q 2563/173 : staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: C12Q 2565/629 : being a microfluidic device
• CPC Class Codes: G01N 1/34 : Purifying; Cleaning processes or apparatus for extracting or separating nucleic acids from biological samples C12N15/1003
• CPC Class Codes: G01N 2001/4088 : filtration
• CPC Class Codes: G01N 2035/00237 : Handling microquantities of analyte, e.g. microvalves, capillary networks
• CPC Class Codes: G01N 35/08 : using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis

Abstract

The invention provides an apparatus that can be used in methods of preparing, amplifying, detecting, and/or optionally selecting for further analysis the genomic material from an organism for the rapid detection and/or classification of an organism in a sample (e.g., screening for, identifying, quantifying, and/or optionally further analyzing, e.g., sequencing, the genomic material of the organism). The invention further provides methods of using the apparatus, e.g., in combination with novel SGP primers for improved use in waveform-profiling methods of DNA amplification. It is an object of the invention to provide an apparatus for fully automated analysis of genomic material, and multiple methods of using the apparatus that are beneficial to society, e.g., the apparatus may be used in methods of screening for, identifying, quantifying, and/or selecting genomic material for further analysis (e.g., sequencing) in relation to monitoring a source for the presence of contaminating organisms.

Claims

1. A microfluidic device, comprising:

at least one sipper;

at least one fluid reservoir connected to at least one microfluidic inline reaction channel, wherein the at least one microfluidic inline reaction channel runs through a reagent assembly area, an amplification area within a first temperature-controlled area, and a detection area within a second temperature-controlled area; and

at least one metal trace for heating of and/or fluid movement within the microfluidic inline reaction channel,

wherein detection of amplified DNA products occurs at one or more temperatures.

2. The microfluidic device of claim 1, wherein the at least one microfluidic inline reaction channel further comprises a valve downstream of the detection area.

4. An instrument that controls fluid movement within, heating and cooling of, and data acquisition from, the microfluidic device of claim 1, comprising a cartridge that interfaces between the instrument and the microfluidic device.

3. A microfluidic device, comprising:

at least one sipper;

at least one fluid reservoir connected to at least one microfluidic inline reaction channel, wherein the at least one microfluidic inline reaction channel runs through a reagent assembly area, an amplification area, and a detection area, and wherein the microfluidic inline reaction channel further comprises a valve downstream of the detection area; and

at least one metal trace for heating of and/or fluid movement within the microfluidic inline reaction channel.

5. An instrument that controls fluid movement within, heating and cooling of, and data acquisition from, the microfluidic device of claim 3, comprising a cartridge that interfaces between the instrument and the microfluidic device.

6. An apparatus comprising the microfluidic device of claim 3 and an instrument that controls fluid movement within, heating and cooling of, and data acquisition from, the microfluidic device.

7. The apparatus of claim 6, further comprising a cartridge that interfaces between the instrument and the microfluidic device.

13. A method of detecting the absence or presence of an organism in a sample, comprising the steps of:

(a) acquiring the sample;

(b) isolating at least one copy of the genomic material of the organism, if present in the sample;

(c) aspirating at least one sample droplet into a microfluidic reaction channel;

(d) forming at least one sample plug by mixing the at least one sample droplet with a primer plug, wherein the primer plug comprises amplification reagents that comprise at least one primer, nucleotides, DNA polymerase, and intercalators;

(e) heating the at least one sample plug to a first temperature that will cause each copy of the genomic DNA, if present, to denature into a first and second genomic DNA template;

(f) cooling the at least one sample plug to a second temperature that will cause the primers to anneal to each genomic DNA template;

(g) reheating the at least one sample plug to a third temperature that is between the first and second temperatures as to allow (1) the primers to remain annealed to the genomic DNA and (2) the DNA polymerase to elongate nucleic acid polymers originating from the annealed primers;

(h) maintaining the third temperature for a first length of time;

(i) repeating steps (e)-(h) at least once; and

(j) detecting the resulting amplified products,

wherein at least steps (c)-(j) occur within the apparatus of claim 6.

14. The method of claim 13, wherein the primer plug comprises a PCR primer pair.

15. The method of claim 14, wherein the detecting step of step (j) occurs at one temperature.

16. The method of claim 13, wherein the primer plug comprises a waveform profiling primer.

17. The method of claim 16, wherein the detecting step of step (j) occurs at a range of temperatures.

18. The method of claim 17, further comprising after step (i) and before step (j), the steps of:

(1) repeating steps (e)-(g);

(2) maintaining the third temperature for a length of time equal to about 40-60% of the first length of time; and

(3) cooling the at least one sample plug to a fourth temperature lower than or equal to that of the second temperature to allow formation of higher-order structures containing intercalators.

19. The method as in any one of claims 13-18, further comprising a last step of selecting a sample plug for further analysis, wherein the step of selecting occurs at the valve within the apparatus.

20. A method of screening a sample supply for contamination, comprising the steps of:

(a) continuously acquiring sample droplets from the sample supply into at least one microfluidic reaction channel;

(b) forming sample plugs by mixing each sample droplet with a primer plug, wherein the primer plug comprises amplification reagents;

(c) amplifying DNA in sample plugs comprising genomic material; and

(d) detecting the absence or presence of amplified products,

wherein steps (a)-(d) occur within the apparatus of claim 6.

21. A method of identifying an organism, the method comprising the steps of:

(a) preparing at least one DNA sample droplet comprising a DNA molecule isolated from the organism;

(b) acquiring the at least one DNA sample droplet into at least one microfluidic inline reaction channel;

(c) forming at least one DNA sample plug by mixing at least one DNA sample droplet with a primer plug, wherein the primer plug comprises amplification reagents that comprise at least one known first primer;

(d) subjecting the at least one DNA sample plug to at least one amplification cycle such that the at least one DNA sample plug has detectable amplified DNA products;

(e) detecting amplified DNA products;

(f) identifying the organism based on detection of the amplified products; and

(g) optionally repeating steps (a)-(f) with amplification reagents comprising a known primer that is different from the first known primer to increase the accuracy of the identification of the organism, wherein at least steps (b)-(e) occur within the apparatus of claim 6.

22. A method for quantifying the concentration of genomic material in a sample, the method comprising the steps of:

(a) diluting the sample using dilution factors such that the concentration of the genomic material is at most approximately one molecule per sample droplet;

(b) acquiring sample droplets into at least one microfluidic inline reaction channel;

(c) forming at least more than one sample plug by mixing each sample droplet with a primer plug, wherein the primer plug comprises amplification reagents;

(d) subjecting each sample plug to amplification cycles such that each sample plug comprising at least one DNA molecule has detectable amplified DNA products, and each sample plug not comprising a DNA molecule will have zero-detection of amplified DNA products;

(e) detecting the absence or presence of amplified DNA products in each sample plug;

(f) determining the ratio of sample plugs containing detectable amplified DNA products to sample plugs resulting in zero-detection; and

(g) using the dilution factor to calculate the original concentration of genomic material in the sample,

wherein at least steps (b)-(e) occur within the apparatus of claim 6.

23. The method as in any one of claims 20-22, further comprising a last step of selecting a sample plug for further analysis, wherein the step of selecting occurs at the valve within the apparatus.

24. The method as in any one of claims 13-18 and 20-22, further comprising a step wherein the sample plug(s) is surrounded by an immiscible nonaqueous fluid.

8. A method of determining an organism in a sample, the method comprising the steps of:

(a) acquiring the sample;

(b) isolating at least one copy of the genomic DNA of the organism, if present in the sample;

(c) introducing a first mixture comprising SGP primers, nucleotides, DNA polymerase, and intercalators to the genomic DNA of the organism to form a second mixture;

(d) heating the second mixture to a first temperature that will cause the genomic DNA, if present, to denature into a first and second genomic DNA template;

(e) cooling the second mixture to a second temperature that will cause the primers to anneal to each genomic DNA template;

(f) reheating the second mixture to a third temperature that is between the first and second temperatures to allow the primers to remain annealed to the genomic DNA and the DNA polymerase to elongate nucleic acid polymers originating from the annealed primers;

(g) maintaining the third temperature for a first length of time;

(h) repeating steps (d)-(g) at least once;

(i) repeating steps (d)-(f);

(j) maintaining the third temperature for a second length of time equal to about 40-60% of the first length of time;

(k) recooling the second mixture to a fourth temperature lower than or equal to that of the second temperature to allow formation of higher-order structures containing intercalators;

(l) detecting the resulting higher-order structures;

(m) performing melting temperature analysis;

(n) detecting a waveform profile; and

(o) determining a positive waveform profile from the sample if the sample contained the organism.

9. The method of claim 8, wherein the third temperature is maintained for a second length of time about 50% of the first length of time.

10. The method of claim 8, wherein the number of times steps (d)-(g) are repeated in step (h) is 20-50 times.

11. The method of claim 10, wherein the number of times steps (d)-(g) are repeated in step (h) is 22-24 times.

12. The method of claim 8, wherein the method further comprises repeating steps (i)-(j) one or more times prior to step (k).