Methods for generating short tandem repeat (STR) profiles

• US 8,748,165 B2
• Filed: 08/21/2012
• Issued: 06/10/2014
• Est. Priority Date: 01/22/2008
• Status: Active Grant

• Current Assignee: IntegenX Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: IntegenX Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Vangbo, Mattias, Nielsen, William D., Blaga, Iuliu I., Nguyen, Michael Van, Jovanovich, Steven B.
• Primary Examiner(s): Marcheschi, Michael
• Assistant Examiner(s): Doe, Shanta G
• Application Number:
  US13/590,965
• Publication Number:
  US 20130053255A1
• Time in Patent Office: 658 Days
• Field of Search: 204/453, 204/604, 435/6.12, 435/6.16, 435/91.2, 435/287.1, 435/287.2, 435/287.3, 435/288.7, 435/288.5, 436/63, 436/94
• US Class Current: 435/287.2
• CPC Class Codes: B01D 17/06 Separation of liquids from each other by electricity
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/04 Exchange or ejection of cartridges, containers or reservoirs
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• 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 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/087 Multiple sequential chambers
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2400/043 magnetic forces
• CPC Class Codes: B01L 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 3/502738 characterised by integrated valves microvalves F16K99/0001
• 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: G01N 1/34 Purifying; Cleaning processes or apparatus for extracting or separating nucleic acids from biological samples C12N15/1003
• CPC Class Codes: G01N 1/40 Concentrating samples
• CPC Class Codes: G01N 2035/00158 Elements containing microarrays, i.e. "biochip"
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: G01N 35/1095 for supplying the samples to flow-through analysers for a specific analyser see relevant groups, e.g. under G01N15/00, G01N21/00, G01N27/00, G01N30/00, H01J49/00
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]

Abstract

This invention provides a method for generating short tandem repeat (STR) profiles on each of a plurality of samples comprising, for each sample: a) isolating DNA from the sample; b) amplifying STR markers in the isolated DNA and c) analyzing the amplification product by electrophoresis.

Claims

1. A method for generating short tandem repeat (STR) profiles on each of a plurality of samples comprising, for each sample:

a) isolating DNA from the sample by;

delivering a lysis buffer into a lysis chamber of a cartridge, wherein the lysis chamber contains a swab or swipe containing human cells from the sample, to produce a lysate, wherein the cartridge is configured as a disposable single-use device;

transporting the lysate from the cartridge through a microfluidic channel in a microfluidic microchip to which the cartridge is mated and into a DNA isolation chamber comprising paramagnetic beads in the cartridge, wherein the microfluidic channel

comprises at least one valve that controls movement of a fluid through the channel;

binding the DNA onto the beads;

applying a magnetic field to a side of the DNA isolation chamber to capture the paramagnetic beads; and

washing the beads, to produce purified DNA bound to the beads;

b) amplifying STR markers by;

moving the purified DNA bound to the beads through a microfluidic channel in the microfluidic microchip to a reaction chamber of a thermocycler wherein the reaction chamber is in thermal contact with a temperature modulator, and wherein the reaction chamber is off-chip;

capturing purified DNA bound to the beads in the reaction chamber of the thermocycler by applying a magnetic field;

moving reagents for STR amplification to the reaction chamber of the thermocycler;

performing PCR in the reaction chamber of the thermocycler to amplify STRs to produce amplification product;

and

c) analyzing the amplification product by;

moving the amplification product to a loading channel, wherein the loading channel intersects a gel-filled separation channel, and wherein a cathode and an anode are configured to apply a voltage across the loading channel and the separation channel;

injecting amplification product from the loading channel into the separation channel by applying a voltage across the cathode and the anode;

performing electrophoresis on the amplification product in the separation channel to separate analytes in the amplification product; and

generating an STR profile of the sample from the separation;

wherein all the method is performed on each sample in parallel on an integrated system using software that automates the process.

2. The method of claim 1, wherein separation obtains single base pair resolution out to approximately 330 base pairs in less than 20 minutes.

3. The method of claim 1, wherein isolating DNA from the sample is performed in less than 5 minutes.

4. The method of claim 1, wherein the method is performed on at least 4 samples.

5. The method of claim 1, wherein the magnetic field is applied to the side of the DNA isolation chamber using a movable magnet.

6. The method of claim 1, wherein the cartridge and the microfluidic microchip are clamped together.

7. The method of claim 1, wherein the temperature modulator is a Peltier device.

8. The method of claim 1, wherein the thermocycler further comprises a heat distributing element between the reaction chamber and the temperature modulator.

9. The method of claim 1, wherein the at least one valve is a diaphragm valve that comprises an elastomeric layer and a seat, wherein the valve obstructs flow through the channel when the elastomeric layer is in contact with the seat.

10. The method of claim 9, wherein the at least one diaphragm valve comprises an elastomeric layer that is normally not in contact with the seat.

11. The method of claim 1, wherein the microfluidic channel comprises at least three valves that control movement of a fluid through the channel.

12. The method of claim 1, wherein delivering the lysis buffer into a lysis chamber of the cartridge comprises using an external pressure source.

13. The method of claim 1, wherein moving reagents for STR amplification to the thermocycler comprises using an external pressure source.

14. The method of claim 1, wherein performing PCR in the thermocycler produces a labeled amplification product.

15. The method of claim 14, wherein the labeled amplification product is fluorescently labeled.

16. The method of claim 1, wherein moving the amplification product to the loading channel comprises eluting the amplification product from the beads.

17. The method of claim 1, wherein injecting amplification product from the loading channel into the separation channel comprises field amplified sample stacking.

18. The method of claim 1, wherein injecting amplification product from the loading channel into the separation channel comprises applying a voltage of about 25 to 500 V/cm.

19. The method of claim 1, wherein the microfluidic microchip comprises a fluidics layer, an actuation layer, and a pneumatics layer, and wherein the fluidics layer is adjacent to the cartridge.

20. The method of claim 1, wherein the separation channel comprises a capillary.

21. The method of claim 20, wherein the capillary has an outer diameter of about 150-500 microns and an inner diameter of about 10-100 microns.

22. The method of claim 1, wherein generating the STR profile comprises detecting the analytes in the separation channel, wherein detecting the analytes is performed using a light source and a photodetector.

23. The method of claim 22, wherein the photodetector comprises a CCD.

24. The method of claim 22, wherein the photodetector comprises a CMOS.

25. The method of claim 22, wherein the photodetector comprises a photomultiplier tube (PMT).

26. The method of claim 22, wherein the light source comprises a coherent light source.

27. The method of claim 26, wherein the coherent light source comprises a laser.