Methods for generating short tandem repeat (STR) profiles

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.