Sample Preparation Device and Methods of Use

Abstract

A device for isolating DNA from a sample containing cells, including a cartridge having an entrance port and an exit port, a membrane disposed between the entrance port and the exit port, and a plurality of channels between the membrane and the exit port. Additionally, systems and methods for isolating DNA from a sample containing cells and also systems and methods for amplifying and isolating single-stranded DNA from a sample containing DNA.

What is claimed is: 
     
         1 . A device for isolating DNA from a sample containing cells, comprising
 a cartridge having an entrance port and an exit port;   a membrane disposed between the entrance port and the exit port; and   a plurality of channels between the membrane and the exit port.   
     
     
         2 . The device of  claim 1 , wherein the channels are adjacent to the membrane. 
     
     
         3 . The device of  claim 1 , wherein each of the channels has a channel entrance, a channel exit, and a channel wall, wherein the channel entrance is adjacent to the membrane. 
     
     
         4 . The device of  claim 3 , wherein the channel entrance has a length and width, wherein the length is about 0.1 mm to about 100 mm and the width is about 0.1 mm to about 10 mm. 
     
     
         5 . The device of  claim 1 , having from about 2 to about 1000 channels. 
     
     
         6 . The device of  claim 1 , wherein the channels are arranged in a matrix. 
     
     
         7 . The device of  claim 6 , wherein the channels are adjacent to each other. 
     
     
         8 . The device of  claim 6 , wherein the channels are spaced apart from each other. 
     
     
         9 . The device of  claim 8 , further comprising a solid barrier between each channel entrance. 
     
     
         10 . The device of  claim 1 , wherein the membrane is configured to be permeable to DNA but substantially impermeable to cells. 
     
     
         11 . The device of  claim 1 , wherein the membrane is configured to be permeable to DNA but substantially impermeable to non-DNA cellular material. 
     
     
         12 . The device of  claim 1 , wherein the membrane comprises a cellulose material. 
     
     
         13 . The device of  claim 1 , wherein the membrane has pores. 
     
     
         14 . The device of  claim 13 , wherein the pores are from about 10 nm to 1 micron in diameter. 
     
     
         15 . The device of  claim 1 , further comprising a plurality of particulate material located between the membrane and the entrance port. 
     
     
         16 . The device of  claim 15 , wherein the particulate material comprises glass or stainless steel. 
     
     
         17 . The device of  claim 15 , wherein the particulate material comprises a plurality of microbeads. 
     
     
         18 . The device of  claim 17 , wherein the microbeads have a diameter of from about 1 micron to about 1000 microns. 
     
     
         19 . The device of  claim 1 , wherein the entrance port and exit port comprise standard luer lock fittings. 
     
     
         20 . The device of  claim 1 , wherein the cartridge includes a bottom piece that supports the membrane and the plurality of channels are formed from cavities in the bottom piece of the cartridge. 
     
     
         21 . A system for isolating DNA from a sample containing cells comprising the device of  claim 1 , and a vacuum to pull fluid into the cartridge. 
     
     
         22 . The system of  claim 21 , further comprising a cellular homogenizer to shake the cartridge. 
     
     
         23 . The system of  claim 22 , wherein the cellular homogenizer is capable of shaking the cartridge at a rate of up to 5000 rpm. 
     
     
         24 . The system of  claim 22 , further comprising a thermal cycler configured to subject the DNA to thermal cycling for a polymerase chain reaction (PCR). 
     
     
         25 . The system of  claim 24 , further comprising a magnetic substrate wherein the magnetic substrate is a coating with a binding affinity for double-stranded DNA. 
     
     
         26 . The system of  claim 25 , further comprising a magnet configured to apply a magnetic field to the magnetic substrate. 
     
     
         27 . The system of  claim 25 , wherein the magnetic substrate is a plurality of magnetic beads. 
     
     
         28 . A method of processing DNA from a sample, comprising
 providing a sample containing cells having DNA;   transferring the sample into a cartridge having a membrane and a plurality of channels disposed therein;   lysing the cells to release the DNA and non-DNA cellular material;   passing the DNA through the membrane; and   passing the DNA through the channels;   wherein the DNA is isolated from the sample.   
     
     
         29 . The method of  claim 28 , wherein the sample comprises blood, exudate, water, or food. 
     
     
         30 . The method of  claim 28 , wherein the transferring comprises using a vacuum to transfer the sample into the cartridge. 
     
     
         31 . The method of  claim 28 , wherein the lysing comprises mechanical lysing. 
     
     
         32 . The method of  claim 31 , wherein the mechanical lysing comprises agitating a plurality of particulate material disposed within the cartridge. 
     
     
         33 . The method of  claim 32 , wherein the mechanical lysing substantially destroys the non-DNA cellular material. 
     
     
         34 . The method of  claim 28 , wherein greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the non-DNA cellular material in the sample does not pass through the membrane. 
     
     
         35 . The method of  claim 28 , wherein greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the DNA in the sample is passed through the membrane and the channels. 
     
     
         36 . The method of  claim 28 , further comprising transferring the isolated DNA to a DNA amplification chamber and amplifying the isolated DNA by a polymerase chain reaction (PCR), wherein the PCR is asymmetric, to produce double-stranded DNA and single-stranded DNA. 
     
     
         37 . The method of  claim 36 , wherein a portion of the single-stranded DNA is within a specified range of lengths. 
     
     
         38 . The method of  claim 36 , further comprising binding the double-stranded DNA to a magnetic substrate and applying a magnetic field to the substrate, wherein the magnetic field attracts the substrate bound to the double-stranded DNA, and isolating the single-stranded DNA. 
     
     
         39 . A method of amplifying and isolating single-stranded DNA from a sample containing DNA, comprising
 providing a sample containing DNA;   amplifying the DNA via a polymerase chain reaction (PCR), wherein the PCR is asymmetric PCR, to produce a mixture of double-stranded DNA and single-stranded DNA;   providing a magnetic substrate having an affinity for binding the double-stranded DNA;   binding the magnetic substrate to the double-stranded DNA;   applying a magnetic field to the magnetic substrate, wherein the magnetic field attracts the magnetic substrate bound to the double-stranded DNA; and   isolating the single-stranded DNA from the mixture.   
     
     
         40 . The method of  claim 39 , wherein PCR comprises traditional PCR followed by asymmetric PCR. 
     
     
         41 . The method of  claim 39 , wherein a portion of the single-stranded DNA is within a specified range of lengths. 
     
     
         42 . The method of  claim 41 , wherein at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the single-stranded DNA are within a specified range of lengths. 
     
     
         43 . The method of  claim 41 , wherein a portion of the single-stranded DNA is no more than about 200 bases in length. 
     
     
         44 . The method of  claim 43 , wherein a portion of the single-stranded DNA is no more than about 100 bases in length. 
     
     
         45 . The method of  claim 39 , further comprising an amplification container, placing the sample in the amplification container, and sealing the amplification container with a wax seal. 
     
     
         46 . The method of  claim 39 , wherein the magnetic substrate has a coating with an affinity for binding double-stranded DNA. 
     
     
         47 . The method of  claim 46 , wherein the coating that does not substantially attract single-stranded DNA. 
     
     
         48 . The method of  claim 39 , wherein the magnetic substrate is a paramagnetic substrate. 
     
     
         49 . The method of  claim 39 , wherein the magnetic substrate is a plurality of magnetic beads. 
     
     
         50 . The method of  claim 39 , wherein greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the double-stranded DNA in the mixture is bound to the magnetic substrate and, respectively, less than 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the double-stranded DNA in the mixture is not bound to the magnetic substrate. 
     
     
         51 . The method of  claim 39 , wherein greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the single-stranded DNA in the mixture is isolated. 
     
     
         52 . The method of  claim 39 , wherein the isolated unbound single-stranded DNA as compared to the unbound and bound double-stranded DNA comprises a ratio of greater than 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 99:1, 100:1 or 1000:1.