Sample preparation device and methods of use
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.
1. A method of amplifying and isolating single-stranded DNA from a sample containing DNA, comprising
providing an amplification container containing a magnetic substrate and a sample containing DNA; wherein the magnetic substrate has a coating comprising N-hydroxysuccinimide (NHS);
amplifying the DNA in the amplification container via a polymerase chain reaction (PCR), wherein the PCR comprises asymmetric PCR, to produce a mixture of double-stranded DNA and single-stranded DNA;
binding the magnetic substrate to the double-stranded DNA; wherein the magnetic substrate has an affinity for binding 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.
2. The method of claim 1 , wherein PCR comprises traditional PCR followed by asymmetric PCR.
3. The method of claim 1 , wherein a portion of the single-stranded DNA is within a specified range of lengths.
4. The method of claim 3 , wherein at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the single-stranded DNA are within a specified range of lengths.
5. The method of claim 3 , wherein a portion of the single-stranded DNA is no more than about 200 bases in length.
6. The method of claim 5 , wherein a portion of the single-stranded DNA is no more than about 100 bases in length.
7. The method of claim 1 , further comprising providing an amplification container, placing the sample in the amplification container, and sealing the amplification container with a wax seal.
8. The method of claim 1 , wherein the magnetic substrate is a paramagnetic substrate.
9. The method of claim 1 , wherein the magnetic substrate is a plurality of magnetic beads.
10. The method of claim 9 , wherein the plurality of magnetic beads are silica-based beads.
11. The method of claim 1 , 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.
12. The method of claim 1 , wherein greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the single-stranded DNA in the mixture is isolated.
13. The method of claim 1 , 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.
14. The method of claim 1 , comprising binding the magnetic substrate to the double-stranded DNA during asymmetric PCR.
15. A method of amplifying and isolating single-stranded DNA from a sample containing DNA, comprising
providing an amplification container containing a magnetic substrate and a sample containing DNA; wherein the magnetic substrate has a coating comprising a carboxylic acid linker;
amplifying the DNA in the amplification container via a polymerase chain reaction (PCR), wherein the PCR comprises asymmetric PCR, to produce a mixture of double-stranded DNA and single-stranded DNA;
binding the magnetic substrate to the double-stranded DNA; wherein the magnetic substrate has an affinity for binding 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.
16. A method of amplifying and isolating single-stranded DNA from a sample containing DNA, comprising
providing an amplification container containing a plurality of silica-based magnetic beads and a sample containing DNA, wherein the magnetic beads have a coating comprising N-hydroxysuccinimide (NETS);
amplifying the DNA in the amplification container via a polymerase chain reaction (PCR), wherein the PCR comprises asymmetric PCR, to produce a mixture of double-stranded DNA and single-stranded DNA;
binding the magnetic beads to the double-stranded DNA during asymmetric PCR;
applying a magnetic field to the magnetic beads, wherein the magnetic field attracts the magnetic beads bound to the double-stranded DNA; and
isolating the single-stranded DNA from the mixture.
17. The method of claim 16 , wherein PCR comprises traditional PCR followed by asymmetric PCR.