Nucleic Acid Sample Preparation by Exclusion of DNA
Devices and methods are provided for separation of nucleic acids from waste materials in a biological sample, and then reacting the separated nucleic acids. In some embodiments, the methods comprise mixing a cell lysate having nucleic acids and waste materials with a waste-binding matrix to capture the cellular waste materials from the lysate. The waste-binding matrix can comprise hydrophilic and/or hydrophobic size-exclusion, ion-exchange particles. In some embodiments, the device comprises a substrate comprising a fluid processing pathway in which nucleic acid sample preparation occurs prior to a downstream genotyping reaction.
1 . A device comprising:
a substrate; and
a fluid processing pathway disposed in or on the substrate having a length and comprising
a first end and a second end,
an inlet disposed at the first end,
an outlet disposed at the second end,
a reaction chamber disposed along the pathway between the first end and the second end and comprising a pair of forward and reverse polymerase chain reaction primers for together replicating a target nucleic acid sequence,
a nucleic acid purification chamber disposed along the pathway between the first end and the reaction chamber, and
a polymerase chain reaction product purification chamber disposed along the fluid processing pathway between the reaction chamber and the second end,
wherein the nucleic acid purification chamber comprises a nucleic acid purification material disposed therein and the polymerase chain reaction product purification chamber comprises purification material disposed therein.
2 . The device of claim 1 further comprising a lysing agent disposed in the fluid processing pathway between the first end and the nucleic acid purification chamber.
3 . The device of claim 1 , wherein the reaction chamber further comprises a magnesium catalyst pre-loaded therein.
4 . The device of claim 1 , wherein the reaction chamber further comprises a polymerase enzyme pre-loaded therein.
5 . The device of claim 1 , wherein the purification material disposed in the nucleic acid purification chamber comprises hydrophilic purification beads and hydrophobic purification beads.
6 . The device of claim 1 , wherein the purification material disposed in the nucleic acid purification chamber comprises hydrophilic purification beads and hydrophobic purification beads in a ratio of from about one to five to about five to one, respectively, by volume.
7 . The device of claim 1 , further comprising a second nucleic acid purification chamber disposed along the fluid processing pathway between the nucleic acid purification chamber and the reaction chamber.
8 . The device of claim 7 , further comprising a hydrophilic purification material disposed in the nucleic acid purification chamber and a hydrophobic purification material disposed in the second nucleic acid purification chamber.
9 . The device of claim 7 , further comprising a hydrophobic purification material disposed in the nucleic acid purification chamber and a hydrophilic purification material disposed in the second nucleic acid purification chamber.
10 . A method for isolating nucleic acids from cellular waste materials comprising:
directing a biological sample comprising one or more cells into an inlet of a fluid processing pathway;
lysing the one or more cells in the biological sample to form a lysate comprising nucleic acids and cellular waste materials;
contacting the lysate with a nucleic acid purification material that is disposed within a nucleic acid purification chamber;
capturing the cellular waste materials with the nucleic acid purification material, to form purified nucleic acids; and
directing the purified nucleic acids from the nucleic acid purification chamber into a reaction chamber pathway pre-loaded with reaction reagents.
11 . The method of claim 10 , wherein the nucleic acid purification material comprises a hydrophilic size-exclusion ion-exchange material.
12 . The method of claim 10 , further comprising contacting the purified nucleic acids with hydrophobic size-exclusion particles.
13 . The method of claim 10 , further comprising combining a protease with the lysate.
14 . The method of claim 10 , wherein the pre-loaded reaction reagents comprise polymerase chain reaction reagents.
15 . The method of claim 10 , wherein the pre-loaded reaction reagents comprise a pair of forward and reverse polymerase chain reaction primers for together replicating a target nucleic acid sequence.
16 . The method of claim 10 , further comprising pre-loading the reaction chamber with a polymerase enzyme.
17 . The method of claim 10 , wherein the directing the lysate comprises spinning the substrate.
18 . The method of claim 10 , wherein the directing the purified nucleic acids comprises spinning the substrate.
19 . The method of claim 10 , wherein the directing the purified nucleic acids comprises opening a valve along the fluid processing pathway adjacent the nucleic acid purification chamber.
20 . The method of claim 10 , wherein the reaction chamber is disposed along the fluid processing pathway.