AUTOMATED ISOLATION AND CHEMICAL REACTION(S) OF NUCLEIC ACIDS
The present teachings relate to methods, kits and devices for performing automated sequential nucleic acid isolation and conversion/purification in a single closed system. In various embodiments, the present teaching enable a user to (i) load a device with test samples, reagents and consumables; (ii) select or program the device for the desired nucleic acid isolation and subsequent chemical treatment and/or conversion reaction(s) without further user intervention; and recovering the isolated and treated and/or converted nucleic acid at the conclusion of the program once the device is activated.
1 . A method for performing sequential nucleic acid isolation and conversion/purification reaction(s) in a single closed system, wherein an isolated nucleic acid is prepared for modification detection, comprising:
a. reacting the nucleic acid-containing sample with an extraction reagent, wherein an isolated nucleic acid is obtained from the sample, and
b. reacting said isolated nucleic acid with a conversion reagent .wherein the said nucleic acid having the modification is prepared for further detection.
2 . The method of claim 1 , wherein said modification is methylation.
3 . The method of claim 2 , wherein the single closed system is built in an automated device.
4 . The method of claim 3 , wherein the single closed system can be a sterile environment.
5 . The method of claim 1 , wherein the conversion is a chemical conversion.
6 . The method of claim 5 , wherein the chemical conversion is bisulfite conversion with an optional desulfonation step, and wherein reagents have an over-layer of mineral oil.
7 . The method of claim 6 , wherein the bisulfite conversion is a deamination of cytosine into uracil of said isolated nucleic acid.
8 . The method of claim 1 , wherein the isolation and conversion/purification of the nucleic acid occurs in less than about four hours.
9 . The method of claim 1 , wherein the isolation and purification of said converted nucleic acid utilizes magnetic beads.
10 . The method of claim 1 , wherein the sample is selected from the group consisting of whole blood, plasma, serum, buffy coat, saliva, cheek swab, sputum, stool, urine, cerebral spinal fluid, a cell, a tissue and a formalin fixed paraffin embedded (FFPE) sample.
11 . The method of claim 1 , wherein the nucleic acid is DNA, RNA, cDNA or a combination thereof.
12 . The method of claim 1 , wherein the reacted nucleic acid is further suitable for PCR, qPCR, sequencing, restriction enzyme digestion, ligation, transfection, hybridization, genotyping, forensics testing, quality control, disease detection, prognosis, and molecular diagnosis.
13 . A method for performing sequential nucleic acid isolation and bisulfite conversion reaction in a single closed automated system wherein an isolated nucleic acid is prepared for modification detection, comprising:
a. introducing a nucleic acid-containing sample into a reaction tube, wherein said reaction tube is within a device for performing sequential nucleic acid isolation and conversion/purification in a single closed system;
b. reacting the nucleic acid-containing sample with an extraction reagent;
c. binding the extracted nucleic acid to magnetic beads subsequently added after the extraction reaction, wherein an isolated nucleic acid is obtained from the sample;
d. reacting said isolated nucleic acid with a bisulfite conversion reagent followed by an optional desulfonation reagent, and
e. binding the bisulfite-converted nucleic acid to magnetic beads subsequently added after the conversion reaction, wherein said nucleic acid is obtained for further modification detection.
14 . A kit according to the method of claim 1 comprising:
a. an automated platform,
b. an extraction reagent that isolates nucleic acid from the nucleic acid-containing sample in a sealed cartridge,
c. a bisulfite reagent with optional desulfonation reagent in a sealed cartridge;
d. an optional desulfonation treatment; and
e. a plurality of magnetic particles.
15 . A device for performing sequential nucleic acid isolation and conversion/purification in a single closed device comprising:
a. at least one sealed cartridge containing a plurality of reagents wherein the reagents are purification reagents and conversion reagents,
b. a heating unit capable of contacting a plurality of reaction tubes, and
c. a magnet configured to the reaction tubes.
16 . The device of claim 15 , wherein said single closed device is an automated system.
17 . The device of claim 15 , wherein the single closed device is sealed from the external environment.
18 . The device of claim 15 , wherein the conversion reagent is a bisulfite conversion reagent.
19 . The device of claim 18 , wherein the bisulfite conversion reagent further comprises an optional desulfonation reagent.
20 . The device of claim 18 , wherein the bisulfite conversion is deamination of cytosine into uracil, wherein 5-methylcytosine is not converted.