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 closed automated device which includes:
(A) a plurality of reaction reservoirs;
(B) a heating unit configured to heat the plurality of reaction reservoirs;
(C) a magnet configured to be elevated and lowered relative to the reaction reservoirs;
the method comprising:
a. contacting the nucleic acid-containing sample with an extraction reagent in a first reservoir of the plurality of the reaction chambers to thereby extract the nucleic acid from the sample;
b. binding the extracted nucleic acid with a first plurality of magnetic beads in the first reservoir;
c. while holding the magnetic beads down by elevating the magnet relative to the first plurality of magnetic beads, removing liquid supernatant formed in the first reservoir;
d. eluting the bound extracted nucleic acid from the first plurality of magnetic beads using a first elution buffer while subjecting the first reservoir to a first elevated temperature by the heating unit for a first predetermined duration of time to thereby obtain an isolated nucleic acid eluent;
e. while holding the magnetic beads down by elevating the magnet relative to the first plurality of magnetic beads, transferring the isolated nucleic acid eluent to a second reservoir of the plurality of reaction reservoirs, the second reaction reservoir containing a conversion reagent;
f. reacting said isolated nucleic acid with the conversion reagent in the second reservoir while subjecting the second reservoir to a second elevated temperature by the heating unit for a second predetermined duration of time, to thereby obtain a modified nucleic acid;
g. binding the modified nucleic acid with a second plurality of magnetic beads;
h. while holding the magnetic beads down by elevating the magnet relative to the second plurality of magnetic beads, removing liquid supernatant formed;
j. eluting the bound modified nucleic acid from the second plurality of magnetic beads using a second elution buffer or a second series of elution buffers to thereby obtain a purified modified nucleic acid eluent.
2. The method of claim 1 , wherein said conversion is methylation.
3. The method of claim 2 , wherein the methylation comprises bisulfite conversion.
4. The method of claim 3 , wherein the bisulfite conversion is a deamination of cytosine into uracil of said isolated nucleic acid.
5. The method of claim 3 , wherein the conversion reagent comprises bisulfite.
6. The method of claim 3 , further comprising a step of desulfonation.
7. 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.
8. The method of claim 1 , wherein the nucleic acid is DNA, RNA, cDNA or a combination thereof.