Nucleic acid purification chemistry
The present invention generally relates to the field of nucleic acid isolation on silica solid support. In particular, a novel silica-solid support nucleic acid binding buffer chemistry is hereby disclosed, which is based on the use of a small quaternary organic compounds, e.g. tetramethylammonium chloride (TMAC), at acidic conditions. This novel nucleic acid purification chemistry purifies not only RNA but also DNA and has the potential for being implementable in a wide variety of commercial kits ranging from the spin columns to integrated Lab-On-A-Chip (LOC) devices such as disposable cartridges that make use of a solid-phase extraction technology. Furthermore, the present methods may be performed using relatively small volumes of binding buffer and consequently in such integrated or closed molecular diagnostic devices, they have the potential of allowing increased volumes of sample input, which for liquid biopsy samples such as plasma or urine, can enhance the chances of detecting rare nucleic acid targets.
1 . A double-stranded DNA (dsDNA) extraction method comprising contacting a liquid biopsy sample with a silica solid support at pH value between 3 and 6 and in the presence of a salt consisting of:
a small quaternary organic compound, defined as a quaternary compound consisting of a central positively charged atom with four organic substituents (R1, R2, R3, and R4), wherein the number of carbon atoms in each organic substituent (R1, R2, R3, and R4) does not exceed 2; and
a bromide or a chloride anion to extract dsDNA of 10 to 300 base pairs;
wherein the central positively charged atom of the small quaternary organic compound is nitrogen, and wherein the concentration of the small quaternary organic compound is 0.1 M-2 M.
2 . The method according to claim 1 , wherein the anion is chloride.
3 . The method according to claim 2 , wherein the small quaternary organic compound is tetramethylammonium chloride (TMAC).
4 . The method according to claim 1 , wherein the concentration of the small quaternary organic compound is 0.5 M-1.8 M.
5 . The method according to claim 1 , wherein the pH value is 4-5.8.
6 . The method according to claim 1 , wherein the contacting with the silica solid support is preceded by a protease treatment.
7 . The method according to claim 1 , wherein the liquid biopsy sample is selected from plasma, serum, whole blood, or urine.
8 . The method according to claim 1 , wherein the dsDNA is cell free dsDNA.
9 . The method according to claim 1 , wherein the dsDNA is circulating tumor dsDNA.
10 . The method according to claim 1 , wherein the contacting is further in the presence of a detergent.
11 . The method according claim 10 , wherein the detergent is a quaternary ammonium compound detergent.
12 . The method according claim 11 , wherein the quaternary ammonium compound detergent is cetyltrimethylammonium bromide (CTAB).
13 . The method according to claim 1 , wherein the method is performed using a cartridge.
14 . The method according to claim 1 , wherein the concentration of the small quaternary organic compound is about 1.2 M.
15 . The method according to claim 1 , wherein the pH value is 4.2-5.6.
16 . The method according to claim 1 , wherein the pH value is 4.4-5.4.
17 . The method according to claim 1 , wherein the pH value is about 5.