Enhanced amplifiability of minute fixative-treated tissue samples, minute stained cytology samples, and other minute sources of DNA
The specification provides materials and methods for enhancing the amplifiability of nucleic acids that are present in minute amounts. Preferably, the materials and methods are utilized in combination with pathology techniques to amplify nucleic acids obtained from fixative and/or stained tissue or cell samples. The amplified nucleic acids can then be utilized for rendering a diagnosis or prognosis to the subject from which the tissue or cellular sample was obtained.
1 . A kit for amplification of a nucleic acid from a sample with low nucleic acid concentration comprising: (a) a buffer comprising a nonionic detergent; (b) a proteinase; (c) XCl 2 , wherein X is magnesium or manganese; and (d) sucrose; and wherein XCl 2 and sucrose when admixed with the sample for amplification of DNA have a final concentration of about 5 mM to about 10 mM XCl 2 and about 5 gram percent to about 15 gram percent sucrose.
2 . The kit of claim 1 , wherein the sample is fixative-treated, fixative-treated and stained, a liquid cytology, or a liquid biological sample.
3 . The kit of claim 1 , wherein the nucleic acid is DNA.
4 . The kit of claim 1 , wherein the nonionic detergent is Nonidet P40 (NP-40), Tween, Triton X, or Nikkol.
5 . The kit of claim 4 , wherein the nonionic detergent is present in the amount of about 0.1% to about 3.0%.
6 . The kit of claim 5 , wherein the nonionic detergent is Nonidet P40.
7 . The kit of claim 4 , wherein the nonionic detergent is Nonidet P40 and is present in an amount of about 1.0%.
8 . The kit of claim 1 , wherein the proteinase is proteinase K, pronase, subtilisin, thermolysin, papain, or a combination thereof.
9 . The kit of claim 8 , wherein the proteinase or proteinase combination is present in the amount of about 0.1% to about 3.0%.
10 . The kit of claim 9 , wherein the proteinase is proteinase K.
11 . The kit of claim 8 , wherein the proteinase is proteinase K and is present in an amount of about 1 mg/mL to about 5 mg/mL.
12 . The kit of claim 11 , wherein the proteinase K is present in the amount of about 2 mg/mL.
13 . The kit of claim 1 , wherein XCl 2 is MgCl 2 .
14 . The kit of claim 1 , wherein XCl 2 is MgCl 2 and is present in the amount of about 6.0 mM to about 10.0 mM.
15 . The kit of claim 14 , wherein the MgCl 2 is present in the amount of about 8.0 mM.
16 . The kit of claim 1 , wherein the sucrose is present in the amount of about 12 gram percent.
17 . A method for amplification of DNA of a sample with low DNA concentration comprising:
(a) combining a sample of cellular containing material and an aqueous buffer comprising a nonionic detergent;
(b) adding a proteinase to the buffer and the sample and incubating the buffer, the sample, and proteinase at a temperature suitable for proteinase digestion of protein;
(c) deactivating the proteinase;
(d) adding sucrose to the sample being amplified to a final concentration of about five gram percent to about 15 gram percent; and
(e) adding XCl 2 to the sample being amplified to a final concentration of about 6.0 mM to about 10.0 mM.
18 . The method of claim 17 , wherein about a 1 μL to about a 5 μL sample is amplified.
19 . The method of claim 17 , wherein the buffer further comprises sodium chloride, EDTA, and Tris.
20 . The method of claim 17 , wherein the sample is fixative-treated, stained, fixative-treated and stained, a liquid cytology, or a biological liquid specimen.
21 . The method of claim 17 , wherein the nonionic detergent is Nonidet P40 (NP-40), Tween, Triton X, or Nikkol.
22 . The method of claim 21 , wherein the nonionic detergent is NP-40 and is present in the amount of about 1.0%.
23 . The method of claim 17 , wherein the proteinase is proteinase K, pronase, subtilisin, thermolysin, papain, or a combination thereof.
24 . The method of claim 23 , wherein the proteinase is proteinase K and is present in an amount of about 1.0 mg/mL to about 5.0 mg/mL.
25 . The method of claim 24 , wherein the proteinase K is present in the amount of about 2.0 mg/mL.
26 . The method of claim 17 , wherein said XCl 2 is MgCl 2 or MnCl 2 .
27 . The method of claim 26 , wherein the XCl 2 is MgCl 2 and is present in the amount of about 5.0 mM to about 10.0 mM.
28 . The method of claim 17 , wherein sucrose is present in the amount of about 12.0 gram percent.
29 . The method of claim 17 , wherein the proteinase is proteinase K and is present in the amount of about 2 mg/mL; the sucrose is present in the amount of about 12.0 gram percent; the nonionic detergent is nonidet P-40, which is present in the amount of about 1%; and XCl 2 is MgCl 2 and is present in the amount of about 8.0 mM.