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Methods for extracting nucleic acids from tissue samples and paraffin-embedded tissues

Imported: 23 Feb '17 | Published: 22 Oct '02

Robert T. Belly, Gary J. Chilson

USPTO - Utility Patents

Abstract

The present invention provides rapid and highly effective methods for extracting nucleic acids suitable for PCR amplification from tissue samples and paraffin-embedded tissue samples. Extraction is accomplished within a few minutes using a composition comprising a buffer, at least one nonionic surfactant, and a protease enzyme. The sample is then heated at alkaline pH and after a centrifugation step, the DNA is the supernatant can be used directly in known amplification methods, such as PCR.

Claims

1. A method of extracting nucleic acids from tissue samples comprising contacting said sample with a buffer, at least one nonionic surfactant, and a protease enzyme under conditions sufficient to release said nucleic acids from said sample;

2. The method of claim 1 wherein the tissue is selected from fresh tissue and paraffin-embedded tissue.

3. The method of claim 1 wherein the nonionic surfactant is selected from the group consisting of polyoxyethylene sorbitan derivatives, polyoxyethylene ethers, polyglycol ethers, perfluoroalkyl polyoxyethylenes, fluorinated alkyl alkoxylates and fluorinated alkyl ester compounds.

4. The method of claim 3 wherein the nonionic surfactant is a polyoxyethylene sorbitan derivative.

5. The method of claim 4 wherein the polyoxyethylene sorbitan derivative is Tween-20.

6. The method of claim 1 wherein the protease enzyme is selected from the group consisting of serine proteases, thiol proteases, carboxy proteases, metalloproteases, Protease K, protease from Streptomyces griseus, and PRETAQ.

7. The method of claim 6 wherein the protease enzyme is a thermostable protease.

8. The method of claim 7 wherein the thermostable protease is PreTAQ.

9. The method of claim 1 wherein the heating step is done at about 70 degrees C to about 100 degrees C for 5 minutes to 3 hours.

10. The method of claim 9 wherein the heating is done at from about 90 degrees C to about 100 degrees C for about 10-15 minutes.

11. A method of extracting nucleic acids from a paraffin-embedded tissue sample comprising the steps of:

12. The method of claim 11 wherein the alkali is sodium hydroxide.

13. The method of claim 12 wherein the centrifugation is performed for about 2 minutes at about 14,000 rpm.

14. The method of claim 11 wherein the buffer of step (a) also contains a nonionic surfactant.

15. The method of claim 14 wherein the nonionic surfactant is Tween-20.