ONE-STEP METHOD OF ELUTION OF DNA FROM BLOOD SAMPLES

• US 20120156683A1
• Filed: 12/16/2011
• Published: 06/21/2012
• Est. Priority Date: 12/17/2010
• Status: Active Grant

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Baker, Mei Wang
• Granted Patent: US 9,206,468 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence

Abstract

Described are reagents, methods, and kits for eluting, and amplifying and/or characterizing DNA from liquid and dried blood samples. A one-step DNA elution buffer has been developed that simplifies purification of DNA from blood samples. The purified DNA is suitable for use in subsequent widely used techniques such as enzymatic DNA amplification and quantitative analysis such as real-time PCR.

Claims

1. A one step method of eluting DNA from a blood sample, consisting of

adding a one-step elution buffer having a pH of about 9 to about 13 to the blood sample to form a mixture,

heating the mixture at 90°

C. to 99°

C. for a time sufficient to elute the DNA from the blood sample to form an eluted DNA solution, and

optionally cooling the eluted DNA solution at a temperature of at least about 4°

C. for at least 5 minutes,

wherein the eluted DNA solution is suitable for direct use in an enzymatic DNA amplification reaction.

2. The method of claim 1, wherein the one-step elution buffer consists of 5 to 22.5 mM potassium in the form of KOH, KCl, or a combination thereof, and 7.5 to 30 mM of a base having a buffering range of 7.0 to 9.5, wherein the pH of the one-step elution buffer is about 9 to about 13.

3. The method of claim 2, wherein the base having a buffering range of 7.0 to 9.5 is selected from the group consisting of Tris base, DIPSO (3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxypropanesulfonic Acid Sodium Salt), MOBS (3-(N-morpholino)propanesulfonic acid), TAPSO (3-[N-Tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic Acid), HEPPSO (4-(2-Hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid), POPSO (piperazine-1,4-bis(2-hydroxypropanesulfonic acid) dihydrate), TEA (triethanolamine), EPPS (4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid), TRICINE (N-[Tris(hydroxymethyl)methyl]glycine, 3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonic acid), Glycylglycine, BICINE (N,N-Bis(2-hydroxyethyl)glycine), HEPBS (N-(2-Hydroxyethyl)piperazine-N′

(4-butanesulfonic acid), TAPS(N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic Acid), AMPD (2-Amino-2-methyl-1,3-propandiol), TABS (N-tris[hydroxymethyl]methyl-4-aminobutanesulfonic acid), AMPSO(N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxy-propanesulfonic acid), CHES (N-Cyclohexyl-2-aminoethanesulfonic acid), and combinations thereof.

4. The method of claim 1, wherein the one-step elution buffer consists of 2.5 to 10 mM KOH, 7.5 to 30 mM Tris base and 2.5 to 12.5 mM KCl, and wherein the pH is 11 to 12.

5. The method of claim 1, wherein the mixture is heated at 94°

C. to 99°

C.

6. The method of claim 1, wherein the blood sample is a dried blood sample on an adsorbent matrix.

7. A method of analyzing DNA from a blood sample, consisting essentially of

adding a one-step elution buffer having a pH of about 9 to about 13 to the blood sample to form a mixture,

heating the mixture at 90°

C. to 99°

C. for a time sufficient to elute the DNA from the blood sample to form an eluted DNA solution,

optionally cooling the eluted DNA solution at a temperature of at least about 4°

C. for at least 5 minutes, and

analyzing the eluted DNA solution without further processing after elution.

8. The method of claim 7, wherein analyzing the eluted DNA solution consists essentially of adding the eluted DNA solution directly to an enzymatic DNA amplification reaction.

9. The method of claim 8, wherein the enzymatic DNA amplification reaction is a quantitative PCR reaction or a multi-primer PCR reaction.

10. The method of claim 8, wherein the enzymatic DNA amplification reaction contains primers for the amplification of a marker for a genetic disorder.

11. The method of claim 10, wherein the genetic disorder is SCID and the primers are SEQ ID NO:

13 and SEQ ID NO;

14.

12. The method of claim 7, wherein the one-step elution buffer consists of 5 to 22.5 mM potassium in the form of KOH, KCl, or a combination thereof, and 7.5 to 30 mM of a base having a buffering range of 7.0 to 9.5, wherein the pH of the one-step elution buffer is about 9 to about 13.

13. The method of claim 12, wherein the base having a buffering range of 7.0 to 9.5 is selected from the group consisting of Tris base, DIPSO (3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxypropanesulfonic Acid Sodium Salt), MOBS (3-(N-morpholino)propanesulfonic acid), TAPSO (3-[N-Tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic Acid), HEPPSO (4-(2-Hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid), POPSO (piperazine-1,4-bis(2-hydroxypropanesulfonic acid) dihydrate), TEA (triethanolamine), EPPS (4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid), TRICINE (N-[Tris(hydroxymethyl)methyl]glycine, 3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonic acid), Glycylglycine, BICINE (N,N-Bis(2-hydroxyethyl)glycine), HEPBS (N-(2-Hydroxyethyl)piperazine-N′

(4-butanesulfonic acid), TAPS(N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic Acid), AMPD (2-Amino-2-methyl-1,3-propandiol), TABS (N-tris[hydroxymethyl]methyl-4-aminobutanesulfonic acid), AMPSO(N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxy-propanesulfonic acid), CHES (N-Cyclohexyl-2-aminoethanesulfonic acid), and combinations thereof.

14. The method of claim 7, wherein the one-step elution buffer consists of 2.5 to 10 mM KOH, 7.5 to 30 mM Tris base and 2.5 to 12.5 mM KCl, and wherein the pH is 11 to 12.

15. The method of claim 7, wherein the mixture is heated at 94°

C. to 99°

C.

16. The method of claim 7, wherein the blood sample is a dried blood sample on an adsorbent matrix.

20. The method of claim 16, wherein the mixture is heated at 94°

C. to 99°

C.

17. A method of analyzing DNA from a plurality of blood samples consisting essentially of

disposing the each of the plurality of blood samples into a different well of a first multi-well plate,

adding a one-step elution buffer having a pH of about 9 to about 13 to each well of the first multi-well plate to form a mixture in each well of the multi-well plate,

heating the plurality of mixtures at 90°

C. to 99°

C. for a time sufficient to elute the DNA from the plurality of blood samples to form an eluted DNA solution in each well of the multi-well plate,

optionally cooling the eluted DNA solution in each well of the multi-well plate at a temperature of at least about 4°

C. for at least 5 minutes, and

transferring each eluted DNA solution to a different well of a second multi-well plate without washing, wherein each well of the second multi-well plate contains a reaction mixture and primers for an enzymatic DNA amplification reaction, and

performing an enzymatic DNA amplification reaction in each well of the second multi-well plate that contains an eluted DNA solution.

18. The method of claim 17, wherein the one-step elution buffer consists of 5 to 22.5 mM potassium in the form of KOH, KCl, or a combination thereof, and 7.5 to 30 mM of a base having a buffering range of 7.0 to 9.5, wherein the pH of the one-step elution buffer is about 9 to about 13.

19. The method of claim 17, wherein the one-step elution buffer consists of 2.5 to 10 mM KOH, 7.5 to 30 mM Tris base and 2.5 to 12.5 mM KCl, and wherein the pH is 11 to 12.

21. The method of claim 17, wherein the blood sample is a dried blood sample on an adsorbent matrix.

23. The one-step elution buffer of claim 21, consisting of 15 mM Tris-Base, 10 mM KCl, and 5 mM KOH, wherein the pH is about 11.5.

22. A one-step DNA elution buffer, consisting of 2.5 to 10 mM KOH, 7.5 to 30 mM Tris base and 2.5 to 12.5 mM KCl, and wherein the pH is 11 to 12.

24. A kit for the elution of DNA from blood samples consisting essentially of

a one-step elution buffer having a pH of about 9 to about 13,

a primer pair for amplification of a genetic marker, and

a standard for identification of the genetic marker or a set of calibrators for quantitation of gene copy numbers, and

instructions for use.

25. The kit of claim 24, wherein the one-step elution buffer consists of 5 to 22.5 mM potassium in the form of KOH, KCl, or a combination thereof, and 7.5 to 30 mM of a base having a buffering range of 7.0 to 9.5, wherein the pH of the one-step elution buffer is about 9 to about 13.

26. The kit of claim 25, wherein the one-step elution buffer consists of 2.5 to 10 mM KOH, 7.5 to 30 mM Tris base and 2.5 to 12.5 mM KCl, and wherein the pH is 11 to 12.

27. The kit of claim 24 wherein the genetic marker is a marker for SCID.

28. The kit of claim 27, wherein the wherein the primers are SEQ ID NO:

13 and SEQ ID NO;

14.