Mesylate based master mix

Patent metadata

Field	Value
Publication number	US-11293059-B2
Application number	US-201816623975-A
Country	US
Kind code	B2
Filing date	Jun 20, 2018
Priority date	Jun 22, 2017
Publication date	Apr 5, 2022
Grant date	Apr 5, 2022

Abstract

Disclosed herein are mesylate containing compositions, methods of use of such compositions and kits of components encompassing such compositions for improving the amplification of nucleic acids, especially in the presence of enzymatic inhibitors. These find particular use in forensic and environmental analyses. The instant disclosure provides new compositions, methods and kits of components for overcoming PCR inhibition. In some embodiments disclosed herein is a PCR master mix, the PCR master mix encompassing a mesylate.

First claim

We claim:

1. A PCR master mix comprising:
Tris-mesylate at a concentration of 10-50 mM;
potassium methanesulfonate at a concentration of 30-80 mM;
bovine serum albumin at a concentration of 1.2-4 mg/ml; and
glycerol at a concentration of 3-8% by volume.

2. The PCR master mix of claim 1 , further comprising deoxynucleoside triphosphate.

3. The PCR master mix of claim 1 , further comprising a DNA polymerase.

4. The PCR master mix of claim 1 , further comprising a deoxynucleoside triphosphate, a non-ionic detergent and a DNA polymerase.

5. A kit comprising a first capped tube comprising a PCR master mix as in claim 1 , and a second capped tube comprising a primer.

6. The kit of claim 5 , wherein the master mix further comprises a polymerase.

7. The kit of claim 6 , further comprising a third capped tube comprising an allelic ladder.

8. A method, the method comprising contacting a sample with a PCR master mix and thereby forming a contacted sample, the PCR master mix comprising Tris-mesylate (10-50 mM), potassium methanesulfonate (30-80 mM), bovine serum albumin (1.2-4 mg/ml), glycerol (3-8% volume), MgSO4 (1.4-2.4 mM), Sodium Azide (0.01-0.04%), dATP, dCTP, dGTP, dTTP, at least two primers and a DNA polymerase, subjecting the contacted sample to a thermocycling protocol, thereby forming a cycled sample and subjecting the cycled sample to an electrophoretic separation and thereby detecting a presence or absence of an amplicon.

9. A PCR master mix comprising one or more of:
Tris-mesylate at a concentration of 10-50 mM,
potassium methanesulfonate at a concentration of 30-80 mM, or
magnesium methanesulfonate at a concentration of 1.2-1.8 mM,
the PCR master mix further comprising a stabilizer compound, wherein the stabilizer compound comprises glycerol at a concentration of 3-8% by volume.

10. The PCR master mix of claim 9 , further comprising a proteinaceous compound.

11. The PCR master mix of claim 10 , wherein the proteinaceous compound is bovine serum albumin.

12. The PCR master mix of claim 11 , wherein the bovine serum albumin is provided at a concentration of 1.2-4 mg/ml.

13. The PCR master mix of claim 9 , further comprising deoxynucleoside triphosphate.

14. The PCR master mix of claim 9 , further comprising a DNA polymerase.

15. A PCR master mix comprising one or more of:
Tris-mesylate at a concentration of 10-50 mM,
potassium methanesulfonate at a concentration of 30-80 mM, or
magnesium methanesulfonate at a concentration of 1.2-1.8 mM,
the PCR master mix further comprising a proteinaceous compound, wherein the proteinaceous compound is bovine serum albumin.

16. The PCR master mix of claim 15 , further comprising a stabilizer compound.

17. The PCR master mix of claim 16 , wherein the stabilizer compound comprises glycerol or sucrose.

18. The PCR master mix of claim 15 , wherein the bovine serum albumin is provided at a concentration of 1.2-4 mg/ml.

19. The PCR master mix of claim 15 , further comprising deoxynucleoside triphosphate.

20. The PCR master mix of claim 15 , further comprising a DNA polymerase.

21. A PCR master mix comprising one or more of:
Tris-mesylate at a concentration of 10-50 mM,
potassium methanesulfonate at a concentration of 30-80 mM, or
magnesium methanesulfonate at a concentration of 1.2-1.8 mM,
the PCR master mix further comprising deoxynucleoside triphosphate.

22. The PCR master mix of claim 21 , further comprising a stabilizer compound.

23. The PCR master mix of claim 22 , wherein the stabilizer compound comprises glycerol or sucrose.

24. The PCR master mix of claim 21 , further comprising a proteinaceous compound.

25. The PCR master mix of claim 24 , wherein the proteinaceous compound is bovine serum albumin and wherein the bovine serum albumin is provided at a concentration of 1.2-4 mg/ml.

26. The PCR master mix of claim 21 , further comprising a DNA polymerase.

27. A PCR master mix comprising one or more of:
Tris-mesylate at a concentration of 10-50 mM,
potassium methanesulfonate at a concentration of 30-80 mM, or
magnesium methanesulfonate at a concentration of 1.2-1.8 mM,
the PCR master mix further comprising a DNA polymerase.

28. The PCR master mix of claim 27 , further comprising a stabilizer compound.

29. The PCR master mix of claim 28 , wherein the stabilizer compound comprises glycerol or sucrose.

30. The PCR master mix of claim 27 , further comprising a proteinaceous compound.

31. The PCR master mix of claim 30 , wherein the proteinaceous compound is bovine serum albumin, and wherein the bovine serum albumin is provided at a concentration of 1.2-4 mg/ml.

Assignees

• Life Technologies Corp

Inventors

• Zhong Chang
• Mulero Julio
• Menchen Steven

Classifications

• C12Q1/6848

  characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction · CPC title

• C12Q2527/125

  Specific component of sample, medium or buffer · CPC title

• C12Q1/686 Primary

  Polymerase chain reaction [PCR] · CPC title