Method for direct amplification from crude nucleic acid samples
The present teachings relate to improved methods, kits, and reaction mixtures for amplifying nucleic acids. In some embodiments a novel direct buffer formulation is provided which allows for the direct amplification of the nucleic acids in a crude sample with minimal sample purification.
1. A method of performing a polymerase chain reaction (PCR) comprising;
providing a crude sample comprising deoxyribonucleic acid;
incubating the crude sample with a direct buffer to form a nucleic acid-containing solution; and
performing a polymerase chain reaction (PCR) on said nucleic acid-containing solution, wherein the direct buffer comprises at least 5 PCR primer pairs, Tris-HCl at 10-16 mM, KCl at 50-75 mM, dNTPs at 200-400 uM each dNTP, BSA at 800-960 ug/ml, AmpliTaq Gold polymerase at 2U-8U, MgCl2 at 1.6-2.2 mM, and single-stranded binding protein (ssb) of at least 20 ng/ul.
2. The method according to claim 1 , wherein the direct buffer comprises, Tris-HCl at 10 mM pH 8.3, KCl at 50 mM, dNTPs at 200 uM each dNTP, BSA at 800 ug/ml, AmpliTaq Gold polymerase at 0.16 units/ul, MgCl2 at 1.6 mM, ssb at 60 ng/ul.
3. The method according to claim 2 , wherein the direct buffer further comprises Sodium Azide at 0.02 percent.
4. The method according to claim 2 , wherein the ssb is T4 gene 32 protein from bacteriophage T4.
5. A method of determining the identity of a human comprising:
providing a crude sample comprising deoxyribonucleic acid from the human;
incubating the crude sample with a direct buffer to form a nucleic acid-containing solution, wherein the direct buffer comprises a plurality of primer pairs,
wherein each primer pair flanks a genomic locus containing a short tandem repeat (STR);
wherein the solution comprises the deoxyribonucleic acid from the crude sample;
performing a PCR on the deoxyribonucleic acids from the crude sample to form a plurality of PCR amplicons, wherein each PCR amplicon has an ascertainable size; and,
identifying the human by reference to size of the PCR amplicons, wherein the direct buffer further comprises Tris-HCl at 10-16 mM, KCl at 50-75 mM, dNTPs at 200-400 uM each dNTP, BSA at 800-960 ug/ml, AmpliTaq Gold polymerase at 2U-8U, MgCl2 at 1.6-2.2 mM, and ssb of at least 20 ng/ul.
6. The method according to claim 5 , wherein the direct buffer comprises, Tris-HCl at 10 mM pH 8.3, KCl at 50 mM, dNTPs at 200 uM each dNTP, BSA at 800 ug/ml, AmpliTaq Gold polymerase at 0.16 units/ul, MgCl2 at 1.6 mM, and ssb at 60 ng/ul.
7. The method according to claim 6 , wherein the direct buffer further comprises Sodium Azide at 0.02 percent.
8. The method according to claim 6 wherein the ssb is T4 gene 32 protein from bacteriophage T4.
9. A method of preparing nucleic acids for an enzymatic manipulation comprising:
providing a crude sample comprising deoxyribonucleic acid;
incubating the crude sample with a direct buffer to form a nucleic acid-containing solution;
wherein the solution comprises the deoxyribonucleic acids from the crude sample; and
wherein the direct buffer comprises Tris-HCl at 10-16 mM, KCl at 50-75 mM, dNTPs at 200-400 uM each dNTP, BSA at 800-960 ug/ml, AmpliTaq Gold polymerase at 2U-8U, MgCl2 at 1.6-2.2 mM, and ssb of at least 20 ng/ul, and said nucleic acid-containing solution is used in the enzymatic manipulation.
10. The method according to claim 9 wherein the enzymatic manipulation is a PCR.
11. The method according to claim 9 wherein the direct buffer comprises, Tris-HCl at 10 mM pH 8.3, KCl at 50 mM, dNTPs at 200 uM each dNTP, BSA at 800 ug/ml, AmpliTaq Gold polymerase at 0.16 units/ul, MgCl2 at 1.6 mM, and ssb at 60 ng/ul.
12. The method according to claim 11 wherein the direct buffer further comprises Sodium Azide at 0.02 percent.
13. The method according to claim 11 wherein the ssb is T4 gene 32 protein from bacteriophage T4.
14. A kit for analysis of a crude sample comprising:
a plurality of primer pairs, wherein each primer pair flanks a genomic locus containing a short tandem repeat (STR); and, a direct buffer, wherein the direct buffer comprises Tris-HCl at 10-16 mM, KCl at 50-75 mM, dNTPs at 200-400 uM each dNTP, BSA at 800-960 ug/ml, AmpliTaq Gold polymerase at 2U-8U, MgCl2 at 1.6-2.2 mM, and ssb of at least 20 ng/ul.
15. The kit according to claim 14 wherein the direct buffer comprises, Tris-HCl at 10 mM pH 8.3, KCl at 50 mM, dNTPs at 200 uM each dNTP, BSA at 800 ug/ml, AmpliTaq Gold polymerase at 0.16 units/ul, MgCl2 at 1.6 mM, and ssb at 60 ng/ul.
16. The kit according to claim 15 wherein the direct buffer further comprises Sodium Azide at 0.02 percent.
17. The kit according to claim 15 wherein the ssb is T4 gene 32 protein from bacteriophage T4.
18. A reaction mixture comprising a direct buffer and a plurality of primer pairs, wherein each primer pair flanks a genomic locus of a crude sample containing a short tandem repeat (STR); and wherein the direct buffer comprises Tris-HCl at 10-16 mM, KCl at 50-75 mM, dNTPs at 200-400 uM each dNTP, BSA at 800-960 ug/ml, AmpliTaq Gold polymerase at 2U-8U, MgCl2 at 1.6-2.2 mM, and ssb of at least 20 ng/ul.
19. The reaction mixture according to claim 18 wherein the direct buffer comprises, Tris-HCl at 10 mM pH 8.3, KCl at 50 mM, dNTPs at 200 uM each dNTP, BSA at 800 ug/ml, AmpliTaq Gold polymerase at 0.16 units/ul, MgCl2 at 1.6 mM, and ssb at 60 ng/ul.
20. The reaction according to claim 19 wherein the direct buffer further comprises Sodium Azide at 0.02 percent.
21. The reaction mixture according to claim 19 , wherein the ssb is T4 gene 32 protein from bacteriophage T4.