Method for direct amplification from crude nucleic acid samples
View Patent ↗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;
optionally incubating the crude sample with NaOH;
mixing the crude sample with a direct buffer to form a nucleic acid containing solution; and
performing a PCR on the nucleic acid containing solution, wherein the direct buffer comprises at least 0.2%-0.9% polysorbate, 3%-8% glycerol, and 1000-3000 ug/ml BSA, wherein said direct buffer comprises at least one primer pair.
2. The method according to claim 1 , wherein the direct buffer further comprises, 10-50 mM Tris-HCI (pH 8.3), 30-80 mM KCI, 1.4-2.4 mM MgCl 2 , 0.01%-0.04% Sodium azide, 100-350 uM of each dNTP, and 0.10-0.35 U/ul of DNA polymerase.
3. The method according to claim 1 , wherein the polysorbate is polysorbate 20.
4. A method of determining the identity of a human comprising:
providing a crude sample comprising deoxyribonucleic acid from the human;
optionally incubating the crude sample with NaOH;
mixing 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);
performing a PCR on the nucleic acid containing solution to form a plurality of PCR amplicons, wherein each PCR amplicon has an ascertainable size; and
identifying the human by reference to the size of the PCR amplicons, wherein the direct buffer further comprises 0.2%-0.9% polysorbate, 3%-8% glycerol, and 1000-3000 ug/ml BSA.
5. The method according to claim 4 , wherein the direct buffer further comprises 10-50 mM Tris-HCI (pH 8.3), 30-80 mM KCI, 1.4-2.4 mM MgCl 2 , 0.01%-0.04% Sodium azide, 100-350 uM of each dNTP, and 0.10-0.35 U/ul of DNA polymerase.
6. The method according to claim 4 , wherein the polysorbate is polysorbate 20.
7. A method of preparing nucleic acids comprising:
providing a crude sample comprising deoxyribonucleic acid;
optionally incubating the crude sample with NaOH;
mixing the crude sample with a direct buffer to form a nucleic acid containing solution; and
performing a downstream enzymatic manipulation on the solution, wherein the direct buffer comprises 0.2%-0.9% polysorbate, 3%-8% glycerol, and 1000-3000 ug/ml BSA, wherein said direct buffer comprises at least one primer pair.
8. The method according to claim 7 , wherein the downstream enzymatic manipulation is a PCR.
9. The method according to claim 7 , wherein the direct buffer further comprises 10-50 mM Tris-HCI (pH 8.3), 30-80 mM KCI, 1.4-2.4 mM MgCl 2 , 0.01%-0.04% Sodium azide, 100-350 uM of each dNTP, and 0.10-0.35 U/ul of DNA polymerase.
10. The method according to claim 7 , wherein the direct buffer further comprises 10-50 mM Tris-HCI (pH 8.3), 30-80 mM KCI, 1.4-2.4 mM MgCl 2 , 0.01%-0.04% Sodium azide, 100-350 uM of each dNTP, 1200-3000 ug/ml BSA, and 0.10-0.35 U/ul of DNA polymerase.
11. The method according to claim 7 , wherein the polysorbate is polysorbate 20.