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;
optionally incubating with NaOH,
mixing crude sample with a direct buffer; and
performing a PCR on the deoxyribonucleic acid, wherein the direct buffer comprises at least 5 PCR primer pairs, Tris-HCl at 10-16 mM, KCl at 25-75 mM, dNTPs at 200-400 uM each dNTP, BSA at 160-960 ug/ml, AmpliTaq Gold polymerase at 2 U-8 U, MgCl2 at 1.25-2.2 mM, and DMSO at 0-4%.
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, DMSO at 2%
3 . The method according to claim 2 wherein the direct buffer further comprises Sodium Azide at 0.02 percent.
4 . A method of determining the identity of a human comprising;
providing a crude sample comprising deoxyribonucleic acid from the human;
optionally incubating with NaOH;
mixing the crude sample with a direct buffer, 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 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 25-75 mM, dNTPs at 200-400 uM each dNTP, BSA at 160-960 ug/ml, AmpliTaq Gold polymerase at 2 U-8 U, MgCl2 at 1.25-2.2 mM, and DMSO at 0-4%.
5 . The method according to claim 4 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 DMSO at 0-4%.
6 . The method according to claim 5 wherein the direct buffer further comprises Sodium Azide at 0.02 percent.
7 . A method of preparing nucleic acids for a downstream enzymatic manipulation comprising;
providing a crude sample comprising deoxyribonucleic acid;
optionally incubating crude sample with NaOH;
mixing the crude sample with a direct buffer; and
performing a downstream enzymatic manipulation on the mixture, wherein the direct buffer comprises Tris-HCl at 10-16 mM, KCl at 25-75 mM, dNTPs at 200-400 uM each dNTP, BSA at 160-960 ug/ml, AmpliTaq Gold polymerase at 2 U-8 U, MgCl2 at 1.25-2.2 mM, and DMSO at 0-4%.
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 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 DMSO at 2%.
10 . The method according to claim 9 wherein the direct buffer further comprises Sodium Azide at 0.2 percent.
11 . A kit 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 25-75 mM, dNTPs at 200-400 uM each dNTP, BSA at 160-960 ug/ml, AmpliTaq Gold polymerase at 2 U-8 U, MgCl2 at 1.25-2.2 mM, and DMSO at 0-4%.
12 . The kit according to claim 11 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 DMSO at 0-4%.
13 . The kit according to claim 12 wherein the direct buffer further comprises Sodium Azide at 0.02 percent.
14 . A reaction mixture comprising a direct buffer and a plurality of primer pairs, wherein each primer pair flanks a genomic locus containing a short tandem repeat (STR); and wherein the direct buffer comprises Tris-HCl at 10-16 mM, KCl at 25-75 mM, dNTPs at 200-400 uM each dNTP, BSA at 160-960 ug/ml, AmpliTaq Gold polymerase at 2 U-8 U, MgCl2 at 1.25-2.2 mM, and DMSO at 0-4%.
15 . The reaction mixture 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 DMSO at 0-4%.
16 . The reaction according to claim 15 wherein the direct buffer further comprises Sodium Azide at 0.02 percent.