Method for Direct Amplification from Crude Nucleic Acid Samples
AssigneeApplera Corporation
InventorsChien-Wei Chang, Dennis Wang, Lori K. Hennessy
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Abstract
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.
Claims21
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;
- removing the eluate from the incubated direct buffer wherein the eluate comprises the deoxyribonucleic acid from the crude sample;
- and
- performing a PCR on the deoxyribonucleic acid, 1 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 2U-8U, MgCl2 at 1.25-2.2 mM, and ssb of at least 20 ng/ul.
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, wherein the direct buffer comprises a plurality of primer pairs, wherein each primer pair flanks a genomic locus containing a short tandem repeat (STR);
- removing the eluate from the incubated direct buffer, wherein the eluate 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 25-75 mM, dNTPs at 200-400 uM each dNTP, BSA at 160-960 ug/ml, AmpliTaq Gold polymerase at 2U-8U, MgCl2 at 1.25-2.2 mM, and ssb of at least 20 ng/ul.
A method of preparing nucleic acids for a downstream enzymatic manipulation comprising;
- providing a crude sample comprising deoxyribonucleic acid;
- incubating the crude sample with a direct buffer to form an eluate;
- removing the eluate from the incubated direct buffer, wherein the eluate comprises the deoxyribonucleic acids from the crude sample;
- and
- performing a downstream enzymatic manipulation on the eluate, 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 2U-8U, MgCl2 at 1.25-2.2 mM, and ssb of at least 20 ng/ul.
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 ssb of at least 20 ng/ul.
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 ssb of at least 20 ng/ul.
Dependencies are inferred from the claim text.