# US 9,518,302 B2 · Method for direct amplification from crude nucleic acid samples

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- Number: US 9,518,302 B2
- Document ID: US9518302B2
- Status: Granted
- Kind: B2
- Application number: 14/739,722
- Filed: 2015-06-15
- Published: 2015-12-10
- Granted: 2016-12-13
- Earliest claimed: 2008-06-30
- Assignees: Life Technologies Corporation
- Applicants: Life Technologies Corporation
- Inventors: Chien-Wei Chang; Lori Hennessy; Dennis Wang
- Web page: https://patentgrep.com/patent/US9518302B2

## CPC

- C12Q 1/6888: for detection or identification of organisms
- C12Q 1/68: involving nucleic acids
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## 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.

## Claims

### Claim 1

A method comprising:
mixing a crude sample with a direct buffer comprising a plurality of primer pairs and thereby forming a reaction mixture, each primer pair flanking a short tandem repeat locus;
performing a PCR on the reaction mixture to form a plurality of PCR amplicons;
subjecting the reaction mixture to capillary electrophoresis; and
identifying a human by reference to the size of the PCR amplicons,
wherein the direct buffer comprises at least 3%-8% glycerol, 0.2%-0.9% polysorbate, and 1000-3000 μg/ml BSA.

### Claim 2 (depends on 1)

The method of claim 1, wherein the direct buffer further comprises 10-50 mM Tris-HCl (pH 8.3), 30-80 mM KCl, 1.4-2.4 mM MgCl2, 0.01%-0.04% Sodium azide, 3%-8% glycerol, 100-350 μM of each dNTPs and 0.10-0.35 U/μl of DNA polymerase.

### Claim 3 (depends on 1)

The method of claim 1, wherein the polysorbate is polysorbate 20.