US RE49,835 E1Granted

Mouse cell line authentication

AssigneeThe United States of America as represented by the Secretary of Commerce

InventorsJamie L Almeida, Kenneth D. Cole

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Abstract

A multiplex polymerase chain reaction assay that targets nine tetranucleotide short tandem repeat (STR) markers in the mouse genome. Unique profiles were obtained from seventy-two mouse samples that were used to determine the allele distribution for each STR marker. Correlations between allele fragment length and repeat number were determined with DNA Sanger sequencing. Genotypes for L929 and NIH3T3 cell lines were shown to be stable with increasing passage numbers as there were no significant differences in fragment length with samples of low passage when compared to high passage samples. In order to detect cell line contaminants, primers for two human STR markers were incorporated into the multiplex assay to facilitate detection of human and African green monkey DNA. This multiplex assay is the first of its kind to provide a unique STR profile for each individual mouse sample and can be used to authenticate mouse cell lines.

Claims10

Dependent claims

A method of determining the alleles present in a DNA sample, the method comprising:

obtaining a DNA sample to be analyzed;
selecting a set of STR marker loci of the DNA sample to be analyzed that can be amplified together in a multiplex amplification reaction, wherein the set of STR marker loci are selected from the group consisting of:
18-3, 4-2, 6-7, 9-2, 15-3, 6-4, 12-1, 5-5 and X-1;
providing a set of oligonucleotide primer pairs, wherein each oligonucleotide primer pair in the set flanks a single locus in the set of STR marker loci, and wherein each oligonucleotide primer pair is capable of amplification of a single locus from the set of STR marker loci in a multiplex amplification reaction;
co-amplifying the set of STR marker loci in a multiplex amplification reaction, wherein the product of the multiplex amplification reaction comprises a mixture of amplified alleles from each of the co-amplified loci in the set of STR marker loci;
evaluating the products of the co-amplification reaction to determine the alleles present at each of the loci analyzed in the set of STR marker loci within the DNA sample;
and
wherein the source of the DNA sample to be analyzed is at least one of a mouse and a cell line derived from a mouse.

5 more elements

Dependencies are inferred from the claim text.

Facts

Application: 16/263,835Filed 31 Jan 2019 · reissue
Priority: 3 Jul 2013
Granted: 13 Feb 2024
Term ends: Not estimatedThe source records don’t support an estimate.

Classification4 codes

C12Q 1/6881
Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes: for tissue or cell typing, e.g. human leukocyte antigen [HLA]…
C12Q 2600/156
Oligonucleotides characterized by their use: polymorphic or mutational markers
C12Q 2600/16
Oligonucleotides characterized by their use: primer sets for multiplex assays
C12Q
Measuring or testing processes involving enzymes, nucleic acids or microorganisms