Kits and methods for quantitative assessment and enrichment of extrachromosomal circular dna
AssigneeCedars-Sinai Medical Center
InventorsHisashi Tanaka, Lila Montoya, Armando E. Giuliano
Life of this patent
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Abstract
Methods and reagents for related kits are provided for assessing enrichment of extrachromosomal circular DNA (ec-cDNA) isolated from biological samples. The methods include spiking the biological samples with a known proportion of linearized and circular plasmid DNA molecules and measuring their relative amounts post-exonuclease treatment as a benchmark to validate enrichment of eccDNA in the isolation process. Alternatively, or in combination, the method includes measuring the relative amounts of endogenous mitochondrial DNA and chromosomal DNA post-exonuclease treatment to assess the enrichment of eccDNA in the isolation process.
Claims33
An assessment kit for use with a sample containing circular DNA, comprising:
- a. a first plasmid DNA molecule which is linearized, or a first plasmid DNA molecule and one or more restriction endonucleases to linearize the first plasmid DNA;
- and
- one or more of:
- b. a second plasmid DNA molecule having a size between 1,000 bp and 9,999 bp;
- c. a third plasmid DNA molecule having a size between 10,000 bp and 99,999 bp;
- and
- d. a fourth plasmid DNA molecule having a size between 100,000 bp and 300,000 bp.
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A method for enriching and quantifying extrachromosomal circular DNA (eccDNA) in a cell or tissue, wherein the cell or tissue comprises chromosomal DNA and the eccDNA, and the eccDNA comprises mitochondrial DNA and non-organelle eccDNA, the method comprising:
- (i) lysing the cell or tissue, or obtaining a sample wherein the cell or tissue is lysed;
- and
- (ii) adding to the lysed cell or tissue, or to the sample:
- a. a first plasmid DNA molecule which is linearized, and
- one or more of:
- b. a second plasmid DNA molecule having a size between 1,000 bp and 9,999 bp;
- c. a third plasmid DNA molecule having a size between 10,000 bp and 99,999 bp;
- and
- d. a fourth plasmid DNA molecule having a size between 100,000 bp and 300,000 bp;
- to obtain a plasmid-spiked sample;
- treating the plasmid-spiked sample with a proteinase to degrade protein from the plasmid-spiked sample,
- and subsequently treating with an exonuclease to digest linear polynucleotides but not circular DNA, followed by recovery of DNA to obtain the eccDNA;
- and
- (iii) quantifying respective amounts of the first plasmid DNA molecule and the one or more of the second, the third, and the fourth plasmid DNA molecules after the treatment with the exonuclease;
- and/or quantifying an amount of the mitochondrial DNA and an amount of the chromosomal DNA.
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A method for enriching and quantifying extrachromosomal circular DNA (eccDNA) in a cell or tissue, wherein the cell or tissue comprises chromosomal DNA and the eccDNA, and the eccDNA comprises mitochondrial DNA and non-organelle eccDNA, the method comprising:
- (i) lysing the cell or tissue, or obtaining a sample wherein the cell or tissue is lysed;
- and
- (ii) adding to the lysed cell or tissue, or to the sample:
- a first plasmid DNA molecule which is linearized, and
- one or more of:
- a second plasmid DNA molecule having a size between 1,000 bp and 9,999 bp;
- a third plasmid DNA molecule having a size between 10,000 bp and 99,999 bp;
- and
- a fourth plasmid DNA molecule having a size between 100,000 bp and 300,000 bp,
- to obtain a plasmid-spiked sample;
- embedding the plasmid-spiked sample into agarose;
- digesting the agarose with b-agarese I;
- removing any remaining carbohydrate;
- precipitating DNA to obtain eccDNA;
- and
- (iii) quantifying respective amounts of the first plasmid DNA molecule and the one or more of the second, the third, and the fourth plasmid DNA molecules;
- and/or quantifying an amount of the mitochondrial DNA and an amount of the chromosomal DNA.
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Dependencies are inferred from the claim text.