Cell free DNA diagnostic testing standards

• US 10,577,655 B2
• Filed: 01/14/2016
• Issued: 03/03/2020
• Est. Priority Date: 09/27/2013
• Status: Active Grant

• Current Assignee: Natera Incorporated (Natera)
• Original Assignee: Natera Incorporated (Natera)
• Inventors: Babiarz, Joshua, Zimmermann, Bernhard, Constantin, Tudor Pompiliu
• Primary Examiner(s): Dauner, Joseph G.
• Application Number:
  US14/996,097
• Publication Number:
  US 20160244838A1
• Time in Patent Office: 1,510 Days
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2537/16 Assays for determining copy number or wherein the copy number is of special importance
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: C12Q 2600/166 Oligonucleotides used as internal standards, controls or normalisation probes
• CPC Class Codes: Y10T 436/10 Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing

Abstract

Embodiments of the invention include methods and compositions for producing standards for noninvasive prenatal genetic diagnostics and for the detection and monitoring of cancer. The compositions can include a plurality of different nucleosomal DNA fragments derived from either primary cells or cell lines and can include one or more synthetic oligonucleotides. The amount of the different nucleosomal DNA fragments can be varied so as to simulate naturally occurring cell free DNA samples obtained from the blood of the pregnant woman or naturally occurring cell free DNA samples obtained from the blood of cancer patients.

Claims

1. A kit, comprising two or more nucleic acid standard compositions for analyzing circulating tumor DNA, each standard composition comprising a mixture of a first nucleosomal ladder nucleic acid preparation generated in vitro from a cancer cell source and a second nucleosomal ladder nucleic acid preparation generated in vitro from a matched non-cancer cell source, wherein the first nucleosomal ladder nucleic acid preparation and the second nucleosomal ladder nucleic acid preparation are generated in vitro by MNase treatment, wherein the ratio of the quantity of the first nucleosomal ladder nucleic acid preparation and the quantity of the second nucleosomal ladder nucleic acid preparation in each nucleic acid standard composition is different, wherein the first nucleosomal ladder nucleic acid preparation comprises between 0.01% to 20% of the total nucleosomal nucleic acids in at least two of the nucleic acid standard compositions, and wherein a copy number of a chromosomal region known to exhibit copy number variation in cancer is different in the first nucleosomal ladder nucleic acid preparation compared to the second nucleosomal ladder nucleic acid preparation.

2. The kit of claim 1, wherein the kit comprises between 2 and 20 nucleic acid standard compositions.

3. The kit of claim 1, wherein the kit comprises between 5 and 10 nucleic acid standard compositions.

4. The kit of claim 1, wherein the kit comprises at least one nucleic acid standard composition in which the quantity of the first nucleosomal ladder nucleic acid preparation comprises between 0.01% to 10% of the total nucleic acids of the nucleic acid standard composition.

5. The kit of claim 1, wherein the kit comprises at least two nucleic acid standard composition in which the quantity of the first nucleosomal ladder nucleic acid preparation comprises between 0.01% to 1% of the total nucleic acids of the nucleic acid standard composition.

6. The kit of claim 1, wherein the kit comprises at least two nucleic acid standard composition in which the quantity of the first nucleosomal ladder nucleic acid preparation comprises between 1% to 10% of the total nucleic acids of the nucleic acid standard composition.

7. The kit of claim 1, wherein at least one of the nucleic acid standard compositions further comprises between 0.01 and 10% of total nucleic acids in the nucleic acid standard composition, of a synthetic oligonucleotide between 50 and 500 base pairs in length comprising at least 50 contiguous nucleotides having a sequence that is at least 90% identical to a genomic sequence comprising or flanking a mutation correlated with cancer selected from a single nucleotide variant, an indel, or a gene fusion junction.

8. A kit comprising:

(a) two or more nucleic acid standard compositions each in a separate chamber, tube, or vessel and each standard composition comprising a mixture of a first nucleosomal ladder nucleic acid preparation generated in vitro from a cancer cell source and a second nucleosomal ladder nucleic acid preparation generated in vitro from a matched non-cancer cell source, wherein the first nucleosomal ladder nucleic acid preparation and the second nucleosomal ladder nucleic acid preparation are generated in vitro by MNase treatment, wherein the ratio of the quantity of the first nucleosomal ladder nucleic acid preparation and the quantity of the second nucleosomal ladder nucleic acid preparation in each nucleic acid standard composition is different, wherein the first nucleosomal ladder nucleic acid preparation comprises between 0.01% to 20% of the total nucleosomal nucleic acid in at least two of the nucleic acid standard compositions, and wherein a copy number of a chromosomal region known to exhibit copy number variation in cancer is different in the first nucleosomal ladder nucleic acid preparation compared to the second nucleosomal ladder nucleic acid preparation; and

(b) one or more primers for amplifying at least a portion of the chromosomal region known to exhibit copy number variation in cancer.

9. The kit of claim 8, wherein the kit comprises at least one nucleic acid standard composition in which the quantity of the first nucleosomal ladder nucleic acid preparation comprises between 0.01% and 1% of the total nucleic acids of the nucleic acid standard composition preparations.