US 11,702,697 B2
Quality testing of DNA samples
Sophie Beckert, Neuzelle (DE); and Bernd Hinzmann, Penzberg (DE)
Assigned to Roche Sequencing Solutions, Inc., Pleasanton, CA (US)
Appl. No. 17/55,038
Filed by Roche Sequencing Solutions, Inc., Pleasanton, CA (US)
PCT Filed May 14, 2018, PCT No. PCT/EP2018/062304
§ 371(c)(1), (2) Date Nov. 12, 2020,
PCT Pub. No. WO2019/219157, PCT Pub. Date Nov. 21, 2019.
Prior Publication US 2021/0214790 A1, Jul. 15, 2021
Int. Cl. C12Q 1/68 (2018.01); C12Q 1/6876 (2018.01); C12Q 1/6848 (2018.01); C12Q 1/6851 (2018.01)
CPC C12Q 1/6876 (2013.01) [C12Q 1/6848 (2013.01); C12Q 1/6851 (2013.01)] 4 Claims
OG exemplary drawing
 
1. A method of measuring quality of cell free DNA in a sample comprising the steps of
a) providing (i) a DNA sample comprising cell free DNA and cellular DNA, and (ii) a control sample which has been proven previously to contain no genomic contamination,
b) providing a first pair of amplification primers comprising SEQ ID Nos: 1 and 2, which is capable of generating a first amplicon from a LINE sequence that has a size of less than 80 bp,
c) providing a second pair of amplification primers comprising SEQ ID Nos: 3 and 4 which is capable of generating a second amplicon from a LINE sequence that has a size of more than 160 bp,
d) providing a third pair of amplification primers comprising SEQ ID Nos: 5 and 2 which is capable of generating a third amplicon from a LINE sequence that has a size of more than 300 bp,
e) performing a qPCR with the first and the second pair of amplification primers and determining cq values for the first and the second generated amplicons in the DNA sample and the control sample,
f) determining the relative concentrations of said amplicons in the DNA sample and the control sample using the formula
crel=2−(cqx−cqy)
where cqx is cq of the second amplicon and cqy is cq of the first amplicon, and
g) determining the quality of the cell free DNA in the DNA sample based on a proximity of crel to 1, wherein if the DNA sample is contaminated with cellular DNA, the crel will be closer to 1 as compared to the control sample.