US 12,410,475 B2
Methods and processes for non-invasive assessment of genetic variations
Taylor Jacob Jensen, San Diego, CA (US); Jennifer Geis, San Diego, CA (US); Sung Kyun Kim, San Diego, CA (US); Cosmin Deciu, San Diego, CA (US); and Mathias Ehrich, San Diego, CA (US)
Assigned to Sequenom, Inc., San Diego, CA (US)
Filed by Sequenom, Inc., San Diego, CA (US)
Filed on May 23, 2022, as Appl. No. 17/751,201.
Application 17/751,201 is a continuation of application No. 15/124,324, granted, now 11,365,447, previously published as PCT/US2015/020250, filed on Mar. 12, 2015.
Claims priority of provisional application 61/952,135, filed on Mar. 13, 2014.
Prior Publication US 2022/0411871 A1, Dec. 29, 2022
Int. Cl. C12Q 1/6883 (2018.01); G16B 25/00 (2019.01); G16B 25/10 (2019.01); G16B 25/20 (2019.01); G16B 45/00 (2019.01); H01J 49/16 (2006.01)
CPC C12Q 1/6883 (2013.01) [G16B 25/00 (2019.02); G16B 25/10 (2019.02); G16B 25/20 (2019.02); G16B 45/00 (2019.02); C12Q 2537/143 (2013.01); C12Q 2565/627 (2013.01); C12Q 2600/106 (2013.01); C12Q 2600/158 (2013.01); C12Q 2600/16 (2013.01); H01J 49/164 (2013.01)] 19 Claims
 
1. A method for detecting one, two, three or four copies of a fetal chromosome or portion thereof in a sample, comprising:
(a) contacting a sample comprising circulating cell-free nucleic acid from a human pregnant female bearing a fetus with a methylation sensitive restriction enzyme under cleavage conditions to selectively digest non-methylated maternal nucleic acid from target polynucleotides in each of chromosomes 13, 18 and 21;
(b) the steps of: (i) contacting the nucleic acid of step (a) with a collection of four sets of primer pairs under amplification conditions, wherein at least one of each primer pair is specific for nucleic acid sequences located within at least one of the target polynucleotides in chromosome 13, chromosome 18 and chromosome 21, and one of the four sets of primer pairs is specific for amplifying the target polynucleotide from one of chromosomes 13, 18 or 21, thereby generating amplicons from the target polynucleotides from the undigested polynucleotide targets, wherein the target polynucleotides are in:
chromosome 13 polynucleotides of SEQ ID NOs: 198 and 211, or a complement thereof;
chromosome 18 polynucleotides of SEQ ID NOs: 229 and 231, or a complement thereof; and
chromosome 21 polynucleotides of SEQ ID NOs: 244 and 252, or a complement thereof; thereby generating amplicons; and
(ii) contacting the sample with at least one competitor oligonucleotide under the amplification conditions such that a competitor amplicon comprising the competitor oligonucleotide is generated, wherein the competitor oligonucleotide comprises a sequence substantially identical to a first target sequence but includes at least one nucleotide substitution;
(c) determining an amount of the chromosome 13, chromosome 18 and chromosome 21 amplicons in (b) by comparing a ratio of the first target amplicon to the at least one competitor amplicon; and
(d) determining, based on the relative amounts of target amplicons from step (c), whether there is one, two, three or four copies of one or more of chromosome 13, chromosome 18, and chromosome 21, or a portion thereof, in the fetus.