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US 11,365,447 B2Granted
Methods and processes for non-invasive assessment of genetic variations
AssigneeSequenom, Inc.
InventorsTaylor Jacob Jensen, Jennifer Geis, Sung Kyun Kim, Cosmin Deciu, Mathias Ehrich
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Abstract
Technology provided herein relates in part to methods, processes and apparatuses for non-invasive assessment of genetic variations. In particular the invention relates to methods and kits for detecting aneuploidy of a fetal chromosome by determining the amounts of differentially methylated regions in each of chromosomes 13, 18 and 21 in circulating cell-free nucleic acid from a human pregnant female.
Claims15
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 on each of chromosomes 13, 18 and 21;
- (b) the steps of:
- (i) contacting the nucleic acid of step (a) with a collection 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 a target polynucleotide from one of chromosomes 13, 18 or 21, thereby generating amplicons from the undigested polynucleotide targets, wherein the target polynucleotides are in:
- chromosome 13 polynucleotides of SEQ ID NOs:
- 209 and 214, or complements thereof;
- chromosome 18 polynucleotides of SEQ ID NOs:
- 232 and 222, or complements thereof;
- and
- chromosome 21 polynucleotides of SEQ ID NOs:
- 256 and 253, or complements thereof;
- 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;
- and
- (c) determining amounts 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 the target amplicons from step (c), whether there is aneuploidy for any one of chromosomes 13, 18, or 21, or portion thereof, in the fetus.
12 more elements
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 on each of chromosomes 13, 18 and 21;
- (b) contacting the nucleic acid of step (a) with a collection 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 a target polynucleotide from one of chromosomes 13, 18 or 21, thereby generating amplicons from the undigested polynucleotide targets, wherein the target polynucleotides are in:
- chromosome 13 polynucleotides of SEQ ID NOs:
- 209 and 214, or complements thereof;
- chromosome 18 polynucleotides of SEQ ID NOs:
- 232 and 222, or complements thereof;
- and
- chromosome 21 polynucleotides of SEQ ID NOs:
- 256 and 253, or complements thereof;
- (c) determining amounts of the chromosome 13, chromosome 18 and chromosome 21 amplicons in (b);
- and
- (d) determining, based on the relative amounts of the target amplicons from step (c), whether there is aneuploidy for any one of chromosomes 13, 18, or 21, or portion thereof, in the fetus, wherein step (b) further comprises contacting the sample with at least one competitor oligonucleotide under the amplification conditions such that a competitor specific amplicon comprising the competitor oligonucleotide is generated, and wherein the competitor oligonucleotide comprises a sequence substantially identical to a first target sequence but includes at least one nucleotide substitution, and wherein the competitor oligonucleotides comprises at least one polynucleotide of SEQ ID NOs:
- 273, 278, 286, 296, 317, 320, or a complement thereof, and step (c) further comprises comparing a ratio of the first target specific amplicon to the at least one competitor specific amplicon.
10 more elements
Dependencies are inferred from the claim text.