Non-invasive fetal sex determination
First Claim
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1. A method for measuring a risk for Y chromosomal frequency abnormalities in a fetus comprising the steps of:
- (a) annealing sets of two fixed sequence oligonucleotides specific to selected non-polymorphic nucleic acid regions on the Y chromosome and to polymorphic and non-polymorphic selected nucleic acid regions on at least one non-Y chromosome in maternal samples comprising maternal and fetal nucleic acids, wherein a portion of each of the two fixed sequence oligonucleotides is complementary to one of the selected non-polymorphic or polymorphic nucleic acid regions, at least one of the two fixed sequence oligonucleotides specific to the non-polymorphic nucleic acid regions comprises a locus index, at least one of the two fixed sequence oligonucleotides specific to the polymorphic nucleic acid regions comprises an allele index, at least one of the two fixed sequence oligonucleotides per set comprises an amplification universal primer sequence, and wherein the oligonucleotides specific to the polymorphic selected nucleic acid regions on the at least one non-Y chromosome in the maternal and fetal nucleic acids are specific for different alleles in the selected polymorphic nucleic acid region;
(b) annealing bridging oligonucleotides to the selected non-polymorphic and polymorphic nucleic acid regions, wherein the bridging oligonucleotides hybridize between the two fixed sequence oligonucleotides of each set;
(c) ligating the fixed sequence oligonucleotides and bridging oligonucleotides;
(d) selectively amplifying the selected nucleic acid regions from the Y chromosome and the at least one non-Y chromosome to generate amplified selected nucleic acid regions using the universal primer sequence, wherein distinct labels are incorporated into the amplification products, wherein at least eight selected nucleic acid regions from the Y chromosome and the at least one non-Y chromosome are amplified;
(e) hybridizing the amplification products to an array;
(f) measuring a percent of fetal nucleic acids in the maternal samples by quantifying the selected polymorphic nucleic acid regions from the at least one non-Y chromosome by imaging the array and quantifying the labels specific for the different alleles in each selected polymorphic nucleic acid regions, where the maternal nucleic acids are homozygous for one allele and the fetal nucleic acids are heterozygous; and
(g) measuring a probability that the fetus is a normal male fetus and the risk for Y chromosomal frequency abnormalities by (i) quantifying the selected non-polymorphic nucleic acid regions from the Y chromosome by imaging the array and quantifying the labels and (ii) quantifying the selected non-polymorphic nucleic acid regions from the at least one non-Y chromosome by imaging the array and quantifying the labels, wherein the fetus is a normal male fetus if the non-polymorphic nucleic acid regions on the Y chromosome contribute no more than 1/46th of the percent of fetal nucleic acids in the maternal samples and the fetus is at risk for Y chromosomal frequency abnormalities if the non-polymorphic nucleic acid regions on the Y chromosome contribute 1/23rd or more of the percent of fetal nucleic acids in the maternal samples.
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Abstract
The present invention provides methods for non-invasive determination of sex in a fetus or of Y chromosomal frequency abnormalities—indicative of aneuploidy or sex mosaicisms in a fetus—by detecting and determining the relative contribution genetic sequences from the Y chromosome in view of the percent fetal contribution in a maternal mixed sample.
206 Citations
25 Claims
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1. A method for measuring a risk for Y chromosomal frequency abnormalities in a fetus comprising the steps of:
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(a) annealing sets of two fixed sequence oligonucleotides specific to selected non-polymorphic nucleic acid regions on the Y chromosome and to polymorphic and non-polymorphic selected nucleic acid regions on at least one non-Y chromosome in maternal samples comprising maternal and fetal nucleic acids, wherein a portion of each of the two fixed sequence oligonucleotides is complementary to one of the selected non-polymorphic or polymorphic nucleic acid regions, at least one of the two fixed sequence oligonucleotides specific to the non-polymorphic nucleic acid regions comprises a locus index, at least one of the two fixed sequence oligonucleotides specific to the polymorphic nucleic acid regions comprises an allele index, at least one of the two fixed sequence oligonucleotides per set comprises an amplification universal primer sequence, and wherein the oligonucleotides specific to the polymorphic selected nucleic acid regions on the at least one non-Y chromosome in the maternal and fetal nucleic acids are specific for different alleles in the selected polymorphic nucleic acid region; (b) annealing bridging oligonucleotides to the selected non-polymorphic and polymorphic nucleic acid regions, wherein the bridging oligonucleotides hybridize between the two fixed sequence oligonucleotides of each set; (c) ligating the fixed sequence oligonucleotides and bridging oligonucleotides; (d) selectively amplifying the selected nucleic acid regions from the Y chromosome and the at least one non-Y chromosome to generate amplified selected nucleic acid regions using the universal primer sequence, wherein distinct labels are incorporated into the amplification products, wherein at least eight selected nucleic acid regions from the Y chromosome and the at least one non-Y chromosome are amplified; (e) hybridizing the amplification products to an array; (f) measuring a percent of fetal nucleic acids in the maternal samples by quantifying the selected polymorphic nucleic acid regions from the at least one non-Y chromosome by imaging the array and quantifying the labels specific for the different alleles in each selected polymorphic nucleic acid regions, where the maternal nucleic acids are homozygous for one allele and the fetal nucleic acids are heterozygous; and (g) measuring a probability that the fetus is a normal male fetus and the risk for Y chromosomal frequency abnormalities by (i) quantifying the selected non-polymorphic nucleic acid regions from the Y chromosome by imaging the array and quantifying the labels and (ii) quantifying the selected non-polymorphic nucleic acid regions from the at least one non-Y chromosome by imaging the array and quantifying the labels, wherein the fetus is a normal male fetus if the non-polymorphic nucleic acid regions on the Y chromosome contribute no more than 1/46th of the percent of fetal nucleic acids in the maternal samples and the fetus is at risk for Y chromosomal frequency abnormalities if the non-polymorphic nucleic acid regions on the Y chromosome contribute 1/23rd or more of the percent of fetal nucleic acids in the maternal samples. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 25)
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17. A method for measuring the risk of Y chromosomal frequency abnormalities in a fetus comprising the steps of:
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(a) obtaining at least five maternal samples comprising maternal and fetal nucleic acids; (b) placing each of the maternal samples in a separate reaction vessel; (c) annealing sets of two fixed sequence oligonucleotides specific to selected nucleic acid regions on the Y chromosome and to polymorphic and non-polymorphic selected nucleic acid regions on at least two non-Y chromosomes to the maternal and fetal nucleic acids, wherein at least one of the fixed sequence oligonucleotides comprises a sample index; (d) selectively amplifying the selected nucleic acid regions from the Y chromosome and the at least two non-Y chromosomes to generate amplified selected nucleic acid regions; (e) pooling the at least five maternal samples into a single reaction vessel; (f)sequencing the amplified selected nucleic acid regions; (g) using software executed on a computer, quantifying the sequenced nucleic acid regions; (h) using software executed on a computer, determining a frequency of the quantified selected nucleic acid regions from the Y chromosome and the at least two non-Y chromosomes; (i)(using software executed on a computer, measuring a percent of fetal nucleic acids in the maternal sample by measuring the frequency of the quantified polymorphic selected nucleic acid regions from the at least two non-Y chromosomes; and (j) using software executed on a computer, measuring the probability that the fetus is a normal male fetus and the risk for Y chromosomal frequency abnormalities in the fetus by (i) quantifying the frequency of the selected nucleic acid regions from the Y chromosome; and
(ii) quantifying the non-polymorphic selected nucleic acid regions from the at least two non-Y chromosomes, wherein the fetus is a normal male fetus if the nucleic acid regions on the Y chromosome contribute no more than 1/46th of the percent of fetal nucleic acids in the maternal sample and the fetus is at risk for Y chromosomal frequency abnormalities if the nucleic acid regions on the Y chromosome contribute 1/23rd or more of the percent fetal nucleic acids in the maternal sample. - View Dependent Claims (18, 19, 20, 21, 22, 23, 24)
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Specification