US20120302741A1 - Non-invasive detection of fetal genetic traits - Google Patents
Non-invasive detection of fetal genetic traits Download PDFInfo
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- US20120302741A1 US20120302741A1 US13/557,025 US201213557025A US2012302741A1 US 20120302741 A1 US20120302741 A1 US 20120302741A1 US 201213557025 A US201213557025 A US 201213557025A US 2012302741 A1 US2012302741 A1 US 2012302741A1
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- fetal genetic loci e.g. chromosomal aberrations such as aneuploidies or chromosomal aberrations associated with Down's syndrome, or hereditary Mendelian genetic disorders and, respectively, genetic markers associated therewith, such as single gene disorders, e.g. cystic fibrosis or the hemoglobinopathies
- the reason for this difficulty is that the major proportion (generally >90%) of the extracellular DNA in the maternal circulation is derived from the mother.
- This vast bulk of maternal circulatory extracellular DNA renders it difficult, if not impossible, to determine fetal genetic alternations such as those involved in chromosomal aberrations (e.g. aneuploidies) or hereditary Mendelian genetic disorders (e.g. cystic fibrosis or the hemoglobinopathies) from the small amount of circulatory extracellular fetal DNA.
- This selective enrichment which is based on size discrimination of circulatory DNA fragments of approximately 500 base pairs or less, leads to a fraction which is largely constituted by fetal extracellular DNA.
- Size separation of extracellular fetal DNA in the maternal circulation thus facilitates the non-invasive detection of fetal genetic traits, including paternally inherited polymorphisms which permit paternity testing.
- the present invention provides: a fraction of a sample of the blood plasma or serum (which preferably is substantially cell-free) of a pregnant woman in which, as the result of said sample having been submitted to a size separation, the extracellular DNA present therein substantially consists of DNA comprising 500 base pairs or less; the use of such sample-fraction for the non-invasive detection of fetal genetic traits; and a process for performing non-invasive detection of fetal genetic traits which comprises subjecting a sample of the blood plasma or serum of a pregnant woman to a size separation so as to obtain a fraction of said sample in which the extracellular DNA present therein substantially consists of DNA comprising 500 base pairs or less, and determining in said sample-fraction the fetal genetic trait(s) to be detected.
- Said serum or plasma sample is preferably substantially cell-free, and this can be achieved by known methods such as, for example, centrifugation or sterile filtration.
- the size separation of the extracellular DNA in said serum or plasma sample can be brought about by a variety of methods, including but not limited to: chromatography or electrophoresis such as chromatography on agarose or polyacrylamide gels, ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC, see Hecker K H, Green S M, Kobayashi K, J. Biochem. Biophys. Methods 2000 Nov.
- chromatography or electrophoresis such as chromatography on agarose or polyacrylamide gels
- IP RP HPLC ion-pair reversed-phase high performance liquid chromatography
- the sample-fraction thus obtained not only permits the subsequent determination of fetal genetic traits which had already been easily detectable in a conventional manner such as the fetal RhD gene in pregnancies at risk for HDN (hemolytic disease of the fetus and the newborn), or fetal Y chromosome-specific sequences in pregnancies at risk for an X chromosome-linked disorder such as hemophilia, fragile X syndrome or the like, but also the determination of other, more complex fetal genetic loci, including but not limited to: chromosomal aberrations (e.g aneuploidies or Down's syndrome) or hereditary Mendelian genetic disorders and, respectively, genetic markers associated therewith (e.g. single gene disorders such as cystic fibrosis or the hemoglobinopathies); and fetal genetic traits which may be decisive when paternity is to be determined.
- chromosomal aberrations e.g aneuploidies or Down's syndrome
- hereditary Mendelian genetic disorders
- Such determination of fetal genetic traits can be effected by methods such as, for example, PCR (polymerase chain reaction) technology, ligase chain reaction, probe hybridization techniques, nucleic acid arrays (so-called “DNA chips”) and the like.
- DNA from 5-7 ml plasma was extracted using the QIAgen Maxi kit, according to the manufacturers' protocol. DNA was eluted in a volume of 1.5 ml.
- the DNA was recovered by centrifugation at 20000 g for 30 minutes at 4° C.
- the pellet was air dried and dissolved in 35 ⁇ l distilled water.
- a 1% agarose Gel (Invitrogen, Cat No: 15510-027) was prepared for DNA electrophoresis.
- the Gel was cut into pieces corresponding to specific DNA sizes according to the DNA size markers (New England Biolabs, 100 bp ladder and Lamda Hind III digest).
- the DNA sizes contained by the specific gel fragments were: 90-300 bases, 300-500 bases, 500-1000 bases, 1.0-1.5 kilobases (“kb”), 1.5-23 kb and >23 kb.
- the DNA was purified from the agarose gel pieces using the QIAEX II Gel Extraction kit (Qiagen, Cat No. 20021) and eluted in 35 ⁇ l Tris-HCl (pH 8.0, 10 mM).
- Sequences from the Y chromosome (SRY) and from chromosome 12 (GAPDH gene) were amplified with the Applied Biosystems (ABI) 7000 Sequence Detection System by real-time quantitative PCR to quantify amounts of fetal and total DNA in the size-separated fractions.
- the TaqMan system for SRY consisted of the amplification primers SRY_Fwd: TCC TCA AAA GAA ACC GTG CAT (SEQ ID NO: 1) and SRY Rev: AGA TTA ATG GTT GCT AAG GAC TGG AT (SEQ ID NO: 2) and a FAM labeled TaqMan MGB (Minor Groove Binder) probe SRY_MGB: TCC CCA CAA CCT CTT (SEQ ID NO: 3).
- the TaqMan System for the GAPDH gene consisted of the following primers and probe: GAPDH_Fwd: CCC CAC ACA CAT GCA CTT ACC (SEQ ID NO: 4), GAPDH_Rev: CCT AGT CCC AGG GCT TTG ATT (SEQ ID NO: 5) and GAPDH_MGB: TAG GAA GGA CAG GCA AC (SEQ ID NO: 6).
- TaqMan amplification reactions were set up in a total reaction volume of 25 ⁇ l, containing 6 ⁇ l of the sample DNA solution, 300 nM of each primer (HPLC purified, Mycrosynth, Switzerland) and 200 nM of each probe (ABI) at 1 ⁇ concentration of the Universal PCR reaction mix (ABI). Each sample was analyzed in duplicate for each of the two amplification systems. A standard curve containing known amounts of genomic DNA was run in parallel with each analysis.
- Thermal cycling was performed according to the following protocol: an initial incubation at 50° C. for 2 minutes to permit Amp Erase activity, 10 minutes at 95° C. for activation of AmpliTaq Gold, and 40 cycles of 1 minute at 60° C. and 15 seconds at 95° C.
- Amplification data collected by the 7000 Sequence Detection System was quantified using the slope of the standard curve as calculated by the sequence detection software and the results of a standard DNA solution used in the dilution curve with similar DNA copy numbers as the sample reactions as a reference sample for copy number calculations.
- Table 1 shows that in the five pregnancies examined, DNA fragments originating from the fetus were almost completely of sizes smaller than 500 base pairs with around 70% being of fetal origin for sizes smaller than 300 bases.
- the DNA size chosen for the enrichment of fetal DNA will be smaller than 300 or smaller than 500 bases.
- DNA from the plasma was extracted using a modification of the High Pure DNA template kit from Roche, the whole sample was passed through the filter usually used for 200 ⁇ l using a vacuum. The DNA was eluted in a volume of 50 ⁇ l elution buffer. Paternal DNA was extracted from 400 ⁇ l paternal whole blood, using the High Pure DNA template kit, and eluted into 100 ⁇ l. Maternal DNA was isolated from the buffy coat, using the High Pure DNA template kit, and eluted into 100 ⁇ l.
- the DNA was size-separated by electrophoresis on an agarose gel and purified as described in Example 1.
- sequences from tetranucleotide repeat markers on Chromosome 21 were amplified in a multiplex PCR reaction as described in Li et al. Clinical Chemistry 49, No. 4, 2003. Because of the low concentration of plasma DNA, the fetal DNA in maternal plasma was examined by using a semi-nested PCR protocol.
- the maternal and paternal pairs were genotyped using total genomic DNA to monitor microsatellite markers on chromosome 21.
- the STR markers used were:
- the resulting DNA fragments were then size separated by capillary electrophoresis on a sequencer, and the peak areas representing each allele for a specific marker were measured by the software.
- Analysis of the STR fragments can allow for the detection of paternal alleles that are distinct in length from the maternal repeat sequences, and by calculating the ratios between the peak areas it can be possible to identify patterns that are not consistent with a normal fetal karyotype.
- the identification of paternal allele sizes of STRs in the maternal circulation can allow the detection of certain chromosomal aberrations non-invasively. Also paternity testing can be accomplished prenatal in a non-invasive manner.
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Abstract
Description
- This patent application is a continuation of U.S. Patent Application No. 10/964,726 filed on Oct. 15, 2004, entitled NON-INVASIVE DETECTION OF FETAL GENETIC TRAITS, naming Sinuhe Hahn, Wolfgang Holzgreve, Bernhard Zimmermann, and Ying Li as inventors, and designated by Attorney Docket No. SEQ-5002-UT, which claims the benefit under 35 U.S.C. 119(a) of European Patent Application No. 03405742.2 filed on Oct. 16, 2003, entitled NON-INVASIVE DETECTION OF FETAL GENETIC TRAITS, naming Sinuhe Hahn, Wolfgang Holzgreve, Bernhard Zimmermann, and Ying Li as inventors and designated by Attorney Docket No. SEQ-5002-EP. The entirety of each of these patent applications is incorporated herein by reference.
- The presence of circulatory extracellular DNA in the peripheral blood is a well established phenomenon. In this context, it has been shown that in the case of a pregnant woman extracellular fetal DNA is present in the maternal circulation and can be detected in maternal plasma or serum. Studies have shown that this circulatory fetal genetic material can be used for the very reliable determination, e.g. by PCR (polymerase chain reaction) technology, of fetal genetic loci which are completely absent from the maternal genome. Examples of such fetal genetic loci are the fetal RhD gene in pregnancies at risk for HDN (hemolytic disease of the fetus and newborn) or fetal Y chromosome-specific sequences in pregnancies at risk for an X chromosome-linked disorder e.g. hemophilia or fragile X syndrome.
- The determination of other, more complex fetal genetic loci (e.g. chromosomal aberrations such as aneuploidies or chromosomal aberrations associated with Down's syndrome, or hereditary Mendelian genetic disorders and, respectively, genetic markers associated therewith, such as single gene disorders, e.g. cystic fibrosis or the hemoglobinopathies) is, however, more problematic. The reason for this difficulty is that the major proportion (generally >90%) of the extracellular DNA in the maternal circulation is derived from the mother. This vast bulk of maternal circulatory extracellular DNA renders it difficult, if not impossible, to determine fetal genetic alternations such as those involved in chromosomal aberrations (e.g. aneuploidies) or hereditary Mendelian genetic disorders (e.g. cystic fibrosis or the hemoglobinopathies) from the small amount of circulatory extracellular fetal DNA.
- An examination of circulatory extracellular fetal DNA and circulatory extracellular maternal DNA in maternal plasma has now shown that, surprisingly, the majority of the circulatory extracellular fetal DNA has a relatively small size of approximately 500 base pairs or less, whereas the majority of circulatory extracellular maternal DNA in maternal plasma has a size greater than approximately 500 base pairs. Indeed, in certain instances the circulatory DNA material which is smaller than approximately 500 base pairs appears to be almost entirely fetal. Circulatory extracellular fetal DNA in the maternal circulation has thus been found to be smaller in size (approximately 500 base pairs or less) than circulatory extracellular maternal DNA (greater than approximately 500 base pairs).
- This surprising finding forms the basis of the present invention according to which separation of circulatory extracellular DNA fragments which are smaller than approximately 500 base pairs provides a possibility to enrich for fetal DNA sequences from the vast bulk of circulatory extracellular maternal DNA.
- This selective enrichment, which is based on size discrimination of circulatory DNA fragments of approximately 500 base pairs or less, leads to a fraction which is largely constituted by fetal extracellular DNA. This permits the analysis of fetal genetic traits including those involved in chromosomal aberrations (e.g. aneuploidies or chromosomal aberrations associated with Down's syndrome) or hereditary Mendelian genetic disorders and, respectively, genetic markers associated therewith (e.g. single gene disorders such as cystic fibrosis or the hemoglobinopathies), the determination of which had, as mentioned above, so far proved difficult, if not impossible. Size separation of extracellular fetal DNA in the maternal circulation thus facilitates the non-invasive detection of fetal genetic traits, including paternally inherited polymorphisms which permit paternity testing.
- Clinical Chemistry, 1999, Vol. 45(9), pages 1570-1572 and The Australian & New Zealand
- Journal of Obstetrics & Gynaecology, February 2003 (O.sub.2-2003), Vol. 43(1), pages 10-15 describe a sample of blood plasma of a pregnant woman in which extracellular fetal DNA of less than 500 base pairs is enriched by PCR, is separated by gel electrophoresis and fetal male DNA (fetal Y-chromosome-specific sequence) is detected.
- The present invention provides: a fraction of a sample of the blood plasma or serum (which preferably is substantially cell-free) of a pregnant woman in which, as the result of said sample having been submitted to a size separation, the extracellular DNA present therein substantially consists of DNA comprising 500 base pairs or less; the use of such sample-fraction for the non-invasive detection of fetal genetic traits; and a process for performing non-invasive detection of fetal genetic traits which comprises subjecting a sample of the blood plasma or serum of a pregnant woman to a size separation so as to obtain a fraction of said sample in which the extracellular DNA present therein substantially consists of DNA comprising 500 base pairs or less, and determining in said sample-fraction the fetal genetic trait(s) to be detected.
- Said serum or plasma sample is preferably substantially cell-free, and this can be achieved by known methods such as, for example, centrifugation or sterile filtration.
- The size separation of the extracellular DNA in said serum or plasma sample can be brought about by a variety of methods, including but not limited to: chromatography or electrophoresis such as chromatography on agarose or polyacrylamide gels, ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC, see Hecker K H, Green S M, Kobayashi K, J. Biochem. Biophys. Methods 2000 Nov. 20; 46(1-2): 83-93), capillary electrophoresis in a self-coating, low-viscosity polymer matrix (see Du M, Flanagan J H Jr, Lin B, Ma Y, Electrophoresis 2003 September; 24 (18): 3147-53), selective extraction in microfabricated electrophoresis devices (see Lin R, Burke D T, Burn M A, J. Chromatogr. A. 2003 Aug. 29; 1010(2): 255-68), microchip electrophoresis on reduced viscosity polymer matrices (see Xu F, Jabasini M, Liu S, Baba Y, Analyst. 2003 June; 128(6): 589-92), adsorptive membrane chromatography (see Teeters M A, Conrardy S E, Thomas B L, Root T W, Lightfoot E N, J. Chromatogr. A. 2003 Mar. 7; 989(1): 165-73) and the like; density gradient centrifugation (see Raptis L, Menard H A, J. Clin. Invest. 1980 December; 66(6): 1391-9); and methods utilising nanotechnological means such as microfabricated entropic trap arrays (see Han J, Craighead H G, Analytical Chemistry, Vol. 74, No. 2, Jan. 15, 2002) and the like.
- The sample-fraction thus obtained not only permits the subsequent determination of fetal genetic traits which had already been easily detectable in a conventional manner such as the fetal RhD gene in pregnancies at risk for HDN (hemolytic disease of the fetus and the newborn), or fetal Y chromosome-specific sequences in pregnancies at risk for an X chromosome-linked disorder such as hemophilia, fragile X syndrome or the like, but also the determination of other, more complex fetal genetic loci, including but not limited to: chromosomal aberrations (e.g aneuploidies or Down's syndrome) or hereditary Mendelian genetic disorders and, respectively, genetic markers associated therewith (e.g. single gene disorders such as cystic fibrosis or the hemoglobinopathies); and fetal genetic traits which may be decisive when paternity is to be determined.
- Such determination of fetal genetic traits can be effected by methods such as, for example, PCR (polymerase chain reaction) technology, ligase chain reaction, probe hybridization techniques, nucleic acid arrays (so-called “DNA chips”) and the like.
- The following Examples further illustrate the invention but are not to be construed as limitating its scope in any way.
- Subjects and Sample Processing
- Seven women pregnant in the third trimester with a male fetus were recruited for this study. 16-18 ml blood samples were collected into EDTA tubes. 6-9 ml of plasma were obtained after centrifugation at 1600 g for 10 minutes and a second centrifugation of the supernatant at 16000 g for 10 minutes.
- DNA Isolation
- DNA from 5-7 ml plasma was extracted using the QIAgen Maxi kit, according to the manufacturers' protocol. DNA was eluted in a volume of 1.5 ml.
- DNA Precipitation
- 1. To the plasma DNA were added: 1/10 volume NaAc (3M, pH 5.2), 2 volumes absolute ethanol, MgCl2 to a final concentration of 0.01 M and Glycogen to a final concentration of 50 μg/ml. The solution was thoroughly mixed by vortexing.
- 2. The solution was stored overnight at −70° C.
- 3. The DNA was recovered by centrifugation at 20000 g for 30 minutes at 4° C.
- 4. The supernatant was carefully removed and the pellet washed with 500 μl 70% ethanol.
- 5. The pellet was air dried and dissolved in 35 μl distilled water.
- DNA Separation
- 1. A 1% agarose Gel (Invitrogen, Cat No: 15510-027) was prepared for DNA electrophoresis.
- 2. 28 μl DNA solution were loaded on the gel.
- 3. The gel was electrophoresed at 80 Volt for 1 hour.
- 4. The Gel was cut into pieces corresponding to specific DNA sizes according to the DNA size markers (New England Biolabs, 100 bp ladder and Lamda Hind III digest). The DNA sizes contained by the specific gel fragments were: 90-300 bases, 300-500 bases, 500-1000 bases, 1.0-1.5 kilobases (“kb”), 1.5-23 kb and >23 kb.
- 5. The DNA was purified from the agarose gel pieces using the QIAEX II Gel Extraction kit (Qiagen, Cat No. 20021) and eluted in 35 μl Tris-HCl (pH 8.0, 10 mM).
- Real-Time PCR
- Sequences from the Y chromosome (SRY) and from chromosome 12 (GAPDH gene) were amplified with the Applied Biosystems (ABI) 7000 Sequence Detection System by real-time quantitative PCR to quantify amounts of fetal and total DNA in the size-separated fractions. The TaqMan system for SRY consisted of the amplification primers SRY_Fwd: TCC TCA AAA GAA ACC GTG CAT (SEQ ID NO: 1) and SRY Rev: AGA TTA ATG GTT GCT AAG GAC TGG AT (SEQ ID NO: 2) and a FAM labeled TaqMan MGB (Minor Groove Binder) probe SRY_MGB: TCC CCA CAA CCT CTT (SEQ ID NO: 3). The TaqMan System for the GAPDH gene consisted of the following primers and probe: GAPDH_Fwd: CCC CAC ACA CAT GCA CTT ACC (SEQ ID NO: 4), GAPDH_Rev: CCT AGT CCC AGG GCT TTG ATT (SEQ ID NO: 5) and GAPDH_MGB: TAG GAA GGA CAG GCA AC (SEQ ID NO: 6).
- TaqMan amplification reactions were set up in a total reaction volume of 25 μl, containing 6 μl of the sample DNA solution, 300 nM of each primer (HPLC purified, Mycrosynth, Switzerland) and 200 nM of each probe (ABI) at 1× concentration of the Universal PCR reaction mix (ABI). Each sample was analyzed in duplicate for each of the two amplification systems. A standard curve containing known amounts of genomic DNA was run in parallel with each analysis.
- Thermal cycling was performed according to the following protocol: an initial incubation at 50° C. for 2 minutes to permit Amp Erase activity, 10 minutes at 95° C. for activation of AmpliTaq Gold, and 40 cycles of 1 minute at 60° C. and 15 seconds at 95° C. Amplification data collected by the 7000 Sequence Detection System was quantified using the slope of the standard curve as calculated by the sequence detection software and the results of a standard DNA solution used in the dilution curve with similar DNA copy numbers as the sample reactions as a reference sample for copy number calculations.
- Table 1 shows that in the five pregnancies examined, DNA fragments originating from the fetus were almost completely of sizes smaller than 500 base pairs with around 70% being of fetal origin for sizes smaller than 300 bases.
- These results demonstrate that free DNA of fetal origin circulating in the maternal circulation can be specifically enriched by size separation of the total free DNA in the maternal blood.
- Depending on the downstream application the DNA size chosen for the enrichment of fetal DNA will be smaller than 300 or smaller than 500 bases.
-
TABLE 1 % of fetal DNA % of maternal DNA Size of DNA in each fragment in each fragment <0.3 kb 73.2 (22.22-87.06) 26.8 (12.94-77.78) 0.3-0.5 kb 18.95 (6.43-31.42) 81.05 (68.58-93.57) 0.5-1 kb 2.81 (0.00-7.75) 97.19 (92.25-100) 1.0-1.5 kB 0.00 (0.00-12.50) 100 (87.5-100) 1.5-23 kb 0.00 (0.00-8.40) 100 (100-100) - The abbreviation “kb” appearing in the first column of this table stands for 1000 base pairs, and the figures given in its second and the third column are the median values of the percentages and, in brackets, the ranges.
- Subjects and Samples
- 18 ml blood samples from pregnant women and 9 ml blood from their partners were collected into EDTA tubes and plasma separated by centrifugation as described in Example 1. The maternal buffy coat (i.e. the white colored top layer of the cell pellet obtained after the first centrifugation of 1600 g for 10 min.) was washed twice with PBS.
- DNA Isolation
- DNA from the plasma was extracted using a modification of the High Pure DNA template kit from Roche, the whole sample was passed through the filter usually used for 200 μl using a vacuum. The DNA was eluted in a volume of 50 μl elution buffer. Paternal DNA was extracted from 400 μl paternal whole blood, using the High Pure DNA template kit, and eluted into 100 μl. Maternal DNA was isolated from the buffy coat, using the High Pure DNA template kit, and eluted into 100 μl.
- DNA Separation
- The DNA was size-separated by electrophoresis on an agarose gel and purified as described in Example 1.
- PCR Specific for Short Tandem Repeats
- From the fraction of sizes smaller than 500 bases, sequences from tetranucleotide repeat markers on Chromosome 21 were amplified in a multiplex PCR reaction as described in Li et al. Clinical Chemistry 49, No. 4, 2003. Because of the low concentration of plasma DNA, the fetal DNA in maternal plasma was examined by using a semi-nested PCR protocol.
- The maternal and paternal pairs were genotyped using total genomic DNA to monitor microsatellite markers on chromosome 21.
- The STR markers used were:
-
- D211 S11;
- D21S1270;
- D21S1432; and
- D21S1435
- The resulting DNA fragments were then size separated by capillary electrophoresis on a sequencer, and the peak areas representing each allele for a specific marker were measured by the software.
-
-
TABLE 2 Detection of fetal alleles specific for the microsatellite marker (Short Tandem Repeat) D21S11 on chromosome 21 Maternal Fetal alleles alleles detected detected (D21S11) (D21S11) Maternal genomic 232 bp N/A DNA 234 bp Total extracellulear 232 bp No fetal DNA (unseparated) 234 bp alleles detectable Size-separated 232 bp 228 bp extracellular DNA 234 bp 232 bp (<300 bp) Size-separated 232 bp 228 bp extracellular DNA 234 bp 232 bp (300-500 bp) - Only in the size-separated fractions (<300 by and 300-500 bp) could the fetal alleles for D21S11 be detected, namely the paternally inherited 228 by allele and the maternally inherited 232 by allele, i.e., one allele from each parent.
- Discussion
- Analysis of the STR fragments can allow for the detection of paternal alleles that are distinct in length from the maternal repeat sequences, and by calculating the ratios between the peak areas it can be possible to identify patterns that are not consistent with a normal fetal karyotype. The identification of paternal allele sizes of STRs in the maternal circulation can allow the detection of certain chromosomal aberrations non-invasively. Also paternity testing can be accomplished prenatal in a non-invasive manner.
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US13/557,025 US20120302741A1 (en) | 2003-10-16 | 2012-07-24 | Non-invasive detection of fetal genetic traits |
US13/779,300 US20130190483A1 (en) | 2003-10-16 | 2013-02-27 | Non-invasive detection of fetal genetic traits |
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EP03405742.2A EP1524321B2 (en) | 2003-10-16 | 2003-10-16 | Non-invasive detection of fetal genetic traits |
EP03405742.2 | 2003-10-16 | ||
US10/964,726 US20050164241A1 (en) | 2003-10-16 | 2004-10-15 | Non-invasive detection of fetal genetic traits |
US13/557,025 US20120302741A1 (en) | 2003-10-16 | 2012-07-24 | Non-invasive detection of fetal genetic traits |
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US11/855,558 Active 2026-03-09 US7838647B2 (en) | 2003-10-16 | 2007-09-14 | Non-invasive detection of fetal genetic traits |
US13/029,995 Active US9580751B2 (en) | 2003-10-16 | 2011-02-17 | Non-invasive detection of fetal genetic traits |
US13/029,999 Abandoned US20110245482A1 (en) | 2003-10-16 | 2011-02-17 | Non-invasive detection of fetal genetic traits |
US13/557,025 Abandoned US20120302741A1 (en) | 2003-10-16 | 2012-07-24 | Non-invasive detection of fetal genetic traits |
US13/757,637 Active 2026-02-22 US9738931B2 (en) | 2003-10-16 | 2013-02-01 | Non-invasive detection of fetal genetic traits |
US13/779,300 Abandoned US20130190483A1 (en) | 2003-10-16 | 2013-02-27 | Non-invasive detection of fetal genetic traits |
US15/653,401 Abandoned US20170321279A1 (en) | 2003-10-16 | 2017-07-18 | Non-invasive detection of fetal genetic traits |
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US13/029,995 Active US9580751B2 (en) | 2003-10-16 | 2011-02-17 | Non-invasive detection of fetal genetic traits |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20130190483A1 (en) * | 2003-10-16 | 2013-07-25 | Sequenom, Inc. | Non-invasive detection of fetal genetic traits |
WO2019243328A1 (en) | 2018-06-18 | 2019-12-26 | Baxalta Incorporated | Bottom section for being connected to an assembly with plate settler, and assembly with plate settler |
WO2021116273A2 (en) | 2019-12-12 | 2021-06-17 | Baxalta Incorporated | Method for continuous protein recovering |
Families Citing this family (167)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8529744B2 (en) | 2004-02-02 | 2013-09-10 | Boreal Genomics Corp. | Enrichment of nucleic acid targets |
US10337054B2 (en) | 2004-02-02 | 2019-07-02 | Quantum-Si Incorporated | Enrichment of nucleic acid targets |
JP2007526823A (en) | 2004-02-02 | 2007-09-20 | ザ ユニバーシティ オブ ブリティッシュ コロンビア | Skodaphoresis and method and apparatus for moving and concentrating particles |
US20100216153A1 (en) | 2004-02-27 | 2010-08-26 | Helicos Biosciences Corporation | Methods for detecting fetal nucleic acids and diagnosing fetal abnormalities |
US8024128B2 (en) * | 2004-09-07 | 2011-09-20 | Gene Security Network, Inc. | System and method for improving clinical decisions by aggregating, validating and analysing genetic and phenotypic data |
FR2880897B1 (en) * | 2005-01-18 | 2010-12-17 | Inst Nat Sante Rech Med | METHOD OF DETECTION, NON-INVASIVE, PRENATAL, IN VITRO OF NORMAL HEALTHY CONDITION, HEALTHY CARRIER STATUS OR SICK CARRIER STATUS OF MUCOVISCIDOSIS |
US20090317798A1 (en) * | 2005-06-02 | 2009-12-24 | Heid Christian A | Analysis using microfluidic partitioning devices |
US8532930B2 (en) | 2005-11-26 | 2013-09-10 | Natera, Inc. | Method for determining the number of copies of a chromosome in the genome of a target individual using genetic data from genetically related individuals |
US10083273B2 (en) | 2005-07-29 | 2018-09-25 | Natera, Inc. | System and method for cleaning noisy genetic data and determining chromosome copy number |
US10081839B2 (en) | 2005-07-29 | 2018-09-25 | Natera, Inc | System and method for cleaning noisy genetic data and determining chromosome copy number |
US8515679B2 (en) | 2005-12-06 | 2013-08-20 | Natera, Inc. | System and method for cleaning noisy genetic data and determining chromosome copy number |
US20070027636A1 (en) * | 2005-07-29 | 2007-02-01 | Matthew Rabinowitz | System and method for using genetic, phentoypic and clinical data to make predictions for clinical or lifestyle decisions |
US11111544B2 (en) | 2005-07-29 | 2021-09-07 | Natera, Inc. | System and method for cleaning noisy genetic data and determining chromosome copy number |
US9424392B2 (en) | 2005-11-26 | 2016-08-23 | Natera, Inc. | System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals |
US20070178501A1 (en) * | 2005-12-06 | 2007-08-02 | Matthew Rabinowitz | System and method for integrating and validating genotypic, phenotypic and medical information into a database according to a standardized ontology |
US11111543B2 (en) | 2005-07-29 | 2021-09-07 | Natera, Inc. | System and method for cleaning noisy genetic data and determining chromosome copy number |
US20070122823A1 (en) * | 2005-09-01 | 2007-05-31 | Bianchi Diana W | Amniotic fluid cell-free fetal DNA fragment size pattern for prenatal diagnosis |
EP3599609A1 (en) * | 2005-11-26 | 2020-01-29 | Natera, Inc. | System and method for cleaning noisy genetic data and using data to make predictions |
EP3591068A1 (en) * | 2006-02-02 | 2020-01-08 | The Board of Trustees of the Leland Stanford Junior University | Non-invasive fetal genetic screening by digital analysis |
US20100184043A1 (en) * | 2006-02-28 | 2010-07-22 | University Of Louisville Research Foundation | Detecting Genetic Abnormalities |
US8609338B2 (en) * | 2006-02-28 | 2013-12-17 | University Of Louisville Research Foundation, Inc. | Detecting fetal chromosomal abnormalities using tandem single nucleotide polymorphisms |
AU2007220991C1 (en) * | 2006-02-28 | 2013-08-15 | University Of Louisville Research Foundation | Detecting fetal chromosomal abnormalities using tandem single nucleotide polymorphisms |
US20100184044A1 (en) | 2006-02-28 | 2010-07-22 | University Of Louisville Research Foundation | Detecting Genetic Abnormalities |
WO2007103910A2 (en) * | 2006-03-06 | 2007-09-13 | The Trustees Of Columbia University In The City Of New York | Specific amplification of fetal dna sequences from a mixed, fetal-maternal source |
US8679741B2 (en) * | 2006-05-31 | 2014-03-25 | Sequenom, Inc. | Methods and compositions for the extraction and amplification of nucleic acid from a sample |
US20080050739A1 (en) | 2006-06-14 | 2008-02-28 | Roland Stoughton | Diagnosis of fetal abnormalities using polymorphisms including short tandem repeats |
WO2007147074A2 (en) * | 2006-06-14 | 2007-12-21 | Living Microsystems, Inc. | Use of highly parallel snp genotyping for fetal diagnosis |
US8137912B2 (en) | 2006-06-14 | 2012-03-20 | The General Hospital Corporation | Methods for the diagnosis of fetal abnormalities |
US8372584B2 (en) * | 2006-06-14 | 2013-02-12 | The General Hospital Corporation | Rare cell analysis using sample splitting and DNA tags |
WO2007147063A2 (en) * | 2006-06-16 | 2007-12-21 | Sequenom, Inc. | Methods and compositions for the amplification, detection and quantification of nucleic acid from a sample |
WO2008070862A2 (en) * | 2006-12-07 | 2008-06-12 | Biocept, Inc. | Non-invasive prenatal genetic screen |
WO2008118988A1 (en) | 2007-03-26 | 2008-10-02 | Sequenom, Inc. | Restriction endonuclease enhanced polymorphic sequence detection |
US20100112590A1 (en) | 2007-07-23 | 2010-05-06 | The Chinese University Of Hong Kong | Diagnosing Fetal Chromosomal Aneuploidy Using Genomic Sequencing With Enrichment |
US20090029377A1 (en) | 2007-07-23 | 2009-01-29 | The Chinese University Of Hong Kong | Diagnosing fetal chromosomal aneuploidy using massively parallel genomic sequencing |
US20090053719A1 (en) | 2007-08-03 | 2009-02-26 | The Chinese University Of Hong Kong | Analysis of nucleic acids by digital pcr |
SG10201704689XA (en) | 2008-01-18 | 2017-07-28 | Harvard College | Methods of detecting signatures of disease or conditions in bodily fluids |
WO2009094772A1 (en) | 2008-02-01 | 2009-08-06 | The University Of British Columbia | Methods and apparatus for particle introduction and recovery |
WO2009103110A1 (en) * | 2008-02-18 | 2009-08-27 | Genetic Technologies Limited | Cell processing and/or enrichment methods |
US20110033862A1 (en) * | 2008-02-19 | 2011-02-10 | Gene Security Network, Inc. | Methods for cell genotyping |
AU2009223671B2 (en) * | 2008-03-11 | 2014-11-27 | Sequenom, Inc. | Nucleic acid-based tests for prenatal gender determination |
AU2009228312B2 (en) | 2008-03-26 | 2015-05-21 | Sequenom, Inc. | Restriction endonuclease enhanced polymorphic sequence detection |
US20110092763A1 (en) * | 2008-05-27 | 2011-04-21 | Gene Security Network, Inc. | Methods for Embryo Characterization and Comparison |
EP2128169A1 (en) * | 2008-05-30 | 2009-12-02 | Qiagen GmbH | Method for isolating short chain nucleic acids |
WO2010009440A2 (en) * | 2008-07-18 | 2010-01-21 | Biocept, Inc. | Non-invasive fetal rhd genotyping from maternal whole blood |
CA2731991C (en) | 2008-08-04 | 2021-06-08 | Gene Security Network, Inc. | Methods for allele calling and ploidy calling |
US8962247B2 (en) | 2008-09-16 | 2015-02-24 | Sequenom, Inc. | Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non invasive prenatal diagnoses |
US8476013B2 (en) | 2008-09-16 | 2013-07-02 | Sequenom, Inc. | Processes and compositions for methylation-based acid enrichment of fetal nucleic acid from a maternal sample useful for non-invasive prenatal diagnoses |
EP3103871B1 (en) | 2008-09-16 | 2020-07-29 | Sequenom, Inc. | Processes for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for fetal nucleic acid quantification |
CA3069081C (en) | 2008-09-20 | 2023-05-23 | The Board Of Trustees Of The Leland Stanford Junior University | Noninvasive diagnosis of fetal aneuploidy by sequencing |
EP3211095B1 (en) | 2009-04-03 | 2019-01-02 | Sequenom, Inc. | Nucleic acid preparation compositions and methods |
WO2010121381A1 (en) | 2009-04-21 | 2010-10-28 | The University Of British Columbia | System and methods for detection of particles |
SG175282A1 (en) | 2009-04-21 | 2011-11-28 | Genetic Technologies Ltd | Methods for obtaining fetal genetic material |
ES2640776T3 (en) | 2009-09-30 | 2017-11-06 | Natera, Inc. | Methods for non-invasively calling prenatal ploidy |
FI3783110T3 (en) | 2009-11-05 | 2023-03-02 | Fetal genomic analysis from a maternal biological sample | |
BR112012010708A2 (en) | 2009-11-06 | 2016-03-29 | Univ Hong Kong Chinese | method for performing prenatal diagnosis, and, computer program product |
EP3660165B1 (en) | 2009-12-22 | 2023-01-04 | Sequenom, Inc. | Processes and kits for identifying aneuploidy |
CA2786564A1 (en) | 2010-01-19 | 2011-07-28 | Verinata Health, Inc. | Identification of polymorphic sequences in mixtures of genomic dna by whole genome sequencing |
US20110312503A1 (en) | 2010-01-23 | 2011-12-22 | Artemis Health, Inc. | Methods of fetal abnormality detection |
US8774488B2 (en) | 2010-03-11 | 2014-07-08 | Cellscape Corporation | Method and device for identification of nucleated red blood cells from a maternal blood sample |
AU2011255641A1 (en) | 2010-05-18 | 2012-12-06 | Natera, Inc. | Methods for non-invasive prenatal ploidy calling |
US11408031B2 (en) | 2010-05-18 | 2022-08-09 | Natera, Inc. | Methods for non-invasive prenatal paternity testing |
US20190010543A1 (en) | 2010-05-18 | 2019-01-10 | Natera, Inc. | Methods for simultaneous amplification of target loci |
US11326208B2 (en) | 2010-05-18 | 2022-05-10 | Natera, Inc. | Methods for nested PCR amplification of cell-free DNA |
US11322224B2 (en) | 2010-05-18 | 2022-05-03 | Natera, Inc. | Methods for non-invasive prenatal ploidy calling |
US11939634B2 (en) | 2010-05-18 | 2024-03-26 | Natera, Inc. | Methods for simultaneous amplification of target loci |
US11339429B2 (en) | 2010-05-18 | 2022-05-24 | Natera, Inc. | Methods for non-invasive prenatal ploidy calling |
US9677118B2 (en) | 2014-04-21 | 2017-06-13 | Natera, Inc. | Methods for simultaneous amplification of target loci |
US10316362B2 (en) | 2010-05-18 | 2019-06-11 | Natera, Inc. | Methods for simultaneous amplification of target loci |
US11332793B2 (en) | 2010-05-18 | 2022-05-17 | Natera, Inc. | Methods for simultaneous amplification of target loci |
US11332785B2 (en) | 2010-05-18 | 2022-05-17 | Natera, Inc. | Methods for non-invasive prenatal ploidy calling |
SG185723A1 (en) | 2010-06-07 | 2013-01-30 | Esoterix Genetic Lab Llc | Enumeration of nucleic acids |
MX361944B (en) | 2010-07-23 | 2018-12-19 | President And Fellows Of Harvard College Star | Methods for detecting signatures of disease or conditions in bodily fluids. |
JP2013541323A (en) | 2010-07-23 | 2013-11-14 | プレジデント アンド フェロウズ オブ ハーバード カレッジ | Methods for detecting disease or symptom signatures using phagocytes |
US20130203624A1 (en) | 2010-07-23 | 2013-08-08 | President And Fellows Of Harvard College | Methods of Detecting Prenatal or Pregnancy-Related Diseases or Conditions |
AU2011280997A1 (en) | 2010-07-23 | 2013-02-28 | President And Fellows Of Harvard College | Methods of detecting autoimmune or immune-related diseases or conditions |
US20120034603A1 (en) * | 2010-08-06 | 2012-02-09 | Tandem Diagnostics, Inc. | Ligation-based detection of genetic variants |
AU2011293355A1 (en) * | 2010-08-24 | 2013-03-14 | Bio Dx, Inc. | Defining diagnostic and therapeutic targets of conserved free floating fetal DNA in maternal circulating blood |
ES2770342T3 (en) | 2010-12-22 | 2020-07-01 | Natera Inc | Noninvasive Prenatal Paternity Testing Procedures |
US10131947B2 (en) * | 2011-01-25 | 2018-11-20 | Ariosa Diagnostics, Inc. | Noninvasive detection of fetal aneuploidy in egg donor pregnancies |
CA2824387C (en) | 2011-02-09 | 2019-09-24 | Natera, Inc. | Methods for non-invasive prenatal ploidy calling |
GB2488358A (en) | 2011-02-25 | 2012-08-29 | Univ Plymouth | Enrichment of foetal DNA in maternal plasma |
US9260753B2 (en) | 2011-03-24 | 2016-02-16 | President And Fellows Of Harvard College | Single cell nucleic acid detection and analysis |
AU2012249531B2 (en) | 2011-04-29 | 2017-06-29 | Sequenom, Inc. | Quantification of a minority nucleic acid species |
CA2742327A1 (en) | 2011-05-20 | 2012-11-20 | The University Of British Columiba | Systems and methods for enhanced scoda |
US20140235474A1 (en) | 2011-06-24 | 2014-08-21 | Sequenom, Inc. | Methods and processes for non invasive assessment of a genetic variation |
JP6013332B2 (en) * | 2011-06-27 | 2016-10-25 | オリンパス株式会社 | Target particle counting method |
CA2791118C (en) | 2011-06-29 | 2019-05-07 | Furnan Jiang | Noninvasive detection of fetal genetic abnormality |
US9984198B2 (en) | 2011-10-06 | 2018-05-29 | Sequenom, Inc. | Reducing sequence read count error in assessment of complex genetic variations |
US10196681B2 (en) | 2011-10-06 | 2019-02-05 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
US9367663B2 (en) | 2011-10-06 | 2016-06-14 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
CA2850785C (en) | 2011-10-06 | 2022-12-13 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
US10424394B2 (en) | 2011-10-06 | 2019-09-24 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
JP2014533509A (en) * | 2011-11-17 | 2014-12-15 | セルスケープ・コーポレーション | Method, apparatus and kit for obtaining and analyzing cells |
WO2013104994A2 (en) | 2012-01-13 | 2013-07-18 | The University Of British Columbia | Multiple arm apparatus and methods for separation of particles |
ES2929923T3 (en) | 2012-01-20 | 2022-12-02 | Sequenom Inc | Diagnostic processes that condition the experimental conditions |
EP2820129A1 (en) | 2012-03-02 | 2015-01-07 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
US9892230B2 (en) | 2012-03-08 | 2018-02-13 | The Chinese University Of Hong Kong | Size-based analysis of fetal or tumor DNA fraction in plasma |
WO2013166444A2 (en) | 2012-05-04 | 2013-11-07 | Boreal Genomics Corp. | Biomarker analysis using scodaphoresis |
CA2874343C (en) | 2012-05-21 | 2021-11-09 | Fluidigm Corporation | Single-particle analysis of particle populations |
US9920361B2 (en) | 2012-05-21 | 2018-03-20 | Sequenom, Inc. | Methods and compositions for analyzing nucleic acid |
US10504613B2 (en) | 2012-12-20 | 2019-12-10 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
US11261494B2 (en) | 2012-06-21 | 2022-03-01 | The Chinese University Of Hong Kong | Method of measuring a fractional concentration of tumor DNA |
US10497461B2 (en) | 2012-06-22 | 2019-12-03 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
US20140093873A1 (en) | 2012-07-13 | 2014-04-03 | Sequenom, Inc. | Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non-invasive prenatal diagnoses |
US20160040229A1 (en) | 2013-08-16 | 2016-02-11 | Guardant Health, Inc. | Systems and methods to detect rare mutations and copy number variation |
US10876152B2 (en) | 2012-09-04 | 2020-12-29 | Guardant Health, Inc. | Systems and methods to detect rare mutations and copy number variation |
IL269097B2 (en) | 2012-09-04 | 2024-01-01 | Guardant Health Inc | Systems and methods to detect rare mutations and copy number variation |
US11913065B2 (en) | 2012-09-04 | 2024-02-27 | Guardent Health, Inc. | Systems and methods to detect rare mutations and copy number variation |
US10482994B2 (en) | 2012-10-04 | 2019-11-19 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
US20130309666A1 (en) | 2013-01-25 | 2013-11-21 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
US10494675B2 (en) | 2013-03-09 | 2019-12-03 | Cell Mdx, Llc | Methods of detecting cancer |
US11585814B2 (en) | 2013-03-09 | 2023-02-21 | Immunis.Ai, Inc. | Methods of detecting prostate cancer |
EP2971100A1 (en) | 2013-03-13 | 2016-01-20 | Sequenom, Inc. | Primers for dna methylation analysis |
KR20150132216A (en) | 2013-03-15 | 2015-11-25 | 더 차이니즈 유니버시티 오브 홍콩 | Determining fetal genomes for multiple fetus pregnancies |
US9340835B2 (en) | 2013-03-15 | 2016-05-17 | Boreal Genomics Corp. | Method for separating homoduplexed and heteroduplexed nucleic acids |
PL2981921T3 (en) | 2013-04-03 | 2023-05-08 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
EP3004383B1 (en) | 2013-05-24 | 2019-04-24 | Sequenom, Inc. | Methods for non-invasive assessment of genetic variations using area-under-curve (auc) analysis |
AU2014284180B2 (en) | 2013-06-21 | 2020-03-19 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
EP3968023A1 (en) | 2013-08-19 | 2022-03-16 | Singular Bio Inc. | Assays for single molecule detection and use thereof |
US10262755B2 (en) | 2014-04-21 | 2019-04-16 | Natera, Inc. | Detecting cancer mutations and aneuploidy in chromosomal segments |
US9499870B2 (en) | 2013-09-27 | 2016-11-22 | Natera, Inc. | Cell free DNA diagnostic testing standards |
US10577655B2 (en) | 2013-09-27 | 2020-03-03 | Natera, Inc. | Cell free DNA diagnostic testing standards |
WO2015051163A2 (en) | 2013-10-04 | 2015-04-09 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
CA2925111C (en) | 2013-10-07 | 2024-01-16 | Sequenom, Inc. | Methods and processes for non-invasive assessment of chromosome alterations |
CA2934822A1 (en) | 2013-12-28 | 2015-07-02 | Guardant Health, Inc. | Methods and systems for detecting genetic variants |
GB2524948A (en) * | 2014-03-07 | 2015-10-14 | Oxford Gene Technology Operations Ltd | Detecting Increase or Decrease in the Amount of a Nucleic Acid having a Sequence of Interest |
EP3736344A1 (en) | 2014-03-13 | 2020-11-11 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
RU2717641C2 (en) | 2014-04-21 | 2020-03-24 | Натера, Инк. | Detection of mutations and ploidy in chromosomal segments |
EP2942400A1 (en) | 2014-05-09 | 2015-11-11 | Lifecodexx AG | Multiplex detection of DNA that originates from a specific cell-type |
EP3140421B8 (en) | 2014-05-09 | 2020-05-06 | Eurofins LifeCodexx GmbH | Detection of dna that originates from a specific cell-type and related methods |
KR20160010277A (en) | 2014-07-18 | 2016-01-27 | 에스케이텔레콤 주식회사 | Method for prediction of fetal monogenic genetic variations through next generation sequencing of maternal cell-free dna |
EP4358097A1 (en) | 2014-07-25 | 2024-04-24 | University of Washington | Methods of determining tissues and/or cell types giving rise to cell-free dna, and methods of identifying a disease or disorder using same |
US11783911B2 (en) | 2014-07-30 | 2023-10-10 | Sequenom, Inc | Methods and processes for non-invasive assessment of genetic variations |
WO2016040843A1 (en) | 2014-09-11 | 2016-03-17 | Harry Stylli | Methods of detecting prostate cancer |
US10364467B2 (en) | 2015-01-13 | 2019-07-30 | The Chinese University Of Hong Kong | Using size and number aberrations in plasma DNA for detecting cancer |
PT3256605T (en) | 2015-02-10 | 2022-03-17 | Univ Hong Kong Chinese | Detecting mutations for cancer screening and fetal analysis |
US11739371B2 (en) | 2015-02-18 | 2023-08-29 | Invitae Corporation | Arrays for single molecule detection and use thereof |
US11168351B2 (en) | 2015-03-05 | 2021-11-09 | Streck, Inc. | Stabilization of nucleic acids in urine |
WO2016183106A1 (en) | 2015-05-11 | 2016-11-17 | Natera, Inc. | Methods and compositions for determining ploidy |
US11130986B2 (en) | 2015-05-20 | 2021-09-28 | Quantum-Si Incorporated | Method for isolating target nucleic acid using heteroduplex binding proteins |
ES2796501T3 (en) * | 2015-10-10 | 2020-11-27 | Guardant Health Inc | Methods and applications of gene fusion detection in cell-free DNA analysis |
HUE050491T2 (en) | 2015-11-10 | 2020-12-28 | Eurofins Lifecodexx Gmbh | Detection of foetal chromosomal aneuploidies using dna regions that are differentially methylated between the foetus and the pregnant female |
US20170145475A1 (en) | 2015-11-20 | 2017-05-25 | Streck, Inc. | Single spin process for blood plasma separation and plasma composition including preservative |
SG11201805119QA (en) | 2015-12-17 | 2018-07-30 | Guardant Health Inc | Methods to determine tumor gene copy number by analysis of cell-free dna |
US11708574B2 (en) | 2016-06-10 | 2023-07-25 | Myriad Women's Health, Inc. | Nucleic acid sequencing adapters and uses thereof |
WO2018013837A1 (en) | 2016-07-15 | 2018-01-18 | The Regents Of The University Of California | Methods of producing nucleic acid libraries |
WO2018022890A1 (en) | 2016-07-27 | 2018-02-01 | Sequenom, Inc. | Genetic copy number alteration classifications |
WO2018022991A1 (en) | 2016-07-29 | 2018-02-01 | Streck, Inc. | Suspension composition for hematology analysis control |
EP3518974A4 (en) | 2016-09-29 | 2020-05-27 | Myriad Women's Health, Inc. | Noninvasive prenatal screening using dynamic iterative depth optimization |
US9850523B1 (en) | 2016-09-30 | 2017-12-26 | Guardant Health, Inc. | Methods for multi-resolution analysis of cell-free nucleic acids |
KR20210158870A (en) | 2016-09-30 | 2021-12-31 | 가던트 헬쓰, 인크. | Methods for multi-resolution analysis of cell-free nucleic acids |
US11485996B2 (en) | 2016-10-04 | 2022-11-01 | Natera, Inc. | Methods for characterizing copy number variation using proximity-litigation sequencing |
EP3535415A4 (en) | 2016-10-24 | 2020-07-01 | The Chinese University of Hong Kong | Methods and systems for tumor detection |
US10011870B2 (en) | 2016-12-07 | 2018-07-03 | Natera, Inc. | Compositions and methods for identifying nucleic acid molecules |
JP7237003B2 (en) | 2017-01-24 | 2023-03-10 | セクエノム, インコーポレイテッド | Methods and processes for evaluation of gene fragments |
TWI803477B (en) | 2017-01-25 | 2023-06-01 | 香港中文大學 | Diagnostic applications using nucleic acid fragments |
EP3585889A1 (en) | 2017-02-21 | 2020-01-01 | Natera, Inc. | Compositions, methods, and kits for isolating nucleic acids |
WO2018175907A1 (en) | 2017-03-24 | 2018-09-27 | Counsyl, Inc. | Copy number variant caller |
US20200299677A1 (en) | 2017-10-27 | 2020-09-24 | Juno Diagnostics, Inc. | Devices, systems and methods for ultra-low volume liquid biopsy |
US11584929B2 (en) | 2018-01-12 | 2023-02-21 | Claret Bioscience, Llc | Methods and compositions for analyzing nucleic acid |
AU2019280712A1 (en) | 2018-06-06 | 2021-01-07 | The Regents Of The University Of California | Methods of producing nucleic acid libraries and compositions and kits for practicing same |
US11525159B2 (en) | 2018-07-03 | 2022-12-13 | Natera, Inc. | Methods for detection of donor-derived cell-free DNA |
TWI725686B (en) | 2018-12-26 | 2021-04-21 | 財團法人工業技術研究院 | Tubular structure for producing droplets and method for producing droplets |
JP2022519045A (en) | 2019-01-31 | 2022-03-18 | ガーダント ヘルス, インコーポレイテッド | Compositions and Methods for Isolating Cell-Free DNA |
US20220259637A1 (en) | 2019-06-28 | 2022-08-18 | Qiagen Gmbh | Method for enriching nucleic acids by size |
US20230120825A1 (en) * | 2020-02-28 | 2023-04-20 | Laboratory Corporation Of America Holdings | Compositions, Methods, and Systems for Paternity Determination |
EP4150074A1 (en) | 2020-05-14 | 2023-03-22 | Sequenom, Inc. | Methods, systems, and compositions for the analysis of cell-free nucleic acids |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5599666A (en) * | 1994-03-28 | 1997-02-04 | Promega Corporation | Allelic ladders for short tandem repeat loci |
WO1996027420A1 (en) * | 1995-03-08 | 1996-09-12 | Bioseparations, Inc. | Method for enriching rare cell population |
GB9704444D0 (en) | 1997-03-04 | 1997-04-23 | Isis Innovation | Non-invasive prenatal diagnosis |
MXPA04008477A (en) | 2002-03-01 | 2005-10-26 | Ravgen Inc | Methods for detection of genetic disorders. |
JP2006521086A (en) | 2003-02-28 | 2006-09-21 | ラブジェン, インコーポレイテッド | Methods for detecting genetic diseases |
DE60328193D1 (en) | 2003-10-16 | 2009-08-13 | Sequenom Inc | Non-invasive detection of fetal genetic traits |
-
2003
- 2003-10-16 DE DE60328193T patent/DE60328193D1/en not_active Expired - Lifetime
- 2003-10-16 AT AT03405742T patent/ATE435301T1/en not_active IP Right Cessation
- 2003-10-16 EP EP03405742.2A patent/EP1524321B2/en not_active Expired - Lifetime
-
2004
- 2004-10-15 JP JP2004301575A patent/JP4705774B2/en active Active
- 2004-10-15 US US10/964,726 patent/US20050164241A1/en not_active Abandoned
-
2007
- 2007-09-14 US US11/855,558 patent/US7838647B2/en active Active
-
2010
- 2010-11-12 JP JP2010253675A patent/JP5222926B2/en active Active
-
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- 2011-02-17 US US13/029,995 patent/US9580751B2/en active Active
- 2011-02-17 US US13/029,999 patent/US20110245482A1/en not_active Abandoned
-
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- 2012-07-24 US US13/557,025 patent/US20120302741A1/en not_active Abandoned
-
2013
- 2013-02-01 US US13/757,637 patent/US9738931B2/en active Active
- 2013-02-04 JP JP2013019380A patent/JP5728510B2/en active Active
- 2013-02-27 US US13/779,300 patent/US20130190483A1/en not_active Abandoned
-
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- 2017-07-18 US US15/653,401 patent/US20170321279A1/en not_active Abandoned
-
2021
- 2021-05-11 US US17/317,240 patent/US20210262035A1/en active Pending
Non-Patent Citations (2)
Title |
---|
Shimamura S. et al. International Journal of Biochemistry, Volume 22, Issue 5 (1990) Pages 545-549. * |
Supreme Court of the United States; Association for Molecular Pathology et al v. Myriad Genetics, Inc. et al, Deced June 13, 2013; 569 U. S. ____ (2013), 22 printed pages; from http://www.supremecourt.gov/opinions/12pdf/12-398_8njq.pdf * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
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US20130190483A1 (en) * | 2003-10-16 | 2013-07-25 | Sequenom, Inc. | Non-invasive detection of fetal genetic traits |
US9580751B2 (en) | 2003-10-16 | 2017-02-28 | Sequenom, Inc. | Non-invasive detection of fetal genetic traits |
US9738931B2 (en) | 2003-10-16 | 2017-08-22 | Sequenom, Inc. | Non-invasive detection of fetal genetic traits |
WO2019243328A1 (en) | 2018-06-18 | 2019-12-26 | Baxalta Incorporated | Bottom section for being connected to an assembly with plate settler, and assembly with plate settler |
WO2021116273A2 (en) | 2019-12-12 | 2021-06-17 | Baxalta Incorporated | Method for continuous protein recovering |
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JP2011087584A (en) | 2011-05-06 |
EP1524321B1 (en) | 2009-07-01 |
EP1524321B2 (en) | 2014-07-23 |
JP5728510B2 (en) | 2015-06-03 |
US20170321279A1 (en) | 2017-11-09 |
US9738931B2 (en) | 2017-08-22 |
US20080071076A1 (en) | 2008-03-20 |
US20140193808A1 (en) | 2014-07-10 |
US20110251076A1 (en) | 2011-10-13 |
JP2005160470A (en) | 2005-06-23 |
JP2013121359A (en) | 2013-06-20 |
US7838647B2 (en) | 2010-11-23 |
ATE435301T1 (en) | 2009-07-15 |
US20130190483A1 (en) | 2013-07-25 |
JP5222926B2 (en) | 2013-06-26 |
US20050164241A1 (en) | 2005-07-28 |
EP1524321A1 (en) | 2005-04-20 |
US9580751B2 (en) | 2017-02-28 |
JP4705774B2 (en) | 2011-06-22 |
US20110245482A1 (en) | 2011-10-06 |
DE60328193D1 (en) | 2009-08-13 |
US20210262035A1 (en) | 2021-08-26 |
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