US20050164241A1 - Non-invasive detection of fetal genetic traits - Google Patents

Non-invasive detection of fetal genetic traits Download PDF

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Publication number
US20050164241A1
US20050164241A1 US10/964,726 US96472604A US2005164241A1 US 20050164241 A1 US20050164241 A1 US 20050164241A1 US 96472604 A US96472604 A US 96472604A US 2005164241 A1 US2005164241 A1 US 2005164241A1
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United States
Prior art keywords
fetal
dna
sample
disorder
genetic
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Abandoned
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US10/964,726
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English (en)
Inventor
Sinuhe Hahn
Wolfgang Holzgreve
Bernhard Zimmermann
Ying Li
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Sequenom Inc
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Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=34354635&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US20050164241(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by Individual filed Critical Individual
Publication of US20050164241A1 publication Critical patent/US20050164241A1/en
Assigned to SEQUENOM, INC. reassignment SEQUENOM, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: UNIVERSITY HOSPITAL OF BASEL
Assigned to UNIVERSITY HOSPITAL OF BASEL reassignment UNIVERSITY HOSPITAL OF BASEL ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HAHN, SINUHE, HOLZGREVE, WOLFGANG, LI, YING, ZIMMERMANN, BERNHARD
Priority to US11/855,558 priority Critical patent/US7838647B2/en
Priority to US13/029,999 priority patent/US20110245482A1/en
Priority to US13/029,995 priority patent/US9580751B2/en
Priority to US13/557,025 priority patent/US20120302741A1/en
Priority to US13/757,637 priority patent/US9738931B2/en
Priority to US13/779,300 priority patent/US20130190483A1/en
Priority to US15/653,401 priority patent/US20170321279A1/en
Priority to US17/317,240 priority patent/US20210262035A1/en
Priority to US18/538,488 priority patent/US20240182970A1/en
Abandoned legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • 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
  • 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
  • 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
  • Such determination of fetal genetic traits can be effected by methods such as, for example, PCR (polymerase chain reaction) technology, ligase chain reaction, probe hybridisation 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.
  • 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 and SRY_Rev: AGA TTA ATG GTT GCT AAG GAC TGG AT and a FAM labeled TaqMan MGB (Minor Groove Binder) probe SRY_MGB: TCC CCA CAA CCT CTT.
  • the TaqMan System for the GAPDH gene consisted of the following primers and probe: GAPDH_Fwd: CCC CAC ACA CAT GCA CTT ACC, GAPDH_Rev: CCT AGT CCC AGG GCT TTG ATT and GAPDH_MGB: TAG GAA GGA CAG GCA AC.
  • 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.
  • 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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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measurement And Recording Of Electrical Phenomena And Electrical Characteristics Of The Living Body (AREA)
  • Ultra Sonic Daignosis Equipment (AREA)
US10/964,726 2003-10-16 2004-10-15 Non-invasive detection of fetal genetic traits Abandoned US20050164241A1 (en)

Priority Applications (9)

Application Number Priority Date Filing Date Title
US11/855,558 US7838647B2 (en) 2003-10-16 2007-09-14 Non-invasive detection of fetal genetic traits
US13/029,999 US20110245482A1 (en) 2003-10-16 2011-02-17 Non-invasive detection of fetal genetic traits
US13/029,995 US9580751B2 (en) 2003-10-16 2011-02-17 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
US13/757,637 US9738931B2 (en) 2003-10-16 2013-02-01 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
US15/653,401 US20170321279A1 (en) 2003-10-16 2017-07-18 Non-invasive detection of fetal genetic traits
US17/317,240 US20210262035A1 (en) 2003-10-16 2021-05-11 Non-invasive detection of fetal genetic traits
US18/538,488 US20240182970A1 (en) 2003-10-16 2023-12-13 Non-invasive detection of fetal genetic traits

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP03405742.2 2003-10-16
EP03405742.2A EP1524321B2 (en) 2003-10-16 2003-10-16 Non-invasive detection of fetal genetic traits

Related Child Applications (4)

Application Number Title Priority Date Filing Date
US11/855,558 Division US7838647B2 (en) 2003-10-16 2007-09-14 Non-invasive detection of fetal genetic traits
US13/029,995 Division US9580751B2 (en) 2003-10-16 2011-02-17 Non-invasive detection of fetal genetic traits
US13/029,999 Continuation US20110245482A1 (en) 2003-10-16 2011-02-17 Non-invasive detection of fetal genetic traits
US13/557,025 Continuation US20120302741A1 (en) 2003-10-16 2012-07-24 Non-invasive detection of fetal genetic traits

Publications (1)

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US20050164241A1 true US20050164241A1 (en) 2005-07-28

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Family Applications (10)

Application Number Title Priority Date Filing Date
US10/964,726 Abandoned US20050164241A1 (en) 2003-10-16 2004-10-15 Non-invasive detection of fetal genetic traits
US11/855,558 Active 2026-03-09 US7838647B2 (en) 2003-10-16 2007-09-14 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
US13/029,995 Active US9580751B2 (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
US17/317,240 Pending US20210262035A1 (en) 2003-10-16 2021-05-11 Non-invasive detection of fetal genetic traits
US18/538,488 Pending US20240182970A1 (en) 2003-10-16 2023-12-13 Non-invasive detection of fetal genetic traits

Family Applications After (9)

Application Number Title Priority Date Filing Date
US11/855,558 Active 2026-03-09 US7838647B2 (en) 2003-10-16 2007-09-14 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
US13/029,995 Active US9580751B2 (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
US17/317,240 Pending US20210262035A1 (en) 2003-10-16 2021-05-11 Non-invasive detection of fetal genetic traits
US18/538,488 Pending US20240182970A1 (en) 2003-10-16 2023-12-13 Non-invasive detection of fetal genetic traits

Country Status (5)

Country Link
US (10) US20050164241A1 (ja)
EP (1) EP1524321B2 (ja)
JP (3) JP4705774B2 (ja)
AT (1) ATE435301T1 (ja)
DE (1) DE60328193D1 (ja)

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US9580751B2 (en) 2017-02-28

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