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WO2012075598A2 - Détermination prénatale du sexe par pcr en temps réel chez les humains - Google Patents

Détermination prénatale du sexe par pcr en temps réel chez les humains Download PDF

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Publication number
WO2012075598A2
WO2012075598A2 PCT/CL2011/000075 CL2011000075W WO2012075598A2 WO 2012075598 A2 WO2012075598 A2 WO 2012075598A2 CL 2011000075 W CL2011000075 W CL 2011000075W WO 2012075598 A2 WO2012075598 A2 WO 2012075598A2
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WO
WIPO (PCT)
Prior art keywords
real
sex
sample
humans
dna
Prior art date
Application number
PCT/CL2011/000075
Other languages
English (en)
Spanish (es)
Other versions
WO2012075598A3 (fr
Inventor
Alexis Iván MARTINEZ HERNANDEZ
Original Assignee
Aquainnovo S.A.
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.)
Filing date
Publication date
Application filed by Aquainnovo S.A. filed Critical Aquainnovo S.A.
Priority to CA2821013A priority Critical patent/CA2821013A1/fr
Priority to GB1311623.1A priority patent/GB2500154A/en
Publication of WO2012075598A2 publication Critical patent/WO2012075598A2/fr
Publication of WO2012075598A3 publication Critical patent/WO2012075598A3/fr

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Classifications

    • 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
    • C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present invention relates to a method for determining sex in prenatal form by real-time chain polymerase reaction (PCR), in humans, the splitters and related kit.
  • PCR real-time chain polymerase reaction
  • the real-time polymerase chain reaction (PCR) technique consists of "in vitro" amplification of a specific sequence of interest, whose amplification generates a fluorescent signal detectable by the equipment. Depending on the behavior of the signal, the abundance of the amplified sequence of interest can be established. Therefore, you can have two results with this technique, the first is to establish the presence or absence of a sequence of interest in a sample that is analyzed and second, you can establish a kind of relative quantification of this sequence in the sample.
  • An objective of the present invention is to provide a method for determining prenatal sex in a peripheral blood sample of pregnant women from the 12th week of pregnancy.
  • a probe-splitter set and a kit for the prenatal determination of sex in a peripheral blood sample of pregnant women are provided.
  • DYS14 which comprises the sequence
  • the present invention relates to a method of real-time PCR detection for detecting said multicopy region, in peripheral blood of pregnant women.
  • REPLACEMENT SHEET (Rule 26) So the invention provides a method for determining sex in prenatal form by using the real-time polymerase chain reaction (PCR) in humans comprising the following steps:
  • a) take a peripheral blood sample from a woman 12 or more weeks pregnant and obtain an eluted deoxyribonucleic acid (DNA)
  • step (b) amplifying the eluted DNA obtained from step (a) by polymerase chain reaction (PCR), real - time format using a fluorescent probe DYS14: 5'-TCGGCCTTTCTAGTGGAGAGGTGCT-3 '(BHQ) and a mixture of splitters comprising the DYS14F2 splitter: 5 -CTTCTGGGCCAATGTTGTATCC- 3 ' and the DYS14R2 splitter: 5 ' -CTGCCCATCGGTCACTTACAC-3 ' ; Y
  • c) identify the presence of the sequence CTTCTGGGCCAATGTTGTATCCTTCTCAGTGTTTCTTCGGCCTTTCTAGTGGAGAG GTGCTCTCGGGGAAGTGTAAGTGACCGATGGGCAGCTCGGCGT, as a fluorescence signal, in the amplification resulting from the stage, assigning the color of the sample to the peripheral stage of the sample, assigning the color to the stage of the sample whose DNA was amplified, and in the absence of said fluorescence signal, the determination of female sex in the peripheral blood sample, whose DNA was amplified.
  • the present invention relates to a mixture of splitters for determining sex in prenatal form by using the real-time polymerase chain reaction (PCR) in humans, comprising the synthetic sequence DYS14F2: 5 ' - CTTCTGGGCCAATGTTGTATCC-3 ' and the synthetic sequence DYS14R2 5 ' - CTGCCCATCGGTCACTTACAC-3 ' .
  • the invention provides a kit for determining sex in prenatal form by using the real-time chain polymerase reaction (PCR) in humans, CHARACTERIZED because it comprises:
  • a mixture of primers comprising the synthetic sequence DYS14F2: 5 '- CTTCTGGGCCAATGTTGTATCC-3' and DYS14R2 synthetic sequence 5 '- CTGCCCATCGGTCACTTACAC-3';
  • Figure 1 shows the preliminary real-time PCR results of samples from mothers who are carriers of a fetus of unknown (pregnant) sex for the determination of prenatal sex.
  • the upper date corresponds to a positive control (blood of a man).
  • the middle arrow indicates a clinical sample (blood of a woman carrying a male baby).
  • the lower arrow corresponds to the negative control and a clinical sample of a mother carrying a female baby.
  • a 2 ml sample of peripheral blood is taken from a woman 12 or more weeks pregnant. 1 ml of the sample is taken and placed inside an eppendorf tube and the sample is centrifuged at 13,000 rpm for 5 minutes. In this centrifugation process, plasma is obtained at the top of the tube. Subsequently, a DNA extraction (deoxyribonucleic acid) is performed with a commercial kit, for this example the protocol of the commercial kit QIAamp®DNA Blood Mini Kit (Qiagen; Catalog No. 51104X) will be used, where in the description of the methodology they mention solutions whose abbreviations correspond to the originals exposed in the commercial kit (for example, AL buffer).
  • a 200 ul aliquot of serum is taken and placed in an eppendorf tube that has not been previously used, and 20 ul of Proteinase K (Invitrogen) is added and gently homogenized to be incubated for 30 minutes at 60 ° C. Subsequently, 200 ul of AL buffer is added to the sample. Homogenize by vortex for 15 seconds and incubate at 56 ° C for 12 min. Briefly, centrifuge at low revolution so as not to leave drops (3000 rpm for 0 sec) at the edges. Additional 200 ul of absolute ethanol (96-100%) and homogenize by vortex for 15 seconds. Carefully add the homogenate to a column and centrifuge at 10,000 xg for 1 min.
  • a commercial kit qPCR Supermix Universal Express Kit (Invitrogen) is used, for this purpose a number of reactions are prepared based on the number of samples to be analyzed. For example, to prepare 10 amplification reactions , prepare the microplates corresponding to this number of reactions and prepare the mixture according to what is described in the kit, the following table shows the volumes of each component to prepare the master mix:
  • FAM 6-carboxy-fluorescein (fluorophore)
  • Nuclease-free water water that is free of nucleases that are enzymes that degrade genetic material.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé pour déterminer le sexe de manière prénatale par utilisation de la PCR en temps réel chez les humains. Le procédé comprend les étapes suivantes de prélèvement d'échantillon de sang périphérique d'une femme à 12 semaines de grossesse ou plus et d'obtention d'un éluat d'acide désoxyribonucléique (ADN); de stockage de l'éluat à 4°C; d'amplification de l'éluat d'ADN par PCR, en temps réel, à l'aide d'une sonde fluorescente DYS14: 5'- TCGGCCTTTCTAGTGGAGAGGTGCT-3'(BHQ) d'un mélange d'amorces qui comprend l'amorce DYS14F2: 5'-CTTCTGGGCCAATGTTGTATCC-3' et l'amorce DYS14R2: 5'-CTGCCCATCGGTCACTTACAC-3'; et de détermination de la présence d'une séquence au moyen d'un signal de fluorescence, la présence dudit signal de fluorescence déterminant le sexe masculin et l'absence dudit signal de fluorescence déterminant le sexe féminin dans l'échantillon de sang périphérique, dont l'ADN a été amplifié. L'invention concerne le mélange d'amorces et le kit pour déterminer le sexe de manière prénatale par PCR en temps réel chez les humains.
PCT/CL2011/000075 2010-12-10 2011-12-09 Détermination prénatale du sexe par pcr en temps réel chez les humains WO2012075598A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CA2821013A CA2821013A1 (fr) 2010-12-10 2011-12-09 Determination prenatale du sexe par pcr en temps reel chez les humains
GB1311623.1A GB2500154A (en) 2010-12-10 2011-12-09 Discernment of prenatal sex in humans by real-time PCR

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CL2010001419A CL2010001419A1 (es) 2010-12-10 2010-12-10 Metodo para detectar el sexo de forma prenatal en el plasma de madre, mediante el uso de la reaccion de polimerasa en cadena (pcr) humano en tiempo real.
CL1419-2010 2010-12-10

Publications (2)

Publication Number Publication Date
WO2012075598A2 true WO2012075598A2 (fr) 2012-06-14
WO2012075598A3 WO2012075598A3 (fr) 2012-08-02

Family

ID=46207546

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CL2011/000075 WO2012075598A2 (fr) 2010-12-10 2011-12-09 Détermination prénatale du sexe par pcr en temps réel chez les humains

Country Status (4)

Country Link
CA (1) CA2821013A1 (fr)
CL (1) CL2010001419A1 (fr)
GB (1) GB2500154A (fr)
WO (1) WO2012075598A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112522417A (zh) * 2019-09-19 2021-03-19 公安部物证鉴定中心 一种用于检测男性人类基因组dna的dna片段和引物对

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9704444D0 (en) * 1997-03-04 1997-04-23 Isis Innovation Non-invasive prenatal diagnosis

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112522417A (zh) * 2019-09-19 2021-03-19 公安部物证鉴定中心 一种用于检测男性人类基因组dna的dna片段和引物对

Also Published As

Publication number Publication date
CA2821013A1 (fr) 2012-06-14
GB2500154A (en) 2013-09-11
WO2012075598A3 (fr) 2012-08-02
GB201311623D0 (en) 2013-08-14
CL2010001419A1 (es) 2011-04-25

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