Methods for detecting aneuploidy using microparticle multiplex detection

a technology of multiplex detection and microparticles, applied in the direction of fluorescence/phosphorescence, instruments, biochemistry apparatus and processes, etc., can solve the problems of cytogenetic studies of human oocytes fixed, the most common lethal monosomy, and the abnormality of individuals

Inactive Publication Date: 2011-09-22
WILDENBERG ANDREW PATRICK +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This approach enables rapid, inexpensive, and automatable detection of aneuploidy in embryos, increasing the success rates of IVF by allowing for the pre-implantation screening of aberrant chromosome numbers, thereby improving fertilization and implantation success.

Problems solved by technology

Monosomy is most commonly lethal during prenatal development.
However, birth of a live triploid is extraordinarily rare and such individuals are quite abnormal.
Cytogenetic studies of human oocytes fixed after failing to fertilize in-vitro display a relatively high incidence of chromosomal abnormalities (aneuploidy).
Also, studies of many spontaneous abortions and pre-term embryos show that chromosomal abnormalities may be the main cause of fetal loss.
Many reports strongly indicate that chromosomal aneuploidy is the prime cause of fertilization failure in oocytes and implantation failure of embryos.
Aneuploidy mainly arises during meiotic non-disjunction; but many environmental factors may also disrupt spindle function and eventually lead to the formation of aneuploid embryos.
However, this method is not a practical solution for single cells, and therefore cannot be performed as a pre-implantation screen.

Method used

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  • Methods for detecting aneuploidy using microparticle multiplex detection
  • Methods for detecting aneuploidy using microparticle multiplex detection
  • Methods for detecting aneuploidy using microparticle multiplex detection

Examples

Experimental program
Comparison scheme
Effect test

example 1

12-Plex Detection of Microparticles

[0173]The ability of the method to differentiate between 12 different microparticle classes was tested. Microbeads of 3.0 μm, 4.12 μm, 5.0 μm and 6.8 μm were labeled with tetramethyl rhodamine at three different fluorescence intensity levels, 0, 33% and 100% to give 12 classes of microbead. These beads were then subjected to flow cytometry using a single detection channel. The results are depicted in FIG. 2.

example 2

Exemplary Microparticles for Simultaneous Detection of Aneuploidy in All Human Chromosomes

[0174]Table 3 indicates an exemplary range of potential binding agents suitable for the simultaneous detection of aneuploidy in all human chromosomes. These microparticles comprise, 5 different size microbeads, labeled with a single fluorescent marker at 5 different intensity levels. This combination of bead size and marker intensity yields 25 possible bead classes which accommodates the 24 classes needed to examine all human chromosomes simultaneously.

example 3

Multiplex Analysis Using Number Clustering

[0175]Human hGATA4, exon 4 DNA was produced by PCR with a 5′ phosphate on the forward primer. Allele specific probes were constructed for a Single Nucleotide Polymorphism within the PCR product. After PCR, excess primers and primer dimers were removed by Exol digestion. Forward strand DNA was preferentially degraded by Lambda exonuclease. The ssDNA from the PCR was mixed with AmpaSand™ Beads (Genera Biosystems, Melbourne, Australia) customized with DNA identical to the phosphorylated forward primer in either a 25 bead per test or 50 bead per test configuration. Allele specific probes (SNP—A specific probe labelled with Tetramethylrhodamine (TMR) which emits in the yellow channel; and SNP-G specific probe labelled with Cy5 which emits in the red channel). After competitive hybridisation, experiments from 2 different experiments were combined and run simultaneously on a Becton-Dickinson FACSArray flow cytometer. Data was acquired and analyzed ...

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Abstract

The present invention provides a method for the detection and sorting of microparticles in a mixture of microparticles. The method of the present invention allows for the detection and sorting of many distinct microparticle classes. Detection and sorting is on the basis of microparticle size, the fluorescence spectrum of any attached reporter molecule, the fluorescence intensity of the reporter molecule, and the number of particles in each classification bin. These microparticle classes have particular applications in many genetic or biochemical multiplexing studies and especially as binding agents for the detection of aneuploidy in an organism or embryo of the organism. In humans, the detection and sorting of at least 24 classes of microparticles would be sufficient for a single tube method for the simultaneous detection of aneuploidy in all chromosomes, wherein each distinct microparticle class comprises a polynucleotide sequence complementary to, and specific for, a polynucleotide sequence that is unique to a particular human chromosome. Furthermore, using currently available technology, the present method has application for the simultaneous detection of aneuploidy in all chromosomes for an organism that has 216 or fewer pairs of chromosomes. Kits for the simultaneous detection of aneuploidy in one or more human chromosomes are also provided.

Description

BACKGROUND OF THE INVENTION[0001]1. Field of the Invention[0002]The present invention provides a method for the detection and sorting of microparticles in a mixture of microparticles. The method of the present invention allows for the detection and sorting of many distinct microparticle classes. Detection and sorting is on the basis of microparticle size, the fluorescence spectrum of any attached reporter molecule, the fluorescence intensity of the reporter molecule, and the number of particles in each classification bin. These microparticle classes have particular applications in many genetic or biochemical multiplexing studies and especially as binding agents for the detection of aneuploidy in an organism or embryo of the organism. In humans, the detection and sorting of at least 24 classes of microparticles would be sufficient for a single tube method for the simultaneous detection of aneuploidy in all chromosomes, wherein each distinct microparticle class comprises a polynucleot...

Claims

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Application Information

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Patent Type & AuthorityApplications(United States)
IPC IPC(8): C12Q1/68G01N21/64C12N15/12G01N33/543
CPCC12Q1/6827C12Q1/6883G01N33/54313C12Q2537/143C12Q2563/107C12Q2563/155
InventorWILDENBERG, ANDREW PATRICKPOETTER, KARL
OwnerWILDENBERG ANDREW PATRICK