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Use of highly parallel SNP genotyping for fetal diagnosis

a genotyping and fetal technology, applied in the field of use of highly parallel snp genotyping for fetal diagnosis, can solve the problems of inaccurate calls, time-consuming analysis, and achieve the effect of reducing the risk of today's invasive diagnosis, time-consuming analysis and prone to errors

Inactive Publication Date: 2009-11-26
STOUGHTON ROLAND +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The present invention provides a method for detecting fetal abnormalities by analyzing the ratio of maternal and paternal alleles in a sample of fetal cells. This method uses highly parallel SNP detection and DNA microarray readout to determine the ratios of alleles in a genomic region suspected of trisomy or partial trisomy. The method can be used with as little as 100 fetal cells and can detect fetal abnormalities with a level of confidence that exceeds 90%. The invention also includes a method for estimating the number of fetal cells present in a sample."

Problems solved by technology

However, fetal cells represent a small number of cells against the background of a large number of maternal cells in the blood which make the analysis time consuming and prone to error.
Current technologies and protocols for highly parallel SNP detection with DNA microarray readout result in inaccurate calls when there are too few starting DNA copies or when a particular allele represents a small fraction in the population of input DNA molecules.

Method used

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  • Use of highly parallel SNP genotyping for fetal diagnosis
  • Use of highly parallel SNP genotyping for fetal diagnosis
  • Use of highly parallel SNP genotyping for fetal diagnosis

Examples

Experimental program
Comparison scheme
Effect test

example 1

Separation of Fetal Cord Blood

[0137]FIG. 12A-D shows a schematic of the device used to separate nucleated cells from fetal cord blood.

[0138]Dimensions: 100 mm×28 mm×1 mm

[0139]Array design: 3 stages, gap size=18, 12 and 8 μm for the first, second and third stage, respectively.

[0140]Device fabrication:

[0141]The arrays and channels were fabricated in silicon using standard photolithography and deep silicon reactive etching techniques. The etch depth is 140 μm. Through holes for fluid access are made using KOH wet etching. The silicon substrate was sealed on the etched face to form enclosed fluidic channels using a blood compatible pressure sensitive adhesive (9795, 3M, St Paul, Minn.).

[0142]Device packaging:

[0143]The device was mechanically mated to a plastic manifold with external fluidic reservoirs to deliver blood and buffer to the device and extract the generated fractions.

[0144]Device operation:

[0145]An external pressure source was used to apply a pressure of 2.0 PSI to the buffer...

example 2

Isolation of Fetal Cells from Maternal Blood

[0152]The device and process described in detail in Example 1 were used in combination with immunomagnetic affinity enrichment techniques to demonstrate the feasibility of isolating fetal cells from maternal blood.

[0153]Experimental conditions:

[0154]Blood from consenting maternal donors carrying male fetuses was collected into K2EDTA vacutainers (366643, Becton Dickinson, Franklin Lakes, N.J.) immediately following elective termination of pregnancy. The undiluted blood was processed using the device described in Example 1 at room temperature and within 9 hrs of draw. Nucleated cells from the blood were separated from enucleated cells (red blood cells and platelets), and plasma delivered into a buffer stream of calcium and magnesium-free Dulbecco's Phosphate Buffered Saline (14190-144, Invitrogen, Carlsbad, Calif.) containing 1% Bovine Serum Albumin (BSA) (A8412-100ML, Sigma-Aldrich, St Louis, Mo.). Subsequently, the nucleated cell fraction...

example 3

Confirmation of the Presence of Male Fetal Cells in Enriched Samples

[0159]Confirmation of the presence of a male fetal cell in an enriched sample is performed using qPCR with primers specific for DYZ, a marker repeated in high copy number on the Y chromosome. After enrichment of fnRBC by any of the methods described herein, the resulting enriched fnRBC are binned by dividing the sample into 100 PCR wells. Prior to binning, enriched samples may be screened by FISH to determine the presence of any fnRBC containing an aneuploidy of interest. Because of the low number of fnRBC in maternal blood, only a portion of the wells will contain a single fnRBC (the other wells are expected to be negative for fnRBC). The cells are fixed in 2% Paraformaldehyde and stored at 4° C. Cells in each bin are pelleted and resuspended in 5 μl PBS plus 1 μl 20 mg / ml Proteinase K (Sigma #P-2308). Cells are lysed by incubation at 65° C. for 60 minutes followed by inactivation of the Proteinase K by incubation ...

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Abstract

The present invention provides apparatus and methods for enriching components or cells from a sample and conducting genetic analysis, such as SNP genotyping to provide diagnostic results for fetal disorders or conditions.

Description

CROSS-REFERENCE[0001]This application is a continuation application of U.S. application Ser. No. 11 / 763,133, filed on Jun. 4, 2007, which claims the benefit of U.S. Provisional Application No. 60 / 804,816, filed Jun. 14, 2006, which applications are incorporated herein by reference.BACKGROUND OF THE INVENTION[0002]Analysis of specific cells can give insight into a variety of diseases. These analyses can provide non-invasive tests for detection, diagnosis and prognosis of diseases, thereby eliminating the risk of invasive diagnosis. For instance, social developments have resulted in an increased number of prenatal tests. However, the available methods today, amniocentesis and chorionic villus sampling (CVS) are potentially harmful to the mother and to the fetus. The rate of miscarriage for pregnant women undergoing amniocentesis is increased by 0.5-1%, and that figure is slightly higher for CVS. Because of the inherent risks posed by amniocentesis and CVS, these procedures are offered...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q1/6874C12Q2600/156
Inventor STOUGHTON, ROLANDKAPUR, RAVICOHEN, BARB ARIEL
Owner STOUGHTON ROLAND
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