Methods for multiplex detection of alleles associated with ophthalmic conditions

A genome, gene sequence technology, applied in the field of multiple detection of alleles related to ophthalmic conditions, which can solve problems such as vision loss, poor vision, and omission of potential symptoms of patients

Active Publication Date: 2016-08-24
AVELLINO LAB USA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] Thus, although an accurate diagnosis of Avellino's dystrophy is required to prevent the progression of Avellino's dystrophy due to LASIK surgery, only microscopic observation of corneal...

Method used

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  • Methods for multiplex detection of alleles associated with ophthalmic conditions
  • Methods for multiplex detection of alleles associated with ophthalmic conditions
  • Methods for multiplex detection of alleles associated with ophthalmic conditions

Examples

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Embodiment 1

[0225] Example 1: DNA extraction (DNA Extract All Reagents, ThermoFisher)

[0226] DNA was extracted from oral epithelium or hair roots or whole blood as described below and in accordance with the disclosure provided herein.

[0227] For DNA extraction from oral epithelium or hair roots, first pretreat samples in 300 µL of 1X PBS. Next, 30 μL of lysis solution was added to the tube and the mixture was vortexed. The mixture was then incubated at 95°C for 3 min. Then, 30 μL of DNA stabilization solution (from Life Technologies / Thermo Scientific, USA) was added and the mixture was vortexed. The mixture was then centrifuged at 13,000 RPM for 1 min.

[0228] For DNA extraction from whole blood, start with 3 µL of whole blood and first pretreat the sample in 300 µL of 1X PBS. Next, 30 μL of lysis solution was added to the tube and the mixture was vortexed. The mixture was then incubated at 95°C for 3 min. Then, 30 μL of DNA stabilization solution (from Life Technologies / Thermo S...

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Abstract

Systems and methods for detecting at least two genomic alleles associated with corneal dystrophy in a sample from a human subject are disclosed in which cells (e.g., epithelial) of the subject are adhered to a tip of a substrate. The tip of the substrate is agitated in a lysis solution that lyses cells adhered to the substrate. The substrate is removed from the lysis solution upon completion of this agitation. The resulting lysis solution is incubated and then genomic DNA from the lysis solution is isolated to form a gDNA solution. From this, identity of at least two nucleotides present in the human TGFpI gene is determined using at least two oligonucleotide primer pairs and the gDNA solution. These at least two nucleotides are located at respective independent positions of the TGFpI gene corresponding to respective independent single nucleotide polymorphisms (SNPs) associated with corneal dystrophy.

Description

field of invention [0001] The present application generally relates to methods for isolating and detecting disease-associated genetic alleles. In particular, the present application relates to improved methods for detecting alleles associated with Avellino corneal dystrophy. Background of the invention [0002] Real-time PCR can be used to detect differences between nucleic acid sequences that have substantially identical sequences. By using differentially labeled fluorescent nucleic acid probes (eg, one that binds the wild-type sequence and one that binds the mutant sequence), single nucleotide changes in the human genome can be rapidly and reliably detected. This resolution capability has been applied to medical diagnostics, where single nucleotide polymorphisms (SNPs), single base changes found in the coding and / or non-coding sequences of proteins, are associated with human disease. [0003] However, real-time PCR analysis is highly dependent on the collection and isola...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q2600/156C12Q1/6851C12Q1/6883C12Q2537/143C12Q2537/161C12Q2561/113C12Q2600/158
Inventor C.查奥-谢恩S.乔G.李
Owner AVELLINO LAB USA
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