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Method and device for SNP genotyping

一种基因分型、基因组的技术，应用在生物化学设备和方法、微生物的测定/检验等方向，能够解决复杂等问题

Pending Publication Date: 2018-06-08
ESTAB FR DU SANG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, despite the progress made, these techniques remain complex to perform, especially because of the use of two sequential amplification techniques (PCR followed by primer extension reactions) and / or coupling to each allele to be detected. Production of gene-specific sequence microspheres and deposition of these microspheres on nitrocellulose membranes

Method used

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  • Method and device for SNP genotyping
  • Method and device for SNP genotyping
  • Method and device for SNP genotyping

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0124] (i) said migration buffer comprises a substrate or binding partner capable of interacting with said label to produce a detectable signal, directly or indirectly, or

[0125] (ii) said lateral flow assay device comprises a labeling region comprising a substrate or binding partner specific for said amplification product label, said substrate or binding partner being capable of directly or indirectly producing a detectable signal. The labeling zone is preferably located downstream of the migration buffer application zone and upstream of the amplification product application zone.

[0126] According to a particular embodiment, said amplification product marker is the first member of a ligand / anti-ligand pair. In this case, the migration buffer or labeling region comprises the second member of the pair, optionally coupled to a detectable label. Examples of detectable labels include, but are not limited to, colloidal metals such as gold or silver; non-colloidal metals such a...

Embodiment 1

[0177] Materials and methods

[0178] Sodium Chloride, Sodium Citrate, Methanol, Sucrose, Sodium Dodecyl Sulfate (SDS), 20. Formamide and biotinylated bovine serum albumin were purchased from Sigma-Aldrich. Gold particles (40nm, 9×10 10 particles / mL) were purchased from BBI Solutions (England). Oligonucleotides, primers and probes were synthesized by Eurogentec (Belgium).

[0179] Whole Blood Samples, Phenotyping and Genotyping Techniques

[0180] whole blood samples from du Sang Collected on anticoagulant (EDTA) and extensively phenotyped using standard serological techniques in the Blood Donation Qualification Laboratory (Metz–Tessy, France). The samples were extensively genotyped using previously described blood genotyping techniques (Paris et al., 2014).

[0181] Design of LATE–PCR primers

[0182] The primers were designed to be used at non-stoichiometric concentrations and on the basis that the Tm value was adjusted with the working concentration of the prim...

Embodiment 2

[0212] Materials and methods

[0213] New probes were designed to allow determination of blood group genotypes at lower migration temperatures than those demonstrated in Example 1. Sequences are presented in Table 3. The strips were assembled with HF090 nitrocellulose membrane (80mm x 5mm) and absorbent pad. The films and absorbent pads were cut using a guillotine cutter.

[0214] The concentration is 5mg.mL –1 A biotinylated BSA solution was immobilized on the upper part of the strip to form a control zone (CT) during the assay.

[0215] Use a concentration of 50 μM.L in loading buffer (6X SSC, 0.1% SDS, 2% methanol, 2% sucrose) –1 probe solution, the probes were manually loaded onto the membrane. After loading, the membrane was dried at 80°C for 40-45 minutes.

[0216] Table 3. Probe sequences used during multiple blood group genotyping for JK and FY systems. Polymorphisms (SNPs) for each probe are indicated with bold and underlined nucleotides.

[0217]

[0218] ...

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Abstract

The present invention relates to a method for genotyping single nucleotide polymorphisms (SNPs) using a lateral flow test device. The invention also relates to a kit comprising said lateral flow testdevice and also to the use thereof for genotyping single nucleotide polymorphisms (SNPs).

Description

[0001] The present invention relates to a new method for single nucleotide polymorphism (SNP) genotyping. Background technique [0002] A single nucleotide polymorphism (SNP) is a single base pair variation in the genome between individuals of the same species or between the same pair of chromosomes. SNPs account for 90% of all human genetic variation. They occur approximately every 1000 bases in the human genome, and to date more than 1.8 million SNPs are listed and available in specialized databases. Use of these databases has shown that these SNPs can be used not only for forensic purposes to identify individuals, but also to diagnose or determine susceptibility to certain diseases, predict the severity of a condition or a patient's response to treatment, or determine an individual's blood type. [0003] There are many methods available for detecting SNPs, such as DNA microarrays, allele-specific oligonucleotide (ASO) hybridization, DNA sequencing and single-strand conform...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6827C12Q1/6881
CPCC12Q1/6881C12Q2600/156C12Q2600/16C12Q1/6827C12Q2531/107C12Q2537/143C12Q2565/625
Inventor 让-查里斯·布莱斯朱利安·戈麦斯-马蒂
Owner ESTAB FR DU SANG
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