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Kit for detecting mutations causing genetic disorders

A technology for kits and diseases, applied in the field of kits for detecting mutations that cause genetic diseases, can solve problems such as time-consuming and high cost

Pending Publication Date: 2022-05-10
COUNCIL OF SCI & IND RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] As mentioned above, there are established protocols and kits available for detecting mutations that cause genetic disorders, such as hemoglobinopathies and myopathies; several challenges exist for genetic diagnosis, such as isolation of DNA, transport and storage of samples, precision High costs involved in instrumentation, time-consuming procedures, and specific testing and diagnosis

Method used

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  • Kit for detecting mutations causing genetic disorders
  • Kit for detecting mutations causing genetic disorders
  • Kit for detecting mutations causing genetic disorders

Examples

Experimental program
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Effect test

Embodiment 1

[0061] The ARMS-PCR assay detects sickle cell anemia-causing mutations using unprocessed human dried blood spots (DBS) in a single reaction.

[0062] To test the capabilities of the method developed in the present invention, untreated human dried blood spots were used. Multiple primers were designed to detect wild-type genotype (A / A), mutant genotype (T / T) and heterozygous genotype (A / T) of Cd6 T>A mutation in HBB gene. External forward primer [50nM, 20nt, 5'd (ACC TCA CCC TGT GGA GCC AC) 3'] (SEQ ID NO: 1) and external reverse primer [50nM, 20nt, 5'd (TCA TTC GTC TGT TTC CCA TT)3'] (SEQ ID NO:2), allele-specific primer, internal forward of A allele [50nM, 20nt, 5'd(ATG GTG CAT CTG ACT CCT GA)3']( SEQ ID NO:3), and the internal reverse of the T allele [50nM, 21nt, 5'd(CAG TAA CGG CAG ACT TCT CCA)3'] (SEQ ID NO:4) for detection of specific alleles . Templates for all three genotypes (mutant, heterozygous and wild type) were used. The reaction mixture contained 1X buffer, in...

Embodiment 2

[0065] The ARMS-PCR assay was used to detect 5 common mutations causing β-thalassemia using untreated human dried blood spots separately under a single PCR condition.

[0066] Such as Figure 4 As shown, an ARMS-PCR assay for β-thalassemia mutations was established using untreated human dried blood spots under one common PCR condition. Allele-specific primers have been used so that both alleles can be detected in the same reaction. A number of primers were designed to detect wild-type, mutant and heterozygous genotypes. Used external forward primer 1 [50nM, 20nt, 5'd (ACC TCA CCC TGT GGA GCC AC) 3'] (SEQ ID NO: 1), external forward primer 2 [50nM, 26nt, 5'd (GTA CGG CTG TCA TCA CTT AGA CCT CA) 3'] (SEQ ID NO: 11) and external reverse primer [50nM, 20nt, 5'd(TCA TTC GTC TGT TTC CCA TT) 3'] (SEQ ID NO: 2 ). For the IVS1>5G>C mutation in the HBB gene, an allele-specific primer for G internal forward [50nM, 20nt, 5'd(TGA GGCCCT GGG CAG GTA GG)3'] (SEQ ID NO :6) and an interna...

Embodiment 3

[0069] Allele-specific PCR assay for the detection of spinal muscular atrophy (SMA)-causing exonic deletions using untreated human DBS in a single reaction.

[0070] To detect deletions in the SMN gene for the diagnosis of SMA using unprocessed human dried blood spots, multiple allele-specific primers were designed. This will detect deletions of exon 7 and exon 8 in the SMN gene and simultaneously differentiate between SMN1 and SMN2 copies of the SMN gene in one reaction. Forward primer [50nM, 22nt, 5'd (TTT ATT TTC CTTACA GGG TTT C) 3'] (SEQ ID NO: 22) of exon 7 was used, internal reverse primer [50nM, 24nt, 5'd (GTG AAA GTA TGTTTC TTC CAC GTA)3'] (SEQ ID NO:24), internal forward primer for exon 8 [50nM, 25nt, 5'd(CTG GCATAG AGC AGC ACT AAA TGA C)3'] (SEQ ID NO:27), exon 8 specific reverse primer of SMN1 [50nM, 19nt, 5'd((TGG CCT CCC ACC CCC AAC C)3'](SEQ ID NO:23). As internal As a control, a control forward primer [50nM, 22nt, 5'd(AAG GAC AAT GGG AAC ACT CTC T)3'] (SEQ ID...

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Abstract

The present invention relates to a kit based on ARMS-PCR / AS-PCR in a single tube reaction for detecting mutations causing genetic disorders such as hemoglobinopathy and muscle disorders using untreated human dried blood spots.

Description

technical field [0001] The present invention provides kits for detection from unprocessed human dried blood spots using Amplification Arrested Mutation System (ARMS) / Allele Specific (AS) polymerase chain reaction (PCR). Mutations causing genetic disorders, wherein detection is performed in a single tube / reaction, and diagnostic kits thereof for detecting mutations causing genetic disorders like hemoglobinopathies and musculopathies. Background technique [0002] In the past few years, genetic testing has become available for a large number of genetic conditions, including various hemoglobinopathies, muscle disorders, neurodegenerative disorders, mitochondrial disorders, and bleeding and coagulation disorders. Genetic testing includes molecular diagnostics, carrier testing, predictive analytics, prenatal diagnosis, and genetic counseling. Hemoglobinopathy [including beta thalassemia, sickle cell anemia, hemoglobin E disease (HbE)] and muscle disease [including spinal muscula...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883
CPCC12Q1/6883C12Q2600/156C12Q1/6858
Inventor 吉里拉吉·拉坦·昌达克苏米特·帕里瓦尔斯瓦蒂·巴亚纳维奈·多尼帕迪
Owner COUNCIL OF SCI & IND RES
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