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Primer composition for lysosomal disease gene screening and kit using the same

A primer composition and technology for lysosomal diseases, applied in the direction of recombinant DNA technology, DNA/RNA fragments, etc., can solve problems such as difficulties in differential diagnosis

Inactive Publication Date: 2014-11-05
封志纯
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since lysosomal diseases involve a variety of diseases, and the clinical manifestations of these diseases are mostly the same, and the differential diagnosis is difficult, these methods often have certain limitations when meeting the needs of clinical detection of gene mutations

Method used

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  • Primer composition for lysosomal disease gene screening and kit using the same
  • Primer composition for lysosomal disease gene screening and kit using the same
  • Primer composition for lysosomal disease gene screening and kit using the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Embodiment 1 is used for the primer composition of lysosomal disease gene screening

[0114] According to IDUA (ID: 3425), ARSB (ID: 411), MAN2B1 (ID: 4125), ST3GAL5 (ID: 8869), SMPD1 (ID: 6609), CTNS (ID: 1497), MFSD8 (ID) published in Genbank :256471), IDS(ID:3423), GUSB(ID:2990), GBA(ID:2629), ARSA(ID:410), GNPTAB(ID:79158), SLC17A5(ID:26503), CLN8(ID: 2055), SGSH(ID:6448), HYAL1(ID:3373), PSAP(ID:5660), GNPTAG(ID:2795), PPT1(ID:5538), CTSD(ID:1509), NAGLU(ID:4669 ), FUCA1(ID:2517), GLB1(ID:2720), SUMF1(ID:285362), MCOLN1(ID:57192), TPP1(ID:1200), HGSNAT(ID:138050), NAGA(ID:4668) , GM2A(ID:2760), GALC(ID:2581), LIPA(ID:3988), CLN3(ID:1201), GALNS(ID:2588), NEU1(ID:4758), HEXA(ID:3073), GLA(ID:2717), NPC1(ID:4864), CLN5(ID:1203), MANBA(ID:4126), HEXB(ID:3074), ASAH1(ID:427), NPC2(ID:10577) and CLN6 (ID: 54982) gene sequence, design a primer sequence of 18-40bp length for the exon and exon-intron junction region of the above-mentioned gene sequence annotation; the l...

Embodiment 2

[0115] Embodiment 2 is used for the test kit of lysosomal disease gene screening

[0116] The kit described in this embodiment mainly includes: 2x primer composition, 5x PCR ion amplification mix (PCR buffer, dNTPs, Taq enzyme, etc.), 96-well plate and 96-well plate sealing film.

[0117] The kit also includes the following components (provided by Life technologies):

[0118] Ion Xpress Barcode Adapters 1-16 Kit (Cat. no. 4471250);

[0119] MyOne TM Streptavidin C1 beads (Cat. no. IVGN65001);

[0120] Ion PI TM Template OT2 200 Kit v3 (Cat. no. 4488318);

[0121] Ion Library Quantitation Kit (Cat. no. 4468802).

Embodiment 3

[0122] Example 3 Application of the kit for lysosomal disease genetic screening

[0123] 1. Sample requirements

[0124] More than 30ng of DNA sample is required.

[0125] 2. DNA library construction

[0126] 2.1 PCR amplification of the target region of genomic DNA

[0127] 2.1.1 Mix each DNA and primer composition, and add 5XPCR ion amplification mix (PCR buffer, dNTPs, Taq enzyme, etc.) and deionized water into each reaction well of the PCR96-well plate. For example, to amplify a 20ul system, add 4ul of 5X PCR ion amplification mix, 10ul of primer composition, 5ul of deionized water, and 10ng of DNA sample.

[0128] 2.1.2 Seal the PCR plate with a 96-well plate sealing film, shake it fully, and simply centrifuge the PCR plate to recover as much sample as possible to the bottom of the tube. Then place it in a PCR machine and run the following programs to amplify the target region on the genome.

[0129] 2.2 Digest part of the primer sequence

[0130] 2.2.1 Slightly cen...

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Abstract

The invention provides a primer composition for lysosomal disease gene screening and a kit using the same. The primer composition comprises multiple primer sequences respectively aiming at non-repetitive regions of IDUA, ARSB, MAN2B1, ST3GAL5, SMPD1, CTNS, MFSD8, IDS, GUSB, GBA, ARSA, GNPTAB, SLC17A5, CLN8, SGSH, HYAL1, PSAP, GNPTAG, PPT1, CTSD, NAGLU, FUCA1, GLB1, SUMF1, MCOLN1, TPP1, HGSNAT, NAGA, GM2A, GALC, LIPA, CLN3, GALNS, NEU1, HEXA, GLA, NPC1, CLN5, MANBA, HEXB, ASAH1, NPC2 and CLN6 genes.

Description

technical field [0001] The invention relates to the field of detection kits, in particular to a primer composition and a kit for genetic screening of lysosomal diseases. Background technique [0002] Lysosomal disease is due to the lack of certain hydrolytic enzymes in lysosomes due to genetic defects, so that the corresponding substrates cannot be degraded and accumulated in lysosomes, resulting in cell metabolism disorders and leading to diseases. Lysosomal diseases are a group of disorders that include deficiencies in nearly 50 enzymes. The incidence of a single lysosomal disease is low, but as a group of diseases, its incidence in the population is about 1:6000-7000. According to statistics, about 2,700 of the 16 million newborns in China suffer from lysosomal diseases each year. Most of the lysosomal diseases onset in infants and adolescents. It is of vital significance to clarify the cause of disease and disease-causing genes, as well as early diagnosis and prenatal ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883
Inventor 封志纯王艳杨尧
Owner 封志纯
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