Primer combination, method and kit for constructing targeted library of multiple inherited metabolic liver diseases based on high-throughput sequencing

A primer combination, high-throughput technology, used in biochemical equipment and methods, chemical libraries, combinatorial chemistry, etc., can solve the problems of gene coverage, random missed detection, cumbersome operation, etc., to avoid workload and economic costs, Time saving, simple operation effect

Active Publication Date: 2019-03-15
THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the heterogeneity and individual differences of gene mutations determine that the detection of genetic metabolic liver disease gene mutations needs to cover all exons and intron splicing regions of the disease-causing gene in order to be able to identify accurately, fully and without omission For its pathogenic mutations, simply detecting several hotspot mutation sites of a certain gene will result in a large number of missed detections
At present, there is no genetic testing kit for simultaneous detection of multiple genetic metabolic liver diseases on the market.
[0005] At present, Sanger sequencing is mostly used at home and abroad to detect a single genetic metabolic liver disease. This method can only detect a few hotspot mutations of a gene at a time, and it is difficult to detect potential pathogenic sites and new mutation sites of the entire gene. If all are detected, there may be more than 50% false negatives, and only one gene can be detected at a time. When the direction of suspected diagnosis is vague, it needs to be detected one by one gene by one site, and the operation is cumbersome, time-consuming and labor-intensive
At present, only the method disclosed in the CN 106957901 A document can be retrieved in China to simultaneously detect multiple genetic metabolic liver diseases. This method uses PCR-sequencing method to sequence PCR reactions one by one, and can only detect hyperbilirubinemia A small number of hotspot mutations in , Wilson's disease, and / or hemochromatosis
With the advancement of technology, whole-exome high-throughput sequencing has realized the simultaneous detection of multiple gene variations, but whole-exome high-throughput sequencing is expensive, and it is very wasteful to detect a small number of single-gene diseases. Due to the huge size of the human genome and the limitations of the whole exome sequencing technology itself, there will be a small number of genes that cannot be covered and randomly missed

Method used

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  • Primer combination, method and kit for constructing targeted library of multiple inherited metabolic liver diseases based on high-throughput sequencing
  • Primer combination, method and kit for constructing targeted library of multiple inherited metabolic liver diseases based on high-throughput sequencing
  • Primer combination, method and kit for constructing targeted library of multiple inherited metabolic liver diseases based on high-throughput sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Primer combination

[0021] For SERPINA1, G6PC, SLC37A4, AGL, PYGL, PHKA1, PHKA2, PHKB, PHKG2, GYS2, ASS1, SLC25A13, ASL, HFE, TFR2, SLC40A1, ALAS2, FECH, CFTR, HSD3B7, CYP7B1, AKR1D1, AMACR, CYP27A1, BAAT , GBA, ALDOB, LIPA, UGT1A1, ABCC2, SLCO1B1, SLCO1B3, ATP8B1, ABCB11, ABCB4, TJP2, NR1H4, VPS33B, VIPAS39, JAG1, ATP7B, NOTCH2 and BCHE, a total of 703 exons and splice regions of 43 genes , the UGT1A1 gene also includes the promoter region TATA-box, the phenobarbital enhancer region and the proximal element region, a total of 1119 pairs of primers were designed and synthesized, and another 7 primers were designed and synthesized for the 17th amplicon of the PYGL gene Forward degenerate primer. There are 2245 primers in total, the base sequences of which are shown in SEQ ID No.1-SEQ ID No.2245.

[0022] Genes and regions detected:

[0023]

[0024]

[0025] The optimized primers are as follows:

[0026]

[0027]

[0028]

[0029]

[0030...

Embodiment 2

[0038] Example 2 Detection sample processing and DNA extraction

[0039] The detection sample of the present invention may be whole blood, blood clot, fresh pathological tissue, paraffin-embedded tissue, and this embodiment only uses whole blood sample as an example for illustration.

[0040] In order to reduce the interference of various anticoagulants on the PCR reaction, venous blood should be collected with EDTA-K2 anticoagulant blood collection tubes. The venous blood should be extracted and purified every day. If it cannot be extracted in time on the same day, it should be stored in a 4°C refrigerator. save. Conventional DNA extraction and purification methods such as DNA extraction kits or automatic DNA extractors can be used. The DNA concentration was detected with a Nanodrop 2000 trace nucleic acid and protein detector, and the 260 / 280 ratio and 260 / 230 ratio were determined. The extracted DNA is subjected to 0.5% agarose gel electrophoresis, and a clear electrophor...

Embodiment 3

[0041] Example 3 Construction of various genetic metabolic liver disease targeting libraries

[0042] Standardize the sample DNA extracted in Example 2, adjust the concentration to 40-50ng / ul as the amplification template, and use all or part of the primers from SEQ ID No.1 to SEQ ID No.2245 described in Example 1 The primer mixture pool composed of primers is used for ultra-high multiplex PCR amplification. Perform PCR amplification according to the following amplification system and conditions, and perform ultra-high multiplex PCR amplification according to the multiple ratio volume or equal concentration system of the following system:

[0043]

[0044] Preferably, the ultra-high multiplex PCR amplification conditions are: 95°C for 10 min; 98°C for 15 s, 60°C for 5 min, 10 cycles; 10°C∞. That is, pre-denaturation at 95°C for 10 minutes; denaturation at 98°C for 15 seconds, and annealing at 60°C for 5 minutes, a total of 10 cycles.

[0045] The amplification product is ...

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Abstract

The invention discloses a primer combination, method and kit for constructing a targeted library of multiple inherited metabolic liver diseases based on high-throughput sequencing. The base sequence of the primer combination is shown as SEQ ID No. 1 to SEQ ID No. 2245, and the primer combination covers total 703 exons and cutting regions of 43 genes such as SERPINA1, G6PC, SLC37A4, AGL and the like, and is capable of detecting 41 sub-type gene variation loci of totally 20 inherited metabolic liver diseases comprising alpha-1-antitrypsin deficiency, glycogen storage disease, citrullinemia, aminosuccinuria, hemochromatosis, porphyrinopathy, cystic fibrosis, bile acid synthesis defect, Gaucher disease, hereditary fructose intolerance, cholesterol ester storage disease, Gilbert syndrome, Dubinsyndrome, Rotor syndrome, progressive familial intrahepatic choleatasia and the like. The primer combination disclosed by the invention is simple in operation, high in accuracy and low in time consumption, and the time cost and manual cost used for detecting genes and fragments one by one during Sanger sequencing can be greatly saved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer combination, a method and a kit for constructing multiple genetic metabolic liver disease targeting libraries based on high-throughput sequencing. Background technique [0002] Inherited metabolic liver disease (IMLD) is a type of liver metabolic disorder caused by inherited enzyme defects. There are many kinds of inherited metabolic liver diseases. Currently, more than 600 kinds of genetic metabolic liver diseases have been identified, mainly including carbohydrate metabolism diseases, amino acid metabolism diseases, fatty acid metabolism diseases, organic acid metabolism diseases, mitochondrial liver diseases, lysosomal diseases, excessive Oxisome diseases, metal metabolism disorders and α1-antitrypsin deficiency and other 9 categories. As far as a single disease is concerned, the incidence rate is not high, but the overall incidence rate cannot be ignored. Hereditary me...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6806C12N15/11C12N15/10C40B50/06
CPCC12N15/1093C12Q1/6806C12Q1/6883C12Q2600/16C40B50/06C12Q2531/113C12Q2537/143
Inventor 邓国宏谭文婷但芸婕孙凤明王修华
Owner THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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