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Mitochondrial diseases MELAS and MERRF syndrome related mtDNA mutation site detecting gene chip and detecting method

A technology for detecting gene chips and mutation sites, which is applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of heavy workload, complexity, and high cost of RFLP, achieve high sensitivity, shorten detection time, and shorten The effect of time to diagnosis

Inactive Publication Date: 2008-05-14
ANHUI MEDICAL UNIV +1
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Although many mtDNA mutant detection methods have been established, such as restriction fragment length polymorphism (RFLP), single-strand conformation polymorphism (SSCP), specific primer sequence analysis (SSP), and conformation-sensitive electrophoresis (CSGE). , Transient Temperature Gradient Electrophoresis (TTGE), Denaturing Gradient Gel Electrophoresis (DGGE) and Denaturing High Performance Liquid Chromatography (DHPLC), etc., but there are still many limitations in practical application, such as high cost of RFLP, heavy workload and some important The mutation site cannot be detected because it is not recognized by the restriction endonuclease used, and other methods are more complicated than RFLP
More importantly, most of these methods can only detect one or a few mutation sites, and cannot simultaneously screen mutants with different genes at high throughput. The results of some methods still need to be verified by molecular sequencing

Method used

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  • Mitochondrial diseases MELAS and MERRF syndrome related mtDNA mutation site detecting gene chip and detecting method
  • Mitochondrial diseases MELAS and MERRF syndrome related mtDNA mutation site detecting gene chip and detecting method
  • Mitochondrial diseases MELAS and MERRF syndrome related mtDNA mutation site detecting gene chip and detecting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Probe Design and Chip Preparation

[0059] (1) Design of probes on the chip:

[0060] According to the 31 mtDNA point mutations related to MELAS and MERRF syndromes reported in the literature and the revised Cambridge mtDNA reference sequence (revised Cambridge Reference Sequence, rCRS), 62 oligonucleotide probes were designed with Oligo 6.0 software, so that the different probes The Tm value should be as close as possible to (45±5)°C, so that the length of the probe is 14-19 bp, so that the mutation site detected by each pair of probes is located in or near the middle of the probe sequence, so that the probe is compatible with the corresponding single-stranded target gene. The binding site should be as close as possible between the 5' end and the middle region of the single-stranded target gene to ensure hybridization efficiency. The 5' end of the probe has an amino modification group, so as to combine with the aldehyde group modification chip; the comprehen...

Embodiment 2

[0068] Example 2 Exploration and optimization of chip detection conditions

[0069] The hybridization efficiency and thermal stability of the target molecule and the on-chip oligonucleotide probe have a lot to do with the sequence of the probe and the strict control of the reaction conditions. For example, the length of the probe sequence, G+C content, Tm value; the concentration of the probe and the target molecule, the length of the spacer arm, the temperature, time, pH and salt ion concentration of hybridization and washing, etc. The present invention emphatically explores aspects such as the sample concentration of probe, the coating time of probe, hybridization temperature and time, and elution condition (comprising the concentration of elution buffer, elution temperature and elution time) and so on. Optimization is described as follows:

[0070] Chip preparation:

[0071] Hybridization solution: DIG Easy Hyb, purchased from Roche

[0072] a. Optimization of probe conc...

Embodiment 3

[0091] Embodiment 3 repeatability experiment

[0092] Chip sensitivity experiment: select wild-type and mutant probes (SEQ ID NO.51 and 52) of T8344C, a representative and common mutation site of MERRF-related mtDNA, as probes for chip repeatability experiments. The sample concentration is 12.5uM. Cartesian Tech., PROSYS 5510A chip fabrication system dotted on the surface of aldehyde-modified glass slides, 20 spots for each probe on each chip, and placed at 70% relative humidity and room temperature for 48 hours to fix. After taking it out, place it in a boiling water bath for 30 seconds, fully dry it in the air, and store it at 4°C.

[0093] At the same time in five days, take 5ul of PCR products of the same concentration (primers are SEQ ID NO.67 and 68, DNA template is healthy person N1) mixed with 10ul of hybridization solution and spot on the chip, hybridize at 37°C for 30min, 30°C Wash with 2xSSC+0.1%SDS for 10min, 0.2xSSC+0.1%SDS for 5min, and scan the above-mentione...

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Abstract

The invention relates to a mutation site examination of mitochondrial diseases MELAS and MERRF syndrome correlated mtDNA, disclosing a mutation site detection gene chip correlated with the syndrome mtDNA of the mitochondrial diseases MELAS and MERRF, wherein, a specific oligonucleotide probe is fixed on the surface lattice of the gene chip carrier and is used for detecting the mtDNA mutation sites correlated with mitochondrial diseases syndrome MELAS and MERRF. The invention also further discloses a preparation and application method of the gene chip. The gene chip of the invention can be used for detecting all mutation sites mtDNA concerned with the mitochondrial disease syndrome MELAS and MERRF with high sensibility and specificity and good repeatability; compared with the traditional molecular biological methods, the testing time is greatly shortened, and the purposes of clinical diagnosis and identification diagnosis of the MELAS and MERRF syndrome are realized.

Description

technical field [0001] The invention relates to the detection of mtDNA mutation sites related to mitochondrial disease MELAS and MERRF syndrome, in particular to a gene chip for detecting mtDNA mutation sites related to mitochondrial disease MELAS and MERRF syndrome and its production method, and using the chip to detect clinical samples of patients Detection and screening of mtDNA mutation sites associated with MELAS and MERRF syndromes in China. Background technique [0002] It is now known that mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) and myoclonic epilepsy with ragged red fibers (MERRF) ) are two common mitochondrial diseases mainly caused by mutations in mtDNA. MELAS is a group of mtDNA disorders with high clinical variability and genetic heterogeneity, most MELAS cases are caused by mt-tRNA Leu(UUR) Genetic heterogeneity mutation A3243G caused; a few are caused by other mtDNA point mutations (such as T3271C) or large fragm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 杜卫东张学军曹慧敏赵建龙陈刚李伟
Owner ANHUI MEDICAL UNIV
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