Oligonucleotide chip and its application of detecting muatatonal site of hepatitis B virus

A technology of oligonucleotides and mutation sites, applied in the field of oligonucleotide detection chips, can solve the problems of low detection sensitivity, difficulty in parallel analysis, long operation time, etc.

Inactive Publication Date: 2003-07-23
SHANDONG MEDICAL BIO TECH RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is difficult for them to analyze a large number of samples simultaneously and in parallel, and the required sample volume is large, the detection sensitivity is low, and the operation time is long

Method used

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  • Oligonucleotide chip and its application of detecting muatatonal site of hepatitis B virus
  • Oligonucleotide chip and its application of detecting muatatonal site of hepatitis B virus
  • Oligonucleotide chip and its application of detecting muatatonal site of hepatitis B virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1. Oligonucleotide chip

[0047] The structure is shown in Figure 1: On the glass substrate 1, there are oligonucleotide probes and control dot coating 2 uniformly distributed in an array, which contains known mutation sites and wild sites in the pre-C and S regions of hepatitis B. There are 72 oligonucleotide probes, and each site has a positive control. The glass substrate is a rectangular sheet, which is a silanized glass slide, the length of the oligonucleotide probe is 14-19mer, the difference in Tm value is not more than 10°C, and the percentage of GC bases is 50%-70%.

[0048] The dot coating 2 array of oligonucleotide probes and controls has a total of 8 rows and 9 columns, the pin spot diameter is 100 μm, the pitch is 300 μm, and the total area is 2.2 mm×2.5 mm. 1-6, 7-12, 13-18, 19-24, 25-30, 31-36, 37-42, 43-48, 49-54, 55-60, 61-66, 67-72 represent hepatitis B 12 groups of 72 oligonucleotide detection probes for detection sites 516, 551, 552, 585, 5...

Embodiment 2

[0050] Embodiment 2. The oligonucleotide chip as described in embodiment 1, the difference is that the glass substrate 1 is modified through trimethoxypropyl-ethyleneimine, succinic acid-N-hydroxylated succinyl Imidate-activated glass slides.

Embodiment 3

[0051] Example 3. Oligonucleotide detection chip is used for the detection of mutation site of hepatitis B virus 1. Design and synthesis of oligonucleotide probe for detection of mutation site of hepatitis B

[0052] Oligo5.0 software was used to design probes. The detection probes are oligodeoxynucleotides with a length of 14-19mer. The probes for each mutation site include 2 mutation detection probes, 2 wild detection probes, and 2 positive control probes, which are respectively designed in hepatitis B Sense and antisense strands of DNA. The last bases at the 3' ends of the four detection probes are respectively the mutation sites to be detected on the positive and antisense strands, and the two positive control probes lack the bases to be detected at the 3' ends. (Attached Figure 2 is a schematic diagram of the probe design at the 546 site in the S region) The Tm values ​​of all probes are calculated by GC percentage and are between 65°C and 75°C. See Table 1 for informat...

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Abstract

An oligonucleotide chip for detecting the mutation sites of hepatitis B virus is composed of glass substrate, oligonucleotide probes array and reference point-type coated laeyr. The said probes arrayhas 72 probes, including 12 known mutation sites and relative wild sites. The process for detcting mutation sites of hepatitis B virus includes such steps as mixing the probes as solid primer, hepatitis B virus DNA and template, and three pairs of liquid primers, PCR amplification via fluorescence labeled Cy3-dCTP, and analyzing mutation sites according to intensity of fluorescent signal. Its advantages are high speed, efficiency and correctness.

Description

(1) Technical field [0001] The invention relates to the technical field of gene chips, in particular to an oligonucleotide detection chip for mutation sites of hepatitis B virus. The solid-phase PCR of the oligonucleotide chip is used to detect mutation sites of hepatitis B virus. (2) Background technology [0002] Gene chip technology is a new biotechnology developed in the 1990s, which is the integration of polymerase chain reaction (PCR) and Shaw (Southern) hybridization. It immobilizes a large amount of DNA with a specific sequence on the surface of the carrier in an orderly manner through microelectronic technology and microprocessing technology, forming a DNA array that stores a large amount of information. After the array is hybridized with labeled probes, a large amount of information can be acquired quickly and accurately in a short time. It has the characteristics of high throughput, miniaturization and parallel analysis. There are two main methods for the prepar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 鲁艳芹韩金祥
Owner SHANDONG MEDICAL BIO TECH RES CENT
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