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Method of amplifying dna chip signals
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A DNA chip and signal amplification technology, applied in the field of DNA chip signal amplification, can solve the problems of complex operation, low sensitivity and high cost
Inactive Publication Date: 2003-12-31
三光纯药株式会社
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[0003] At present, this technology has been used for gene expression analysis and detection of mutated genes, etc. However, it is different from the Southern plotting method (E.M.Southern, Detection of Specific sequences among DNA fragments separated by gel lectrophoresis, J.Mol. Biol., 98, 503-517, 1975) compared, there is detection sensitivity low, is 1 / 10, and the problem of long reaction time
[0004] In addition, in order to improve the detection sensitivity of the target gene, there have been the LCR method (USP5,792,607) to amplify DNA using a thermostable DNA ligase and the PCR method to amplify DNA with a thermostable DNA polymerase ( R.Saiki, S.Scharf, F.Faloona, et al., Enzymatic amplification ofβ-globin genomic sequence and restriction site analysis for diagnosis of sicklecell anemia, Science, 230, 1350-1354 (1985)) and other gene amplification methods, Although there is a method to amplify the target gene in advance, it requires complicated operation and high cost
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Embodiment 1～3
[0095] As a hybridization solution, (0.8 μg / μL poly-dA, 0.1 μg / μL yeast tRNA, 0.15 μg / μL human Cot-I DNA, 5×Denhardt's solution, 0.1 mg / mL Sarmon sperm DNA, 0.2% SDS, 6 ×SSC) hybridization solution. (Examples 1 to 3 and Comparative Examples 1 to 3) (1) Purpose
[0096] A Cy3-labeled pair of HCPs (HCP-1 and HCP-2) was used to study the effect of signal amplification. (2) Material (Examples 1-3)
[0097] (a) Capture probes prepared in 500 ng / μL (Example 1), 50 ng / μL (Example 2), and 5 ng / μL (Example 3) respectively used capture probes having a region complementary to the target gene-1. DNA Probe-1a.
[0098] (b) As a primary hybridization solution, dissolve 50 ng of target gene-1 in 10 μL of hybridization solution, heat at 95° C. for 2 minutes, and prepare primary hybridization solution A.
[0099] (c) As a secondary hybridization solution, 25 pmol of Cy3-labeled HCP-1 and 25 pmol of Cy3-labeled HCP-2 were dissolved in 10 μL of hybridization solution, and heated at 95°C for ...
Embodiment 4～6
[0108] (a) Capturing probes The capturing DNA probe-1a prepared at 500 ng / µL (Example 4), 50 ng / µL (Example 5), and 5 ng / µL (Example 6) was used, respectively.
[0109] (b) As the primary hybridization solution, the above-mentioned primary hybridization solution A was used.
[0110] (c) As a secondary hybridization solution, dissolve 25 pmol HCP-1 and 25 pmol HCP-2 in 10 μL of hybridization solution, heat at 95° C. for 2 minutes, and prepare secondary hybridization solution C. (Comparative Examples 4-6)
Embodiment 7～9
[0118] (a) Capturing probes The capturing DNA probe-1a prepared at 500 ng / µL (Example 7), 50 ng / µL (Example 8), and 5 ng / µL (Example 9) was used, respectively.
[0119] (b) As the primary hybridization solution, the above-mentioned primary hybridization solution A was used.
[0120] (c) As the secondary hybridization solution, the above-mentioned secondary hybridization solution C was used. (Comparative examples 7-9)
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Abstract
A signal amplification method for a DNAchip establishes efficient signal amplification for a target gene captured on the DNAchip by the PALSAR method, and further establishes a simple detection by contriving design of a pair of HCPs to be used in the PALSAR method. Sensitivity for detection of the target gene on the DNAchip is improved by making use of a self-assembly reaction which forms a double-stranded self-assembly substance having a regular higher-order structure of oligonucleotides.
Description
technical field [0001] The invention relates to a signal amplification method of a DNA chip. More specifically, it relates to the use of a pair of oligonucleotides that form self-polymers, which can be used as a support or substrate (hereinafter referred to as a support) that binds to a gene for capturing a target gene, using a microplate type, a glass slide, etc. A method for amplifying a gene signal based on the detection sensitivity of a target gene on a DNA chip (hereinafter collectively referred to as a DNA chip) on a support such as a microparticle type, a microparticle type, or a conductive substrate type. Background technique [0002] The DNA chip is to hybridize the nucleic acid (target) after a number of different DNA probes are immobilized at high density on the microplate type, glass slide type, particle type, conductive solid phase substrate type support specially processed on the surface (see M. Judy, W. Geoffrey, Hybridization of Nucleic Acids Immobilized on ...
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