Single molecule dsDNA microarray chip preparation method

Abstract

A process for preparing the unimolecular ds DNA microarray on the surface of solid carrier includes chemically synthesizing target oligonucleotide and universal oligonucleotide, renaturation of them, linking them to the surface of solid carrier to become oligonucleotide microarray, on-chip polymerase elongation reaction to become bimolecular dsDNA microarray, modifying to become ssDNA microarray, renaturation to form hairpin structure, and on-chip polymerase elongation reaction.

Description

1. Technical field: [0001] The present invention proposes a new technical method for preparing unimolecular double-stranded deoxyribonucleic acid microarray (unimolecular dsDNA microarray) on solid supports (solid sustracts). The prepared unimolecular dsDNA microarray chip can be used in basic molecular biology It has important application value in the field of science and biomedicine. In basic molecular biology research, the single-molecule dsDNA microarray chip prepared by the present invention can be used as a high throughput (high throughput) to identify DNA-binding targets of DNA-binding proteins (DNA-binding proteins), screen and detect DNA-binding proteins, A technical platform for analyzing the interaction between DNA-binding proteins and their DNA-binding targets; in biomedical research, the single-molecule dsDNA microarray chip prepared by the present invention can be used as an auxiliary diagnosis of DNA-binding protein-related diseases, research on the mechanism of...

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Problems solved by technology

The advantage of this method is that it can use DNA microarray in situ synthesis technology to prepare high-density dsDNA microarray; but this method also has serious economic and technical problems, technically it relies on photolithographic mask in situ DNA microarray Synthetic patented technology, but the synthesis efficiency of single-stranded oligonucleotides on the surface of the solid phase substrate is not high, and the synthesis efficiency of each nucleotide is 92-96%. Only 4-20% of the oligonucleotides reach the required length of 40 bases. Therefore, the ssDNA and dsDNA microarrays prepared by photolithographic mask in situ synthesis technology are seriously polluted by truncated molecules.

Numerous competing truncated molecules would likely strongly inhibit and mislead protein binding experiments; and there is no reason to believe that every oligonucleotide on a microarray of single-stranded oligonucleotides synthesized in situ by a photolithographic mask could be primed oligonucleotides. Nucleotide binding, so that part of the 40-base full-length oligonucleotide cannot be converted to double-stranded nucleic acid, which not only further reduces the number of ideal double-stranded nucleic acid probes on the chip, but also those that are not converted to double-stranded Single-stranded oligonucleotides also interfere with protein binding experiments

In addition, the dsDNA microarray prepared by this method is a bimolecular (bimolecular) dsDNA microarray, and there is a problem of probe stability, that is, the bimolecular dsDNA is affected by the experimental procedures such as binding and washing, and the double strands will be unzipped and lost. The probe function can only be used once, so its use efficiency is low and the use cost is high

Because the dsDNA microarray prepared by this method relies on the currently very expensive patented technology of photolithography mask in situ DNA microarray synthesis, coupled with the patent protection of this method itself, it is not only very expensive for commercial application

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