Method for detecting nucleic acid molecules based on acrylamide gel chip

Abstract

The invention discloses a method for detecting nucleic acid molecules based on an acrylamide gel chip. The method comprises the following steps: fixing and sectioning tissues or cells according to an ordinary nucleic acid in-situ hybridization process, performing hybridization on single-chain DNA molecules containing closed loops and to-be-detected nuclear molecules by virtue of a base pairing principle, and also combining an amplification primer modified by acrylamide with another site of loop-closed single-chain DNA; adhering sections combined with loop-closed single-chain DNA and the amplification primer to acrylamide modified glass slides, and solidifying the sections by adding an acrylamide liquid above the sections; adding a rolling circle amplification system to ensure that the primer combined with the closed loops of the sections is subjected to rolling circle amplification along the closed loop molecules; and performing detection, calculation and positioning on the quantities and expression positions of to-be-detected nucleic acid molecules after the amplification is completed. The method disclosed by the invention achieves digital quantitative analysis of the nucleic acid molecules, provides a novel method for the quantitative analysis of the nucleic acid molecules, and can be used for establishing a rapid, accurate and cheap nucleic acid detection technique.

Description

technical field [0001] The invention belongs to the technical field of biological engineering, and in particular relates to a method for detecting nucleic acid molecules based on an acrylamide gel chip. Background technique [0002] Fluorescence in situ hybridization (FISH) is a non-radioactive molecular cytogenetic technology developed on the basis of radioactive in situ hybridization technology. method. The basic principle of FISH is to label DNA (or RNA) probes with special nucleotide molecules. The nucleic acid probes used are homologous and complementary to the nucleic acid to be detected, and the two can be denatured-annealed-renatured. Hybridization of target DNA and nucleic acid probes is formed. A certain nucleotide of the nucleic acid probe is labeled with a reporter molecule such as biotin and digoxigenin, and the immunochemical reaction between the reporter molecule and the specific avidin labeled with fluorescein can be used, and the fluorescence detection sys...

AI Technical Summary

Problems solved by technology

Although the fluorescence microscope or laser confocal microscope is used to observe the hybridization results, it still cannot overcome the problem that the hybridization signal is not clearly displayed.

Although some methods of signal amplification have been used for FISH detection, however, the problem is that either a variety of different probes are required, resulting in high cost; or signal amplification is performed by in situ amplification, such as recently There is signal amplification in the original rolling circle amplification, however, the process is too complicated: the target mRNA needs to be reverse-transcribed in situ first, then ligated in situ, followed by rolling circle amplification in situ, the whole process not only operates It is complicated, and it is easy to cause signal loss during the process. At the same time, because the amplification product cannot be well fixed on the position of the original mRNA, it is easy to cause the loss of the product in the subsequent experimental process.