Limited fluorescence labelling method

Abstract

The limited fluorescence labeling process includes the following steps: digesting with limitative endonuclease the cDNA obtained through extracting, purifying and inversely transcribing nucleic acid sample or the proliferated product obtained via multiple PCR proliferation of primer designed for several pairs of target genes to produce many limitative gene segments in the relatively consistent size; designing junction for connection with the above said limitative enzyme incised segments; designing universal PCR primer based on the said limitative endonuclease site and junction sequence and labeling fluorescence molecule in the 5' end of the said primer; PCR proliferation of the sample with the fluorescence labeled universal primer and crossing the purified proliferated sample with DNA chip. The said process can strength the crossing signal obviously, decrease fluorescent matter consumption and raise the sensitivity of chip detection, and is suitable especially for clinical detection chip of pathogen gene.

Description

technical field [0001] The invention relates to a fluorescent labeling method, in particular to a restricted fluorescent labeling method, which can be used to simultaneously amplify and label multiple target genes in a sample. Background technique [0002] According to the properties of the solidified probes on the DNA chip, the DNA chip can be divided into the following types: oligonucleotide chip, cDNA chip or gene chip. The method of DNA chip technology mainly includes the following four aspects: chip preparation, sample preparation, molecular hybridization and detection analysis. The sample preparation includes the separation and purification of sample nucleic acid, amplification and labeling. Due to the limited sensitivity of current chip detection instruments, the isolated and purified nucleic acid needs to be amplified and labeled during the amplification process to detect the hybridization result. At present, the labeling of samples mainly adopts fluorescent labeli...

AI Technical Summary

Problems solved by technology

The above-mentioned methods have the following limitations: ① the fluorescent nucleotide is not a natural substrate of any polymerase, and the fluorescent part connected to the nucleotide is usually relatively large, so the efficiency of polymerase incorporation of nucleotide will be higher than that of incorporation. Incorporation of its natural substrates is much less efficient and thus may affect reverse transcription and PCR product formation

② The fluorescent nucleotides are incorporated into the new strands in a random manner. Therefore, there are certain differences in the amount of fluorescent incorporation of various nucleic acids in the sample. When comparing gene expression levels in this way, there will be a deviation

③The fluorescent nucleotide analogues are quite expensive and require a large amount in reverse transcription or PCR

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