16S rDNA library construction method, 16S rDNA library and application

Abstract

The invention belongs to the technical field of genetic engineering high-throughput sequencing, and discloses a 16S rDNA library construction method, a 16S rDNA library and application, and the 16S rDNA library construction method comprises the following steps: repairing the tail end of a DNA fragment; specific joint connection; and carrying out PCR amplification. According to the 16S rDNA library construction method provided by the invention, the cost can be greatly reduced by a next-generation sequencing technology represented by illumina, and the 16S rDNA library construction method is suitable for large-scale sequencing. The essence of 16S rDNA library establishment is to perform PCR enrichment and screening on a specific region by using enzyme and primers, and the library establishment method is lower in cost compared with methods such as a machine interruption method and the like. The 16S rDNA not only can reflect the difference between different bacteria genus, but also can easily obtain the sequence by utilizing a sequencing technology, and is widely applied to detection and identification of pathogenic bacteria at present. According to the 16SrDNA library building method disclosed by the invention, a library building system is obtained after multiple experiments, so that the library building time and cost can be effectively saved.

Description

technical field [0001] The invention belongs to the technical field of high-throughput sequencing of genetic engineering, and in particular relates to a method for constructing a 16S rDNA library, a 16S rDNA library and an application thereof. Background technique [0002] At present, with the rapid development of molecular biology, the classification and identification of bacteria has entered various genotype classification levels from traditional phenotype, physiological and biochemical classification, such as (G+C) mol%, DNA hybridization, rDNA fingerprint, plasmid Map and 16S rDNA sequence analysis, etc. There are three types of ribosomal RNA in bacteria: 5S rRNA, 16S rRNA, and 23S rRNA. The rRNA gene consists of a conserved region and a variable region. 16S rRNA corresponds to a segment of gene sequence on genomic DNA called 16S rDNA. Although 5S rRNA is easy to analyze, it has too few nucleotides, only tens of bp, and there is not enough genetic information for clas...

AI Technical Summary

Problems solved by technology

Although 5S rRNA is easy to analyze, it has too few nucleotides, only tens of bp, and there is not enough genetic information for classification research; difficulty

[0004] (1) There are too few 5S rRNA nucleotides, only tens of bp, and there is not enough genetic information for taxonomic research

[0005] (2) The number of nucleotides contained in 23S rRNA is twice that of 16S rRNA, the molecular weight is large, and analysis is difficult

[0006] (3) The method for 16S rDNA library construction in the prior art has not been reported yet

[0007] The difficulty in solving the above problems and defects is: because 5S rRNA has too few nucleotides, there is not enough genetic information for classification research, so 5S rRNA is not analyzed; studies have shown that 23S rRNA has a large molecular weight, and its superiority in bacterial diagnosis The specificity is higher than that of 16S rRNA, which is more suitable for the differential diagnosis of common clinical pathogenic microorganisms, but in clinical samples, designing primers for a variety of bacteria for multiple PCR reactions often causes non-specific reactions

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