dna library construction method with low input

Abstract

A method for constructing a DNA library with a low initial amount, the method comprising: taking genomic DNA and adding random primers labeled with biotin for annealing and performing an amplification reaction; separating and purifying the whole genome amplification product; Fill in to obtain linear blunt-ended DNA; add ligase to the filled-in linear blunt-end DNA for ligation reaction; purify to obtain double-stranded circular DNA, and then break the double-stranded circular DNA into linear DNA fragments; use streptomycin Magnetic beads capture linear DNA fragments labeled with biotin; carry out end repair and A tail base reaction on the captured linear DNA fragments; connect adapters at both ends of the linear DNA fragments after adding A tail base reactions; The product was amplified by PCR to obtain a DNA library. The method uses biotin-labeled random primers to randomly amplify a small amount of genomic DNA, and at the same time realizes the amplification, truncation and biotin labeling of genomic DNA, thereby realizing the construction of a large-fragment DNA library with low initial input.

Description

technical field [0001] The invention relates to the technical field of library construction, in particular to a low-input DNA library construction method. Background technique [0002] High-Throughput Sequencing (High-Throughput Sequencing), that is, Nextgeneration Sequencing technology (Nextgeneration sequencing), is to achieve large-scale parallel sequencing on high-density biochips, which has the characteristics of high data output and low cost per unit of data volume. But its disadvantage is that the sequencing read length is short, and the general sequencing length is 2X300bp or 2X150bp. When the obtained short-read sequences have no reference genome alignment and assembly, or the genome contains highly complex structural sequences, the sequence alignment and assembly will be very difficult. At this time, the splicing and assembly of short sequences can be assisted by a large-span large fragment library (mate pair library). In addition, the link algorithm is used to a...

AI Technical Summary

Problems solved by technology

However, with the application of this technology in the detection of clinical chromosomal structural variation, some types of samples may be difficult to obtain or the amount is relatively small, such as tumor puncture samples, amniotic fluid cells, paraffin sections, etc.

At this time, the sample DNA may not be able to meet the needs of conventional large-fragment library construction

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