Low-initial-quantity DNA library construction method

Abstract

The invention discloses a low-initial-quantity DNA library construction method. The method comprises the following steps: taking genome DNA, adding a random primer marked with biotin, and carrying outannealing and an amplification reaction; separating and purifying a whole genome amplification product; carrying out tail end supplement on the separated and purified product to obtain linear blunt end DNA; adding ligase into the filled linear blunt end DNA to carry out connection reaction; carrying out purifying to obtain double-stranded annular DNA, and then breaking the double-stranded annularDNA into linear DNA fragments; capturing a linear DNA fragment labeled with biotin by using streptomycin magnetic beads; carrying out tail end repair on the captured linear DNA fragment and adding anA tail base for reaction; connecting joints to two ends of the linear DNA fragment subjected to the reaction by adding the A tail base; and carrying out PCR amplification on the product of the linkerto obtain a DNA library. According to the method, a small amount of genomic DNA is randomly amplified by using biotin-labeled random primers, and amplification, truncation and biotin labeling of thegenomic DNA are realized at the same time, so that construction of a large-fragment DNA library with low initial input is realized.

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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