Asymmetric deoxyribose nucleic acid (DNA) artificial adapters by using second-generation high-throughput sequencing technology and application thereof

Abstract

The invention provides asymmetric deoxyribose nucleic acid (DNA) artificial adapters by using a second-generation high-throughput sequencing technology, which is composed of two DNA oligonucleotide single strands. The preparation method comprises the following steps: dissolving the two DNA oligonucleotide single strands into a quenching solution; regulating the final concentration to 2mM and volume to 20microlitre; carrying out a quenching reaction to form the DNA artificial adapters which are locally and mutually complemented at a temperature of 95 DEG C for 5 minutes; reducing the temperature of 95 DEG C to 12 DEG C at the speed of 0.1 DEG C per second; keeping the temperature of 12 DEG C, and diluting an adapter solution after quenching to 500 mu M at a ratio of 1:4; and storing at the temperature of minus 20 DEG C. An application of the asymmetric DNA artificial adapters by using the second-generation high-throughput sequencing technology is as follows: A, asymmetric DNA adapters are in ligation with DNA samples; B, non-connected DNA artificial adapters are purified and isolated by using electrophoresis tapping; C, judgment is carried out; and D, a polymerase chain reaction (PCR) amplification reaction is performed based on an asymmetric sequence. The asymmetric DNA artificial adapters by using the second-generation high-throughput sequencing technology has the obvious advantages that: 1) the usage of original DNA samples for establishing a library is reduced to 50 nanogram and the sensitivity of the original DNA samples is improved by 100 times; and 2) 100% of effective sequencing samples are produced in the process of adapter connection for establishing the library.

Description

Technical field: [0001] The invention relates to an asymmetric DNA double link head for second-generation high-throughput sequencing and its application. Background technique: [0002] At the end of 2005, 454 introduced the innovative Genome Sequencer 20 System, an ultra-high-throughput genome sequencing system based on pyrosequencing. In 2007, a second-generation genome sequencing system with better performance: Genome Sequencer FLX System was launched. The high-throughput sequencing technology of 454 uses a plate called "Pico TiterPlate" (PTP) during sequencing, which contains more than 1.6 million holes made of optical fibers, and the holes contain all necessary for chemiluminescence reactions. Kinds of enzymes and substrates. At the beginning of sequencing, the four bases placed in four separate reagent bottles are cycled into the PTP plate in the order of T, A, C, G, and only one base is entered at a time. If base pairing occurs, a pyrophosphate is released. This pyropho...

AI Technical Summary

Problems solved by technology

At the same time, the efficiency of the library preparation technology of DNA samples is low, such as blunt-end ligation itself is inefficient; 50% of the ligation (ligation) products (AA, BB) are invalid; The detection of multiple steps not only requires expensive instruments, like Agilent (US company) 2100Bioanalyzer (instrument name), but also consumes a lot of consumables and reagents

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