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.