Method for performing multi-target-site amplification library construction on plasma free DNA (deoxyribonucleic acid)

Abstract

The invention discloses a method for performing multi-target-site amplification library construction on plasma free DNA (deoxyribonucleic acid). The method sequentially comprises steps as follows: 1), tail end repairing and A addition are performed; 2), linker connection is performed: a linker DNA with an annular hairpin structure formed through annealing is added to a DNA fragment with A addition at the tail end in the step 1), so that double-stranded DNA after being combined with annular hairpin linkers on two sides is obtained; when the amount of plasma free DNA extracted and purified in the step 1) is smaller than or equal to 10 ng, a step 3) is performed; otherwise, when the amount of plasma free DNA extracted and purified in the step 1) is larger than 10 ng, a step 4) is performed; step 3), a specific primer I, Phi29 enzyme and a buffer solution are added to DNA with linker addition in the step 2), and linear amplification is performed; step 4), PCR (polymerase chain reaction) amplification is performed: amplification products constitute a free DNA mutation site enrichment sequencing library.

Description

technical field [0001] The invention belongs to the fields of molecular biology and medicine, and in particular relates to a method for multi-site amplification library of plasma free DNA or short fragment (<300bp) DNA. Background technique [0002] In recent years, it has been found that there are free DNA fragments in the peripheral blood plasma of the human body, with a length of 100-300bp, mainly concentrated around 150bp. In cancer patients, these cell-free DNA may include shed and dead tumor cells, and in pregnant women, may include fetal DNA fragments. Therefore, the mutation of a specific site in a tumor or fetus can be reflected to a certain extent by detecting the mutation of a specific site in the cell-free DNA fragment. However, the free DNA fragments in plasma are relatively short, most of them are around 150-200bp, generally not more than 300bp, and the content is very low. The extraction rate of existing free plasma DNA extraction methods is generally 10n...

AI Technical Summary

Problems solved by technology

[0003] The technical problem to be solved by the present invention is to provide a method for multi-target site amplification and library construction of short fragment DNA such as plasma free DNA. In view of the defects of existing multiple PCR for primer design and DNA fragments, the provided The library construction method can not only greatly reduce the requirements for primer design and DNA input quantity, but also improve the efficiency of library amplification

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