Construction method of plasma free DNA library

Abstract

The invention discloses a 'construction method for a small amount of DNA library'. The method comprises the following steps: carrying out end repairing on plasma free DNA or fragmented DNA and simultaneously carrying out phosphorylation modifying on 5' terminal to obtain modified DNA; later, implementing blunt end linking directly to an artificial linker and purifying to obtain relatively pure linker-containing DNA having a gap on joint; then repairing the gap by virtue of polymerase chain reaction (PCR) polymerase before PCR amplification to obtain repaired complete linker-containing DNA; and carrying out PCR amplification reaction and purifying a finished product to obtain a final high-throughput sequencing library. The method is not only simple, convenient and efficient, and is capable of avoiding a purification step and reducing DNA loss in a library construction process, but also can effectively solve a problem of linker self-linking which is easy to occur in a micro-DNA library construction process by virtue of a special linker and blunt end linking way; and meanwhile, by repairing the gap before amplification, sufficient complete templates are provided before amplification for the amplification reaction.

Description

Technical field [0001] The invention relates to a method for constructing a plasma DNA library for repairing gaps before amplification, in particular to a method for constructing a free DNA library in pregnant women's plasma that can be applied to non-invasive prenatal diagnosis. Background technique [0002] According to medical statistics, about 2 to 3% of newborns born each year suffer from congenital diseases or birth defects, causing long-term sickness, disability, and even premature death of infants and young children, which brings a huge burden to society and families. The main causes of congenital diseases or birth defects are chromosomal abnormalities, single-gene genetic diseases, polygenic genetic diseases, and teratogenic factors, among which chromosomal abnormalities account for a large proportion. In a modern society where eugenic health is emphasized, how to prevent and treat congenital diseases or birth defects has become a very important topic. Prenatal diagnosi...

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method for constructing a high-throughput sequencing library to solve the problems of DNA loss and self-connection of adapters in the process of building a small amount of DNA samples, especially in the process of building a plasma cell-free DNA library.

This method is not only simple and efficient, but also reduces purification steps and DNA loss during library construction, and uses specific adapters and blunt-end connection methods to effectively solve the problem of self-ligation of adapters during the construction of trace DNA libraries.