Nucleic acid preparation and analysis

Abstract

The invention discloses a method and system for preparing and analyzing nucleic acid. The method comprises the following steps: (a), connecting a linker to a single double-chain-target nucleic acid molecule, so as to enable the tail ends of the double-chain-target nucleic acid molecules to be connected, wherein the linker comprises (i) a pairing region and (ii) a non-pairing region; (b), providing (i) a linker primer which is mutually complementary with a primer binding site on a complementary chain of the on-pairing region, (ii) a target specific primer which is mutually complementary with a binding site of target nucleic acid; (c), performing amplified reaction to amplify the double-chain-target nucleic acid molecules with the tail ends being connected with each other in the presence of the linker primer and the target specific primer, so as to produce a first amplified molecule. The method has very high sensitivity for detecting nucleic acid variation, and the limit of detection (LOD) is about 0.01 to 0.001 percent in certain situations.

Description

technical field [0001] The present application relates to the technical field of gene mutation detection, in particular, to a method and system for preparing and analyzing nucleic acid. Background technique [0002] Nucleic acid-based assays detect the presence of specific nucleic acids (DNA or RNA) in a sample. It has been used in various clinical and diagnostic applications. For infectious diseases, nucleic acid-based diagnostics enable the detection of DNA or RNA from the infecting organism. For non-communicable diseases, nucleic acid-based diagnostics can be used to detect the expression of specific genes or genes associated with diseases. A major clinical and diagnostic concern is the ability to detect low-level mutations and small numbers of alleles of clinical importance. Being able to discern mutations is important in many ways, especially for early detection of cancer from tissue samples and body fluids such as plasma or serum; assessment of residual disease afte...

AI Technical Summary

Problems solved by technology

However, this deep sequencing approach has limitations

Although theoretically, DNA subpopulations of any size are detectable when a sufficient number of molecules are deeply sequenced, errors introduced during sample preparation and sequencing limit practical detection

The PCR amplification of the mixture also leads to population skewing due to random and non-random amplification bias, which leads to under-representation or over-representation of specific variants (Kanagawa T. Bias and Artifacts in Multitemplate Polymerase Chain Reactions (PCR) .J Biosci Bioeng. 2003;96:317-23.)

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