Detection method of copy number variations

Abstract

The invention discloses a detection method of copy number variations. The method comprises two steps of performing hybridization and connection (performed at the same time) as well as performing quantitative PCR detection. The hybridization lies in that a probe and a DNA single strand target sequence are combined through complementary pairing, wherein the probe comprises an upstream probe and a downstream probe, a 5' terminal of the upstream probe is a general-purpose sequence, a 3' terminal of the upstream probe is a sequence complementary with the target sequence, a 5' terminal of the downstream probe is a sequence complementary with the target sequence, and a 3' terminal of the downstream probe is a general-purpose sequence; the connection lies in that two segments of hybridized adjacent probes are connected to form a complete nucleic acid single strand through specific DNA ligase; and the quantitative PCR detection is performed through two pairs of universal primers, and then the copy number variations of an original DNA are calculated according to a CT value. Through the adoption of the method disclosed by the invention, detection of the copy number variations can be realized.The method is simple, free from limitation, low in cost and high in specificity, the primers can be general-purpose, and large-scale detection can be realized.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting copy number variation. Background technique [0002] Copy number variations (CNVs) refer to submicroscopic fragment copy number mutations ranging in size from 1kb to 1Mb. The deletion, duplication, inversion, etc. of these copy fragments are collectively referred to as CNVs. [0003] Currently, CNVs detection methods mainly include the following: multiplex ligation-dependent probe amplification (MLPA), microarray-based comparative genomics hybridization (array-CGH), Affymetrix CNV chip technology and Next-generation sequencing technology, real time quantitative polymerase chain reaction (RT-qPCR), fluorescence in situ hybridization (fluorescence in situ hybridization, FISH), etc. [0004] Among them, microarray comparative genomic hybridization (array-CGH), Affymetrix CNV chip technology, fluorescence in situ hybridization technology (FISH) and next-generation...

AI Technical Summary

Problems solved by technology

It was first reported by Schouten et al. in 2002 that the copy number of 50 different fragments can be detected in the same reaction tube, but the probe design is complicated and the process is cumbersome

In terms of probe design, MLPA can only pass through its downstream X-terminus (see figure 1 ) probe length to distinguish, and the length of the probe will have an impact on the synthesis and accuracy, so this method has certain limitations in probe design, which restricts its development and application

In terms of process, MLPA needs to go through steps such as hybridization, PCR, dilution and denaturation, and capillary electrophoresis. The process is complex and has many steps. The complex process and multiple steps will have a certain impact on the accuracy of the verification results.

The disadvantages of the existing MLPA technology are: 1. The preparation of the probe is complicated and has limitations

The shortcomings of the existing fluorescent quantitative PCR technology are: 1. Different primers need to be designed according to different regions, and the designed primers need to be verified for primer efficiency; Poor; 3. The fluorescent quantitative PCR of the probe method needs to synthesize a probe with a fluorescent group, which is costly and can only target one site at a time, which is not conducive to large-scale application

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