Preparation method of long probe capable of being used in multiplex ligation amplification technology

Abstract

The invention discloses a preparation method of a long probe capable of being used in a multiplex ligation amplification technology, which comprises the following steps of: 1) selection of a template nucleotide sequence irrelevant to a target segment to be detected, and design of a primer according to UT and a target nucleotide sequence; 2) synthesis of the primer; 3) PCR amplification; 4) mixing of a PCR product and aidin magnetic beads; 5) denaturation and melting of the PCR product; and 6) separation of a single-chain nucleotide sequence labeled by non-biotin so as to obtain the long probe capable of being used in the multiplex ligation amplification technology. The preparation method of the MLPA long probe can obtain the MLPA long probe by utilizing a simple combining process of the PCR amplification and the aidin magnetic beads, has simple and easy operation steps, avoids the time consuming and complicated operation process for obtaining the long probe through phage infection and the like, greatly reduces the threshold of MLPA technology application, and leads the experiment to be smoothly conducted in a common laboratory.

Description

technical field [0001] The invention relates to the field of preparation of oligonucleotide probes, in particular to a method for preparing long probes which can be used in multiple ligation amplification technology. Background technique [0002] In 2002, Dutch scientists invented a high-throughput gene detection method called multiplex ligation-dependent probe amplification (MLPA). Detect and quantify up to 45 different nucleotide sequences in the same reaction tube. This technology only needs 20ng / μl DNA, and can detect and quantitatively analyze multiple different target genes in the same reaction tube through simple hybridization, ligation, PCR amplification and electrophoresis steps. [0003] The schematic diagram of MLPA technology principle is shown in figure 1 , the technology mainly includes: the hybridization of the probe and the target sequence, the specific ligation of the probe, the amplification of the ligated probe and the detection and analysis of the resul...

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Problems solved by technology

[0005] In this method, long probes and short probes are involved, and the design of the probes is a difficult point. Dutch scientists creatively used the characteristics of the M13 single-stranded phage, Cleverly designed restriction endonuclease (RE) recognition sites at both ends of the sequence derived from the insertion, so that long probes can be obtained by double enzyme digestion, which overcomes the difficulty that the current oligonucleotide synthesis is generally difficult to achieve more than 50mer

However, this method has at least two major defects: 1) Compared with the extremely large laboratories that need molecular biology testing, there are not many laboratories that can cultivate M13 phages, so they can only resort to commercial production with high fees, and the cost Extremely high; 2) The cycle is long and the design is difficult

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