Preparation method for probe used for multiplex ligation-dependent probe amplification (MLPA)

Abstract

The invention discloses a preparation method for a long probe which can be used for multiplex ligation-dependent probe amplification (MLPA). The preparation method comprises the following steps: (1) selecting a vector which is irrelevant with a target sequence to be tested to serve as a template, and designing a primer according to the length of a target nucleotide sequence and the length of a target probe; (2) artificially synthesizing the primer; (3) performing polymerase chain reaction (PCR) amplification; (4) purifying and recovering PCR products; (5) digesting the PCR products by using Lambda exonuclease; and (6) recovering single-stranded deoxyribonucleic acid (ssDNA). The preparation method for the probe for the MLPA is low in cost, and all operations, such as the amplification, enzyme digestion and the like, can be finished only by a PCR instrument; recovery is performed by using a special ssDNA recovery kit after the enzyme digestion is finished, thereby, double-stranded DNA (dsDNA) and nucleotide which are not completely digested are eliminated, and the hybridization efficiency when an MLPA experiment is conducted is increased; and through full Lambda exonuclease digestion, the dsDNA can be changed into the ssDNA to the maximum degree, and thereby, the preparation efficiency of the probe is increased.

Description

technical field

[0001] The invention relates to the preparation of oligonucleotide probes, in particular to the preparation method of long probes which can be used in multiple ligation amplification technique (MLPA). Background technique

[0002] Multiplex ligation-dependent probe amplification (MLPA) is a high-throughput gene detection method. This technology uses nucleic acid hybridization, ligation reaction and PCR amplification reaction, and can detect up to 45 species in the same reaction tube. Different nucleotide sequences are detected and quantified.

[0003] So far, MLPA technology has been applied in various fields of research and genetic diagnosis, and there are continuous new technical improvements, especially in the design of probes, the use of M13 single-stranded phage to insert into both ends of the derived sequence involves restriction internal Dicer recognition sites, in order to obtain long probes by double digestion;

[0004] The application number is 20...

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