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

A technology of multiple ligation and probe, applied in the field of oligonucleotide probe preparation, can solve the problems of dsDNA residue, low probe yield and high cost, achieve low cost, high probe yield and improve hybridization efficiency Effect

Active Publication Date: 2012-07-04
江西南兴医疗科技有限公司 +1
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Problems solved by technology

Although this method is simple to operate, it requires a magnetic bead separation system, which is costly, and incomplete binding of magnetic beads during the separation process causes dsDNA residues, and the yield of probes is not high

Method used

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

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Embodiment Construction

[0028] The preparation method of the present invention is illustrated below in conjunction with the accompanying drawings and specific examples, specifically the preparation and detection of a long probe for detecting human genome CECR1 gene using multiple ligation amplification technology, including the following steps:

[0029] 1) Design a pair of detection primers that have nothing to do with the target fragment to be tested and the nucleotide sequence of the long probe, respectively GP1 (modified with a fluorescent group at the 5' end) and GP2, for the final PCR amplification detection; synthesized at the same time The right probe R-CECR1 with phosphorylation modification at the 5' end is composed of the right probe sequence of CECR1 and the reverse complementary sequence of GP2;

[0030] 2) Selection of a template nucleotide sequence (S) that has nothing to do with the target fragment to be detected, and design primers based on S and the target nucleotide sequence;

[003...

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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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/10
Inventor 黄劭
Owner 江西南兴医疗科技有限公司
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