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Probe preparation method for multiplex ligation amplification technology

A probe and long probe technology, used in recombinant DNA technology, DNA preparation, DNA/RNA fragments, etc., can solve the problems of double-stranded nucleotide residues and low probe yields, reduce non-specific hybridization, Flexibility and easy sequence-sourced effects

Active Publication Date: 2017-05-24
江苏佰龄全基因生物医学技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide a low-cost, high-yield long probe preparation method that can be used in multiple ligation amplification technology to overcome the incomplete binding of magnetic beads in the prior art. Double-stranded nucleotide residues, the defect that the probe yield is not high

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  • Probe preparation method for multiplex ligation amplification technology
  • Probe preparation method for multiplex ligation amplification technology
  • Probe preparation method for multiplex ligation amplification technology

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Embodiment 1

[0044] The preparation method of the present invention is illustrated below in conjunction with accompanying drawings and specific examples, specifically the preparation and detection of the long probe of the amp gene on the pUC18 carrier as the gene to be tested by using multiple ligation amplification technology, comprising the following steps:

[0045] 1) Design a pair of universal detection primers that have nothing to do with the target gene to be tested and the nucleotide sequence of the long probe, respectively upstream primer CF and downstream primer CR, for the final PCR amplification detection, and synthesize the left probe S at the same time, The 5' to 3' sequences are respectively composed of the forward sequence of CF and the sequence TF on the left side of the target fragment;

[0046] 2) Determine a nucleotide sequence T that has nothing to do with the target fragment sequence and the universal primer, and design a pair of primers for amplifying the long probe on...

Embodiment 2

[0109] The preparation method of the present invention is illustrated below in conjunction with accompanying drawings and specific examples, specifically the preparation and detection of the long probe of the amp gene on the pUC18 carrier as the gene to be tested by using multiple ligation amplification technology, comprising the following steps:

[0110] 1) Design a pair of universal detection primers that have nothing to do with the target gene to be tested and the nucleotide sequence of the long probe, namely the upstream primer CF and the downstream primer CR, which are used for the final PCR amplification detection, and synthesize the left probe S' at the same time , the 5' to 3' sequences are respectively composed of the forward sequence of CF and the left sequence TF' of the target fragment;

[0111] 2) Determine a nucleotide sequence T' that is not related to the target fragment sequence and the universal primer, and design a pair of primers for amplifying the long prob...

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Abstract

The invention relates to a preparation method of a long probe for multiplex ligation-dependent probe amplification (MLPA). The preparation method comprises the following steps: 1) designing a pair of universal detection primers independent of a target gene to be detected and the nucleotide sequence of the long probe; 2) artificially synthesizing a short probe; 3) determining a fragment of nucleotide sequence independent of the target gene and the universal detection primers; 4) designing a long probe amplification primer; 5) amplifying double chains of the long probe by virtue of PCR and purifying the product; 6) performing affinity purification by use of streptavidin magnetic beads; 7) preparing and recovering the long probe; 8) performing MLPA verification. The preparation method of the long probe for MLPA is simple and fast, and capable of obtaining the high-quality long probe only by virtue of the processes of PCR amplification and affinity purification by use of the streptavidin magnetic beads as well as a simple enzyme digestion reaction; as a result, the MLPA amplification efficiency is greatly improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular relates to the preparation of oligonucleotide probes, in particular to the preparation method of long probes which can be used in multiple ligation amplification technology (MLPA). Background technique [0002] Multiplex ligation-dependent probe amplification (MLPA) was first reported by Schouten et al. in 2002. It is a new technology developed in recent years for qualitative and semi-quantitative analysis of DNA sequences to be detected. The technology is highly efficient and specific, and can detect changes in the copy number of 45 nucleotide sequences in one reaction. At present, MLPA technology has been applied in many fields, research and gene diagnosis of various diseases, and new technical improvements are constantly being made. [0003] Chinese patent application 200910108977.4 discloses a long probe that can be used in multiple ligation amplification technology by using the PC...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/10
CPCC12N15/1013C12N15/1031
Inventor 潘海波华琴邢楠楠
Owner 江苏佰龄全基因生物医学技术有限公司
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