Method for preparing long probe that can be used for multiple connection dependent amplification technology
Background: In 2002, Dutch scientists invented a high-throughput gene detection method called muitiplex ligation-dependent probe amplication (MLPA), which can detect up to 45 different nuclei in the same reaction tube. The nucleotide sequence was detected and quantitatively analyzed. So far, MLPA technology has been applied in many fields, such as single nucleotide polymorphism and gene mutation detection, gene fragment deletion and duplication, abnormal chromosome number, gene methylation detection and mRNA analysis, etc. Long and short probes are involved in this method, where the preparation of long probes is difficult.
Method improvement: PCR method combined with avidin magnetic bead method (long oligonucleotide preparation by magenetic-beads, LOPM) can be used for multiple ligation-dependent amplification technology of long probes. By designing primers for PCR amplification for target sequences and UT (Unrelated Template, UT), such as plasmids, used to prepare long probes, and one of the primers is labeled with biotin, to The plasmid is used as a template for PCR amplification to obtain a specific length of biotin-labeled nucleic acid sequence, which is purified using avidin magnetic beads, and finally a single-strand oligonucleotide with a specific length (non-biotin label) is prepared.
Results: Using PCR method combined with avidin magnetic bead method to prepare long probes that can be used in multiple junction amplification technology can prepare long probes of any length according to experimental requirements, not only greatly reducing the difficulty of probe design, but also avoiding bacteriophage The time-consuming, high-cost and complicated operation processes such as cultivation and double enzyme digestion enable ordinary laboratories to carry out MLPA detection experiments smoothly.
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