Oligonucleotides for use in allele-specific PCR
一种等位基因特异、寡聚核苷酸的技术，应用在寡聚核苷酸领域，能够解决减弱识别特异性、无法识别出探针中单碱基错配等问题
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[0102] Preparation of polynucleotide templates
[0103] Existing procedures can amplify any specific nucleic acid sequence. The only necessary condition is: the number of known bases at both ends of the target sequence is sufficient, so that a pair of primers can be designed to hybridize with different strands of the target sequence, and the relative positions of the primers are suitable, so that one of the synthetic extensions After the product is separated from its template, it can be used as a template for another primer to extend, thereby amplifying a certain length of nucleic acid. The more sufficient information about the nucleic acids at both ends of the sequence, the higher the specificity of the primers used to amplify the target nucleic acid sequence, and the higher the efficiency of the entire process.
[0104] Any purified or unpurified polynucleotide molecule, as long as it contains the sequence to be detected, can be used as the starting nucleic acid template....
example 1
[0141] In order to illustrate the application of the present invention, a variety of real-time PCR platforms were used to quantitatively analyze the somatic mutation SNP at the G1898T site in the JAK2 gene, and the sensitivity, specificity and repeatability of the method were tested. The primers located at the -1 base of the polymorphic site and the -2 LNA performed well in the detection. In fact, the -2 LNA helps to stabilize the 3' end of the primer, while the -1 base ensures specificity.
[0142] Myeloproliferative disorders (MPDs) are hematological malignancies caused by clonal proliferation of stem cells. The World Health Organization (WHO) originally divided MPDs into the following four typical clinical diseases: polycythemia vera (PV), essential thrombocythemia (ET), myeloid metaplasia with myelofibrosis (MMM), and Chronic myeloid leukemia (CML). Later, other related diseases were gradually added to this classification. Chromosome t(9,22) translocation leads to the f...
example 2
[0193] Example 2: Analysis of cMPL mutations in Philadelphia chromosome-negative myeloproliferative disorders (Ph-MPDs)
[0194] cMPL is the gene encoding the thrombopoietin receptor, which plays an important role in the production of platelets and also in the expansion of multipotent hematopoietic stem cells. A gain-of-function mutation, MPLW515L, in the cMPL gene was first cloned and identified from bone marrow cells of patients with primary myelofibrosis (PMF). In subsequent work, another gain-of-function mutation, MPLW515K, was identified in patients with primary myelofibrosis (PMF) and essential thrombocythemia (ET). W515L is caused by the mutation of TGG→TTG at the corresponding position. W515K is caused by the change of TGG→AAG at the corresponding position.
[0195] Now, a rapid and sensitive real-time quantitative PCR method has been used to enhance the detection resolution of cMPL mutations, which utilizes a primer that introduces a mismatch at the -2 position and ...
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