Method for designing high-sensitivity and high-specificity mismatch nucleic acid sequences

Abstract

The invention discloses a method for designing high-sensitivity and high-specificity mismatch nucleic acid sequences. The method includes computing delta MFE (minimum free energy) values of mismatch sequences, target sequences and homologous interference sequences to determine candidate mismatch nucleic acid sequences. The method has the advantages that the shortcoming of dependence on experienceof mismatch nucleic acid sequence designs can be overcome by the aid of the method, mismatch nucleic acid sequences can be reduced to a great extent, particularly, workload for designing mismatch primers can be relieved to a great extent, a few candidate nucleic acid sequences can be quickly obtained from large quantities of mismatch nucleic acid sequences, and the method is favorable for determining the optimal nucleic acid sequences from the few candidate nucleic acid sequences; the high-sensitivity and high-specificity mismatch nucleic acid sequences still can keep high affinity with the target sequences with low concentration on the basis of mismatch primers designed by the aid of the method, and is poor in affinity with the homologous interference sequences, accordingly, the amplification sensitivity of the target sequences can be effectively improved, and the method can be effectively used for highly homologous nucleic acid sequences and can be particularly effectively used for miRNA [micro-RNA (ribonucleic acid)] amplification.

Description

technical field [0001] The invention relates to a method for designing mismatched nucleic acid sequences with high sensitivity and specificity. Background technique [0002] In order to quantify the amount of the target nucleic acid sequence, it is often necessary to amplify the target nucleic acid sequence. In the amplification of nucleic acid sequences, primers have a crucial influence on the results of amplification. Reasonable primer design can not only effectively improve the amplification efficiency, but also improve the specificity of amplification. Conversely, improperly designed primers not only have poor amplification efficiency, but also relatively poor specificity. When the amplification specificity of the primers is poor, non-target sequences will be amplified, and the amplification results are often unreliable and false results will appear. [0003] The general rule of primer design is to design as complementary sequences as possible according to the amplifi...

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Problems solved by technology

[0009] The above method can solve the problem of low sensitivity and specificity in the amplification of highly homologous sequences to a certain extent, but the method relies heavily on personal experience, and the results are often not extendable to the amplification of other highly homologous sequences

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