Multiplex amplification method
An oligonucleotide and nucleic acid technology, used in biochemical equipment and methods, recombinant DNA technology, DNA/RNA fragments, etc., can solve the problems of decreased amplification efficiency, inconsistent template amplification efficiency, and cumbersome operation steps. Avoid false positives, reduce interference between multiple primers, and achieve high-sensitivity multiplex detection
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Embodiment 1
[0078] Example 1 Basic principle of amplification of the present invention
[0079] The present invention includes two modes: double universal primer mode and single universal primer mode, the principle is shown in Figure 1 (A) and Figure 1 (B).
[0080]In dual universal primer amplification, the capture oligonucleotide includes four regions: a first universal sequence U1s, a folding sequence T1s, a second universal sequence U2s and a binding capture sequence T2a, the capture oligonucleotide first passes through the binding capture sequence T2a The target molecule whose 5'-end sequence is determined is captured, and then the target molecule is used as a template to carry out an extension reaction, and the complementary strand T1a of the folded sequence T1s is added to the 3'-end to obtain an extended capture oligonucleotide. T1a and T1s can be completely complementary or incomplete, as long as T1a and T1s can induce intramolecular self-folding and form a half-hairpin structure...
Embodiment 2
[0082] Example 2 Construction of a product with a clear 5' end sequence
[0083] The method of the present invention requires the use of a sample with a well-defined 5'-end sequence to initiate the extension reaction mediated by the capture oligonucleotide. The strategy for constructing a product with a well-defined 5'-end sequence is as follows: figure 2 Shown: ① For example, a nucleic acid extension blocker is designed for the target molecule to block the extension of DNA polymerase to obtain a product with a 5'-end sequence determined; ② Different cleavage enzymes are used for specific cleavage sites in the target molecule Carry out a cleavage reaction to obtain a product with a 5'-end sequence determined, ③ or design a low-concentration specific primer for the target molecule for specific amplification, thereby obtaining a product with a determined 5'-end sequence.
Embodiment 3
[0084] Example 3 qPCR detection of miRNA21 and let-7a
[0085] MicroRNAs (miRNAs) are a class of endogenous non-coding single-stranded small RNAs with a length of 19-25 nucleotides, which are ubiquitous in eukaryotic cells and control the activity of more than 50% of coding genes.
[0086] In this example, miRNA21 and let-7a are detected, and the steps are as follows:
[0087] (1) Synthesize miRNA21 and let-7a sequences;
[0088] (2) The capture oligonucleotides, universal primers and detection probes of miRNA21 and let-7a genes were simultaneously added to the reaction system containing miRNA21 and let-7a sequences, and miRNA21 and let-7a were detected by PCR. The system is template RNA, 5nM capture oligonucleotide, 150nM first universal primer, 150nM second universal primer, 150nM detection probe, 1U reverse transcriptase, 1U Taq polymerase, 200μM dNTP, 4.5mM MgCl 2 and 2× PCR buffer, the final volume was 20 μL; the PCR reaction program was pre-denaturation at 94 °C for 5 ...
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