Real-time Fluorescence Constant Temperature Exponential Amplification Method
A constant-temperature exponential amplification and real-time fluorescence technology, applied in the fields of life science and biology, can solve problems such as limiting the application value of constant-temperature amplification technology, and achieve the effect of ensuring specificity
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Embodiment 1
[0260] Example 1: Detection of oligonucleotide molecules
[0261] 1. Target-specific oligonucleotides (ODNs) TS ) and signal amplification template (T SA ) design and synthesis:
[0262] Design and synthesize target molecule-specific oligonucleotides (ODN) based on miRNA and other naturally occurring small fragment nucleic acid molecules with 3'-OH TS ) and signal amplification template (T SA ), wherein, SEQ No.1 is a miRNA mimic molecule, which has a 3'-OH that can trigger its own extension, and the nucleotide sequence composition characteristics from the 3'-end to the 5'-end of SEQ No.2 are as follows: ① and ODN TS The nucleotide sequence of the first region complementary to the nucleotide sequence; ②The NERS antisense strand sequence region of Nb.BsrDI nick endonuclease, that is, 5'-GCAATG-3'; ③The first region with the same nucleotide sequence as the first region The nucleotide sequence of the second region, and the fluorescent quenching group BHQ2 is labeled at the c...
Embodiment 2
[0273] Embodiment two: the detection of genomic DNA
[0274] 1. Target-specific oligonucleotides (ODNs) TS ) preparation
[0275] According to the sequence characteristics (SEQ No.3) of the target molecule, it is designed to prepare the target molecule specific oligonucleotide (ODN TS ) signal generation template (T SP ) oligonucleotide (SEQ No.4), wherein the target molecule has the NERS sense strand sequence region of Nt.BstNBI nick endonuclease, ie 5'-GAGTC-3'. The total linear constant temperature amplification system is 20 μl, and the system includes the following components: 1× Reaction Buffer(NEB), 0.5×NEBuffer 3.1(NEB), 0.4 units / μl Nt.BstNBINicking Enzyme(NEB), 0.05 units / μl (exo-)DNA Polymerase (NEB), 2×EvaGreen (Biotium), 400 μM dNTPs (Promega), 10 μg / ml BSA (NEB), 100 nM SEQ No.2, serial final concentration of SEQ No.3. The reaction conditions are: 60°C for 40 minutes, and the fluorescence acquisition is once per minute; 80°C for 20 minutes to inactivate the...
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