Construction method of magnetic microsphere DNA probe for detecting miRNA molecules as well as product and application of magnetic microsphere DNA probe
A technology for DNA probes and construction methods, applied in the direction of recombinant DNA technology, DNA/RNA fragments, biochemical equipment and methods, etc., can solve the problem of inability to directly detect miRNAs levels and non-specific amplification, diagnostic methods cannot reflect miRNAs levels, In order to solve the problems of large sample size and other problems, the separation speed and enrichment efficiency can be improved, non-specific amplification can be avoided, and the method can be simplified.
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Embodiment 1
[0032] 1. Preparation of DNA probes:
[0033] According to the molecular sequence (SEQ ID NO: 1) of the lung cancer marker miRNA-21, four kinds of DNA probes P1, P2, P3 and P4 that are paired with the sequence but have different modified molecules were synthesized, and the specific structures are shown in Table 1 .
[0034] Table 1 DNA probes modified with different groups
[0035] probe molecule Sequence (from 5' to 3' end) modify P1 (SEQ ID NO: 2) TC AAC ATC AGT CTG ATA AGC TA 5'cy3,3'biotin TEG P2 (SEQ ID NO: 3) TC AAC ATC AGT CTG ATA AGC TA 5'cy3,3'biotin (T) P3 (SEQ ID NO: 4) TC AAC ATC AGT CTG ATA AGC TA 5'cy3,3'biotin P4 (SEQ ID NO: 5) TC AAC ATC AGT CTG ATA AGC TA 5'biotin TEG,3'cy3
[0036] 2. Preparation of magnetic microsphere DNA probe:
[0037] (1) Superparamagnetic microspheres and streptavidin are covalently combined to form streptavidin magnetic beads (streptavidin MBs, SMBs). 8.0, 0.5M NaCl, 1mM EDTA) to...
Embodiment 2
[0045] In order to verify the specific detection of the magnetic microsphere DNA probe constructed by the present invention to the specific marker miRNA-21, according to the molecular sequence of miRNA-21, the molecules to be tested with different structures were selected, wherein M1, M2, M3 and miRNA- There are 1 or 2 different bases in 21, and the specific sequences of these six molecules are shown in Table 2.
[0046] Table 2 Sequences of six molecules with different structures
[0047] probe molecule Sequence (from 5' to 3' end) miRNA-21 (SEQ ID NO: 1) UAG CUU AUC AGA CUG AUG UUG A Mismatch 1 (M1) (SEQ ID NO: 6) UAG CUU AUC AGA C C G AUG UUG A
Mismatch 2 (M2) (SEQ ID NO: 7) UA C CUU AUC AGA C C G AUG UUG A
Mismatch 3 (M3) (SEQ ID NO: 8) UA C CUU AUC AGA C C G AUG UUG C
MiRNA-10b (SEQ ID NO: 9) UAC CCU GUA GAA CCG AAU UUG UG NC-MiRNA (NC) (SEQ ID NO: 10) UUG UAC UAC ACA AAA GUA CUG
[0048] The ma...
Embodiment 3
[0051] According to the operation steps of Example 1 and Example 2, real biological samples were selected, and the human serum containing miRNA-21 was diluted into three different concentrations of 10fM, 1pM, and 100pM, and the optimal experimental conditions were used to dilute the human serum in Example 1. The prepared magnetic microsphere DNA probe containing P1 was tested against different concentrations of human serum containing miRNA-21, and the test results are shown in Table 3. For 10fM, 1pM, and 100pM three kinds of test samples, the comparison of test results in serum and buffer solution shows that the RUF value has only a slight change, indicating that the magnetic microsphere DNA probe constructed by the present invention has great application in real human cancer diagnosis. potential.
[0052] Table 3 Test results of different concentrations of human serum containing miRNA-21
[0053] [miRNA], pM RFU%, buffer RFU%, serum Recovery% RSD% 0.01 ...
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