Method for obtaining nucleic acid aptamer with extremely short sequence through screening
A nucleic acid aptamer and candidate sequence technology, which is used in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. Detection costs, reduction of side reactions and interference factors, and elimination of truncation and modification effects
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0022] Example 1: Screening of Vibrio alginolyticus very short sequence nucleic acid aptamers
[0023] 1. Synthesize random oligonucleotide library: Synthesize a random oligonucleotide library with fixed sequences at both ends and random sequence N=35 in the middle as follows: 5'--TCA GTC GCT TCG CCG TCT CCT TC--N35-- GCA CAA GAG GGA GAC CCCAGA GGG--3', the fixed sequences at both ends can be combined with primers P1 and P2 for PCR amplification, and the sequence of P1 is 5'--TCAGTC GCT TCG CCG TCT CCT TC--3', P2 The sequence is 5'--CCC TCT GGG GTC TCC CTC TTG TGC --3'.
[0024] 2. Combination: Combine 100ul of 30pmol of the above random library with 100ul of the target Vibrio alginolyticus (the bacteria content is about 10 8 each) combined at 28°C for 30 minutes;
[0025] 3. Separation: 6000rpm for 10 minutes, discard the supernatant, take the bacterial pellet and heat it to 95°C, then centrifuge at 6000rpm for 10 minutes, take the supernatant, and obtain the oligonucleotid...
Embodiment 2
[0036] Example 2: Screening of Vibrio harveyi very short sequence nucleic acid aptamers
[0037] 1. Synthesize random oligonucleotide library: Synthesize a random oligonucleotide library with fixed sequences at both ends and random sequence N=25 in the middle as follows: 5'--TCA GTC GCT TCG CCG TCT CCT TC--N25-- GCA CAA GAG GGA GAC CCCAGA GGG--3', the fixed sequences at both ends can be combined with primers P1 and P2 for PCR amplification, and the sequence of P1 is 5'--TCAGTC GCT TCG CCG TCT CCT TC--3', P2 The sequence is 5'--CCC TCT GGG GTC TCC CTC TTG TGC --3'.
[0038] 2. Combination: Combine 100ul of 30pmol of the above random library with 100ul of the target Vibrio harveyi (the bacteria content is about 10 8 each) combined at 28°C for 30 minutes;
[0039] 3. Separation: 6000rpm for 10 minutes, discard the supernatant, take the bacterial pellet and heat it to 95°C, then centrifuge at 6000rpm for 10 minutes, take the supernatant, and obtain the oligonucleotide that can b...
Embodiment 1 and Embodiment 2
[0049] In embodiment 1 and embodiment 2, the method for determining the affinity constant of nucleic acid aptamer by single-stranded DNA method is as follows:
[0050] 1. Nucleic acid aptamer treatment: Take the nucleic acid aptamer with a concentration of 10 μM, and dilute it to 10 nM, 20 nM, 40 nM, 60 nM, 80 nM and 100 nM in sequence with 2× binding buffer solution.
[0051] 2. Binding: Take 100 μl (108cfu / ml) of Vibrio liquid and bind to 100 μl of nucleic acid aptamers with different concentrations of 10nM, 20nM, 40nM, 60nM, 80nM and 100nM, and combine on a shaking table (30°C, 100rpm) for 30min.
[0052] 3. Washing: After the binding is completed, centrifuge at 6000 rpm for 5 minutes, remove the supernatant, and wash 3 times with 200 μl 1× buffer solution.
[0053] 4. Heating: heat at 95°C for 5 minutes, centrifuge at 15,000 rpm for 10 minutes, and take the supernatant.
[0054] 5. Detection: Use a K5500 ultra-micro-ultraviolet-visible spectrophotometer to measure the aff...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More