Molecular Marking - AFLP Principles and Procedures - Huaqiang Electronic Network

Molecular Marking - AFLP Principle and Operation Steps I. Principle
AFLP is also a DNA molecular marker technique for detecting DNA polymorphisms by varying lengths of restriction endonuclease fragments. However, AFLP firstly amplifies the fragment by PCR reaction, and then electrophores the amplified fragment on a high-resolution sequential analysis gel, and the polymorphism is detected by the length of the amplified fragment. In the experiment, the fragment was first ligated to an artificial linker containing a co-adhesive end, and the cohesive end sequence and the linker sequence after ligation were used as primer binding sites for subsequent PCR reactions. In the experiment, selective amplification can be achieved by selecting different primers with 1 to 3 selective nucleosides added to the ends as needed. These selective nucleotides allow primers to selectively recognize endonuclease fragments with a specific pairing sequence for binding, resulting in specific amplification.


Second, the experimental reagents
Taq enzyme, EcoRI/MseIEcoRI/MseI linker, E+A primer M+C primer, T4DNALigaseE and M primer, agar, ammonium persulfate, acrylamide, urea, silver nitrate, formamide, dNTPs, xylene cyanide, glacial acetic acid, Glass silane, 50bpMark


Third, the operation steps (a) genomic DNA extraction and purification
A. Refer to Experiment 1 for a large number of DNA extraction methods.
B. Purification of DNA: The size of the fragment was detected by electrophoresis on a 0.8% agarose gel (containing EB 0.5 μg/ml), and 1/3 of the extracted genomic DNA was taken out for purification, first filled with TE buffer to the total volume. 50 ul, then equal volume of phenol / / isoamyl alcohol (25: 24: 1), / isoamyl alcohol (24: 1) were extracted once, centrifuged the supernatant in an Eppendorf tube, adding 1/10 volume of NaAC And two volumes of pre-cooled absolute ethanol, placed at -20 ° C for more than 2 h, centrifuged at 10,000 g for 10 min, rinsed the DNA pellet twice with 70% ethanol, air-dried and dissolved in 30 μl of TE buffer, UV-2401PC (Shimadzu) UV The A260 and A280 values ​​were measured by a spectrophotometer and quantified, and the fragment size was detected by electrophoresis on a 0.8% agarose gel (containing EB 0.5 μg/ml).
Note: 0.1-0.2g tissue can be dissolved in 100ul solution E, 0.5g tissue, solution E can be increased to 300ul, at this time the DNA concentration is about 100ng / ul.
(B) restriction enzyme digestion and ligation in a 0.2ml centrifuge tube: template amount of about 250ng, 2.5μl 10 × digestion buffer, 2.5μl 10 × T4 DNA ligase buffer, 5U EcoRI, 5U MseI, 2U T4 ligase, 50 pmol MseI linker (see Table 2 for the sequence), supplemented to 25 μl with double distilled water. The reaction was set at 37 ° C overnight using a PE PCR instrument, and the enzyme activity was stopped at 65 ° C for 20 min, and stored at -20 ° C as a pre-amplification template.
(3) Pre-amplification 3 μl of restriction enzyme ligation product, adding 75 ng E+A, 75 ng M+C primer, 15 mmol/L Mg2+, 25 mmol/L dNTPs, 1 U tagase, 3 μl 10× PCR buffer, double distilled water Make up to 30μl. The reaction parameters were: 94 ° C for 90 s; 94 ° C for 30 s, 56 ° C for 1 min, 72 ° C for 1 min, 30 cycles; 72 ° C for 10 min (PE company PCR instrument). After the end of the reaction, the amplified product was electrophoresed using a 0.8% agarose gel (containing EB0. 5 μg/ml) (see Fig. 1), and 3 μl of the product was diluted 50-fold to serve as a selective amplification template.
(4) Selective PCR amplification of the diluted product 3μl, add EcoRI selective primer, MseI selective primer 75ng, 15mmol / L Mg2+, 25mmol / L dNTPs, 1UTag enzyme, 3μl 10 × PCR buffer, plus Double steamed water to 30μl. The reaction parameters were: 94 ° C 90 s; 94 ° C 30 s, 65 ° C 1 min, 72 ° C 1 min, 13 cycles (0. 7 ° C per cycle); 94 ° C 30 s, 56 ° C 1 min, 72 ° C 1 min, 25 cycles; 72 ° C 5 min (PE company PCR instrument), first selective amplification products were electrophoresed on a 0.8% agarose gel (containing EB 0.5 μg/ml).
(5) Gel electrophoresis amplification products were separated by electrophoresis using 6% denaturing polyacrylamide gel (thickness 0.5 mm) and 1×TBE electrophoresis buffer (BIO-RAD Sequencing Electrophoresis Apparatus). The comb was pulled out and subjected to 140 W constant power pre-electrophoresis for 30 minutes at a temperature of 47 to 49 °C. Make sure that each well is cleaned of urea. An equal volume of loading buffer (98% formamide, 10 mmol/LEDTA, 0.25% xylene cyanide, 0.25% bromophenol blue) was added to the selective amplification product for 5 min at 94 ° C (PE company PCR instrument). On the ice until the spotting. 8 μl was added to each lane. Initially, electrophoresis was carried out with 100W constant power for about 2 minutes, and the sample was quickly concentrated to the bottom of the well, and then adjusted to 60 W constant power electrophoresis. The temperature was maintained at about 43 ° C, and the temperature was maintained at 2 / 3 of xylene blue FF to the glass plate, and the electrophoresis was terminated.
(6) Silver dye fixing liquid: Take 100ml glacial acetic acid to 900ml deionized water or double distilled water. Dyeing solution: 1g AgNO3, 1.5ml 37% formaldehyde, add deionized water to 1L. Color developing solution: 30 g Na2CO3, 1.5 ml 37% formaldehyde, 2 mg sodium thiosulfate, deionized water to 1 L.
1 After the electrophoresis is completed, the gel-coated glass plate is placed in a plastic tray for silver staining.
2 Fixation: Add the fixative and shake gently for 30 minutes on the shaker. After the fixation is completed, the fixing solution is retained.
3 Rinse with deionized water for 3 times for 2 min each time.
4 Staining: Place the gel into the staining tray, pour in the staining solution (4 ° C), and shake gently on the shaker for 30 min. The gel was rinsed with deionized water for 10 seconds and placed in a color developing tray.
5 Color development: Add color solution (4 ° C) and shake gently on the shaker until the number of bands no longer increases.
6 Termination: Add the fixing solution after the b step and float back and forth for a few minutes. After achieving the best results, rinse with distilled water for a few minutes.
7 Remove the water droplets from the gel and glass plate and place a photo on a white light box with a Nikon digital camera.
(7) Data analysis