Molecular Markers - AFLP Principles and Procedures
Molecular Markers - AFLP Principles and Procedures
AFLP can be used for (1) construction of genetic linkage maps; (2) rapid identification of molecular markers closely linked to target genes using AFLP; (3) AFLP-assisted rotational selection breeding; (4) study of gene expression and regulation; (5) taxonomic and evolutionary studies; and (6) methylation studies, among others.
Operation method
Molecular marker-AFLP
Principle
AFLP is a PCR reaction in which enzyme fragments are first amplified and then electrophoresed on a high-resolution sequential analyzer gel, and polymorphisms are detected by the difference in the lengths of the amplified fragments. In the experiment, the enzyme fragments are firstly connected with artificial junctions containing common sticky ends with them, and the sequence of the sticky ends and the junction order are used as the primer binding site for the subsequent PCR reaction. In the experiment, selective amplification can be achieved by choosing different primers with one to three selective nucleotides on the ends as needed. These selective nucleotides enable the primers to selectively recognize endonuclease fragments with specific pairing order for binding, leading to specific amplification.
Materials and Instruments
DNA Samples Move I. Genomic DNA extraction and purification 1. Extraction of DNA in large quantities (experimental methods vary depending on the source of DNA, omitted here). 2. DNA purification: (1) Detect the fragment size by electrophoresis with 0.8% agarose gel (containing EB 0.5 μg/ml), and remove 1/3 of the extracted genomic DNA for purification. (2) Firstly, replenish with TE buffer to a total volume of 50ul, and then equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) and chloroform/isoamyl alcohol (24:1) were extracted once each. (3) Centrifugation aspirate the supernatant in an Eppendorf tube, add 1/10 volume of NaAC and diploid accumulation of pre-cooled anhydrous ethanol, and leave at -20°C for more than 2h. (4) Centrifuge at 10,000xg for 10min, rinse the DNA precipitate with 70% ethanol for 2 times , air dry and dissolve in 30μl TE buffer. (5) Detect and quantify the A260 and A280 values by UV spectrophotometer, and then detect the fragment size by electrophoresis on 0.8% agarose gel (containing EB 0.5μg/ml). Note: 0.1-0.2g of tissue can be dissolved in 100ul of solution E. For 0.5g of tissue, solution E can be increased to 300ul, at which time the DNA concentration is about 100ng/ul. Restriction enzyme digestion and ligation 1. In a 0.2ml centrifuge tube, add the following: 250ng of template, 2.5μl of 10× digestion buffer, 2.5μl of 10× T4 DNA ligase digestion buffer, 5U of EcoRⅠ, 5U of MseⅠ, 2U of T4 ligase, 50pmol of MseⅠjoint, and make up 25μl of double-distilled water. 2. After overnight reaction with PCR amplifier set at 37℃, inactivate the enzyme at 65℃ for 20min, and store at -20℃ as pre-amplification template. Pre-amplification 1. Take 3μl of ligation product, add 75ng E+A, 75ng M+C primer, 15mmol/L Mg2+, 25mmol/L dNTPs, 1U Tag enzyme, 3μl of 10×PCR buffer, and add double-distilled water to make up to 30μl. The reaction parameters were: 94°C for 90s; 94°C for 30s, 56°C for 1min, 72°C for 1min, 30 cycles; 72°C for 10min. 2. At the end of the reaction, the amplification product was detected by electrophoresis on a 0.8% agarose gel (containing EB 0.5 μg/ml), and 3 μl of the product was diluted 50-fold and used as a selective amplification template. Selective PCR amplification 1. Take 3μl of the diluted product, add EcoRⅠselective primer, MseⅠselective primer 75ng each, 15 mmol/L Mg2+, 25mmol/L dNTPs, 1UTagase, 3μl of 10×PCR buffer, and add double-distilled water to make up to 30μl. The reaction parameters were: 94℃ for 90s; 94℃ for 30s, 65℃ for 1min, 72℃ for 1min, 13 cycles (0.7℃ drop per cycle); 94℃ for 30s, 56℃ for 1min, 72℃ for 1min, 25 cycles; 72℃ for 5min. 2. At the end of the reaction, electrophoresis was performed on a 0.8% agarose gel (containing EB 0.5 μg/ml) to detect the selective amplification products. V. Gel electrophoresis 1. The amplification products were separated by electrophoresis with 6% denaturing polyacrylamide gel (thickness 0.5 mm) and 1×TBE electrophoresis buffer). Remove the comb and pre-electrophoresis at 140W constant power for 30 minutes to a temperature of 47--49°C. Be sure to make each well wash out urea. 2. Selective amplification products are added to an equal volume of upwelling buffer (98% formamide, 10 mmol/L EDTA, 0.25% xylene cyanide, 0.25% bromophenol blue) denatured at 94°C for 5 min, and at the end of this time are quickly placed on ice until spotting. 3. 8μl of sample was added to each lane. 100W constant power electrophoresis was used at the beginning for about 2 minutes to make the sample concentrate to the bottom of the wells quickly, and then adjusted to 60W constant power electrophoresis, and the temperature was kept at about 43℃, and the electrophoresis was ended when the xylene cyanine was swept to the 2/3 of the glass plate. Sixth, silver dyeing 1. Fixing solution preparation: take 100ml glacial acetic acid into 900ml deionized water or double-distilled water. Staining solution: 1g AgNO3, 1.5ml 37% formaldehyde, add deionized water to 1L. Color development solution: 30g Na2CO3, 1.5ml 37% formaldehyde, 2mg sodium thiosulfate, add deionized water to 1L. 2. Specific operation process (1) After electrophoresis is completed, place the glass plate with gel adhered to it into the plastic disk used for silver staining. (2) Fixation: add fixing solution and shake slightly on a shaker for 30 min. after fixation, retain the fixing solution. (3) Add deionized water and rinse 3 times for 2min each time. (4) Staining: put the gel into the staining plate, pour in the staining solution (4℃), and shake slightly on the shaking table for 30min. rinse the gel with deionized water for 10 seconds, and then put it into the color development plate. (5) Color development: add the color development solution (4℃), and shake slightly on the shaking table until the number of bands no longer increases. (6) Termination: Add the fixative solution used in step (2) and bleach back and forth for a few minutes. After achieving the best results, rinse with distilled water for a few minutes. (7) After removing the water droplets on the gel and glass plate, put it on a white light box and take pictures with a digital camera. VII. Data analysis Quantity One software of BIO-RAD was used for statistics, and then NTSYS software was used to calculate the genetic similarity coefficient, and UPGMA method was used for cluster analysis to construct the cluster diagram. For more product details, please visit Aladdin Scientific website.
Taq enzymes EcoRI MseIEcoRI MseI junctions E+A primers M+C primers T4DNALigaseE and M primers Agar Ammonium persulfate Acrylamide Urea Silver nitrate Formamide dNTPs Xylene cyanide Glacial acetic acid Glass silane 50bpMark
Electrophoresis apparatus Centrifuge Eppendorf tubes Gel imager UV spectrophotometer 37°C water bath PCR apparatus