Denaturing Urea-Polyacrylamide Gel Electrophoresis (PAGE) Based Microsatellite Analysis

Introduction

Denaturing polyacrylamid gel is very useful technique which has been used for various application of biology such as analysis of milk proteins, various recombinant proteins and also used for the separation and purification of single stranded fragment of DNA and RNA. This technique generally used for detection of microsattellite markers. Microsatellites are simple sequence repeats amplified by PCR as a new kind of polymorphic marker. These are tandemely repeated motifs of 1-6 nucleotides that are densely and evenly distributed through out the genome and often exhibit substantial variation in the number of repeats. The length of each allele determines by PCR using specific oligonucleotides primers flanking the repeated sequence. The DNA products visualize after electrophoresis. PCR based microsatellites detection facilitates the construction of genome maps in most livestock species because it’s abundance in the genome, the specificity of the primers, its high degree of polymorphism with several alleles and their easy detection. Microsatellites are currently being used in many different fields including behavior genetics, population structure analysis, medical studies and forensics.

Principle

Polyacrylamide gels are chemically cross-linked gels forming by the polymerization of acrylamide with a cross-linking agent, usually N, N’-methylene bisacrylamide (Bis). The polymerization initiates by free radical formation usually carrying out with ammonium per sulfate as the initiator and N, N, N’, N’-tetramethylene diamine (TEMED) as a catalyst. The length of the chain may be determined by the concentration of acrylamide in the polymerization reaction. One molecule of crosslinker includes for every 29 monomers of acrylamide. Denaturing gels polymerized in the presence of an agent (urea or, less frequently, formamide) suppresses base pairing in nucleic acids. Denatured DNA migrates through these gels at a rate that is almost completely independent of its base composition and sequence.

Composition of Denaturing PAGE Gels

Warm the solution at 60°C until urea dissolved completely and filter through Whatman filter paper.
 

Procedure

Setting up and casting a polyacrylamide gel using sequencing apparatus involves the following steps.

1. Wash both the glass plates thoroughly with warm water and liquid detergent. Rinse the glass plates thoroughly with deionized water to remove detergent residues and wipe with tissue paper soaked in 70 percent alcohol. Air dry the glass plates by laying them on a whatman filter paper.
   Note: Detergent microfilm left on the glass plate may result in a high background (brown colored) upon staining the gels.
2. Apply 200 μl of Repel-silane ES on the inner surface of larger glass plate with tissue paper. Remove excess silane by wiping with tissue paper soaked in distilled water.
3. Treat small glass plate by applying the freshly prepared bind saline at the edge of clean plate and leaved it for 4-5 min. (This step is essential to prevent tearing of the gel during silver staining as it chemically adheres gel to the glass plates after 1-2 min). Wipe off excess binding saline with 70% ethanol through tissue paper.
   Note: Rubbing hard will remove excessive amount of bind silane and gel may not adhere as well.
4. Lay the longer glass plate on flat on the bench and place spacers on large plate then second plate was placed on it. Align the edges and cover borders on three sides with sealing tape and clamp them using bulldog clamps.
5. Add 400 μl APS and 40 μl of TEMED to 80 ml PAGE mix and draw the above mixed solution into a barrel of 120ml of syringe and invert the syringe to expel any trapped air that has entered the barrel.
6. Introduce the nozzle of the syringe into the notched region the plates. Expel the mixed solution from the syringe, filling the space almost to the top.
7. Once the solution is filled up insert the comb in gel to the edge of the plate. Clamp with clips and keep in appropriate position till the gel gets polymerize (approximately 1 hr).
8. After acrylamide has polymerized, remove the clamp holding the comb and casting stand pulled out comb straight by wriggling it gently and smoothly.
9. Place the stand with assembly in the lower buffer reservoir of the page apparatus tank. Fill the upper and lower tank with 1x TBE buffer.
10. Check the leakage by marking the level of the buffer in the upper chamber.
11. Fix the safety cover on top on the upper buffer chamber to prevent evaporation of buffer and pre run for 40-45 min at constant watts.
12. Flush the wells thoroughly with buffer and gently fix the comb.
13. Stuck the gel temperature indicator on to the outer plate near the center of gel.
14. Allow to pre-run for 60 minutes at constant Watts (80 W).
15. After completion of pre-run remove the upper reservoir lid and insert the shark toothcomb between the glass plates with teeth facing downwards.
16. Use a needle attached to the syringe filled with 1x-TBE buffer to flush out all the wells.
17. Denature PCR products (5 μl) along with 10-bp ladder mixed in 2X loading dye (20 mM EDTA, 0.05% Xylene cyanole, prepared in 95%formamide) for 5 minutes at 95ºC.
18. Immediately, transfer the denatured samples to ice to prevent annealing.
19. Load the samples (approx. 2.5 μl) into each well and also load two extreme wells with 10bp DNA ladder.
20. After loading the denatured sample into the wells, replace the lid on the upper buffer chamber.
21. Allow the gel to run at 80 W for one and half hour to two and half hours according to the expected size of the PCR products.
22. Note: Run time depends as per the size of PCR products.
23. Remove the plates carefully from apparatus and keep on blotting paper. Remove spacers and separate plates carefully using spatula so that gel should retained on smaller plate.
24. Subject the gel to silver stain to visualize the bands.