Denaturing Urea-Polyacrylamide Gel Electrophoresis (PAGE) Based Microsatellite Analysis
|Author: Sanjeev Sharma1, BR Yadav1,|
|Affiliation: 1 Livestock Genome Analysis Lab, 3 Animal Biochemistry Division, National Dairy Research Institute, Karnal-132001, India|
2Meerut Institute of Engeenering and Technology, Meerut, U.P., India
|Date Added: Mon Feb 02 2009|
|Date Modified: Mon Feb 02 2009|
|Abstract: This protocol will give very good information for researchers working on microsatelite markers with brief introduction and principle of method|
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.
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
|Gel %||Acrylamide (g)||Bisacry lamide (g)||Urea (g)||TBE 10X Buffer (ml)||Demonized water (ml)||10% APS (μl)||TEMED (μl)|
Warm the solution at 60°C until urea dissolved completely and filter through Whatman filter paper.
Setting up and casting a polyacrylamide gel using sequencing apparatus involves the following steps.