Agarose gel electrophoresis is extensively used for the analysis of nucleic acid. Observations made by running DNA or RNA samples on agarose gel are very helpful to determine quantity and quality of nucleic acids. These powerful features make agarose gel electrophoresis an important essential step in many well known techniques such as northern and southern blotting, RFLP, RAPD, PCR, molecular cloning etc. Since agarose gel allows the elution of biomolecules, it can also be used for the purification of nucleic acids and large protein molecules and complexes. In addition, it can also be used to analyse biological processes such as apoptosis.
Broadly, the applications of agarose gel electrophoresis can be divided into following categories:
- Qualitative analysis (Size determination, Purity check, degradation of sample, quality of plasmid prep)
- Quantitative analysis (comparative analysis among samples, semi-quantitative PCR)
- Preparative applications (isolation of DNA fragment of particular size)
- A necessary step of other DNA/RNA analysis techniques (Northern and southern hybridization, RFLP, RAPD)
- Analysis of biological processes such as apoptosis
Quality of Plasmid DNA
The quality of plasmid DNA after isolation can be checked by agarose gel electrophoresis. Agarose gel electrophoresis can distinguish all three forms of plasmid DNA (supercoiled, nicked circular and linear plasmid). One can also observe the denatured plasmid DNA which often appears upon plasmid isolation by alkaline lysis method. Denatured plasmid DNA is resistant to restriction enzyme digestion and appears as a ghost band in agarose gel. The quality of plasmid DNA is of great importance for transfection in cell culture. Supercoiled plasmid DNA has the highest transfection efficiency.
Quality check of PCR reaction
The PCR amplified products can also be checked for the presence of unspecific amplifications or presence of primer dimers by agarose gel electrophoresis. Since we can analyze the size of DNA fragments, any band which does not correspond to expected size DNA fragments could be an unspecific amplification. Primer dimers are generally smaller in size and migrate close to bromophenol blue. In case of unexpected results, qPCR reactions can be analyzed by agarose gel electrophoresis to ensure the fidelity of the reaction.
Determination of DNA fragment size
Agarose gel electrophoresis is extensively used to determine the approximate size of DNA fragment(s). A DNA ladder which contains DNA fragments of known size is run in a parallel well. By comparing the size of DNA fragments with known size fragments of DNA from ladder, one can get the approximate size of the unknown DNA fragment(s). Since the resolution of agarose gel electrophoresis is low, the exact base pair size of DNA fragments can not be calculated by agarose gel electrophoresis.
Detection of nucleic after isolation
Often after isolation of DNA (plasmid or genomic DNA), a small quantity of the sample is run in agarose gel to ensure the presence of DNA in the sample. It also helps to know the quality and approximate quantity of the DNA (see below). RNA can also be checked by regular agarose gel electrophoresis after isolation (total RNA) from biological sources.
Determination of RNA size and expression
Agarose gel electrophoresis is an important step in northern blotting to determine the size and expression level of a gene. Total RNA fractionated on a denaturing agarose gel is blotted to a nylon membrane which is subsequently probed using gene specific probes to determine the size of RNA as well as compare the gene expression under different experimental conditions or among different types of cells and tissue. It is also a powerful technique to analyze the splicing variants of mRNA.
Verifying the accuracy of the spectrophotometric RNA quantitation data
After quantitation of RNA samples, equal quantities of samples are loaded on agarose gel to verify the spectrophotometric RNA quantitation data compared with each other. This is especially important if RNA is to be used for the expression analysis of a gene by qPCR, semiquantitative PCR, northern analysis or any other techniques. This analysis also ensure the quality of RNA among different samples especially degradation of RNA that is often judge by 28S and 18S rRNA band (for mammalian cell total RNA)
ESSENTIAL INTERMEDIARY STEP IN OTHER TECHNIQUES
Standardization of PCR reaction
Agarose gel electrophoresis is used to standardize PCR reaction. PCR reaction products, generated under different sets of conditions, are analyzed by agarose gel electrophoresis. Depending on the specific fragments and intensity of the band, one can decide the optimum condition of PCR reaction. The important parameters that can be monitored by agarose gel electrophoresis are: unspecific amplification, amount of primer dimers, and no amplification of specific band.
Semiquantitative PCR is a PCR-based method to analyze and compare the expression level of genes under different experimental conditions. In this method, the total RNA collected from cells is first reverse-transcribed and then amplified by PCR. The intensity of PCR amplified products on agarose gel is used as an indication of the expression level of the particular genes in different sets of conditions.
ANALYSIS OF BIOLOGICAL PROCESSES
Detection of apoptosis
Fragmentation of genomic DNA into discrete size fragments (180 bp and multiples) is one of the extensively used assays to detect most forms of apoptosis. DNA fragments appear as a ladder which are analyzed by agarose gel electrophoresis.
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