In biotechnology applications and many experiments require the use of cloned DNA. Therefore analyzing required gene of interest and also its transcripts are very important to the experiments. Some of the methods used to analyze the gene and its transcripts are described in this article.
1. Restriction sites can be mapped in the DNA of interest. Restriction maps are used for subcloning and also for comparing the genes with the cDNA.
2. Cloned DNA can also be used as a probe to characterize transcription of the same gene in cells or in vivo condition. This technique can be used to get the information such as:
a. Size of the initial gene of interest.
b. Steps involved in the mature mRNA production or post transcriptional steps can be studied.
c. The amount of gene being transcribed.
d. Timing of gene expression in the cell cycle can also be studied.
e. Pattern of expression of gene of interest in different tissues or during different stages of development.
3. DNA sequence present in the computer database can be used to compare the complete DNA sequence of gene of interest. This method can be used to understand following things:
a. This technique can be used to examine similarity between related genes.
b. Can get clues about the functions of uncharacterized genes or unknown genes.
c. This technique can also be used to predict the protein or polypeptide chain produced by a gene.
Analysis of Cloned DNA Sequence by Restriction Enzyme:
1. Cloned DNA or gene of interest can be digested using restriction enzymes. Digested DNA is electrophored on agarose gel and is visualized using ethidium bromide. This helps in construction of maps of restriction enzyme sites.
2. Cloned DNA is digested with several different types of restriction enzymes, and each cut is loaded in a lane of an agarose gel. Electrical current is applied; current drives the negatively charged DNA fragments through positive pole via agarose gel. Small fragments of the DNA molecules more quickly than the large ones. Therefore restricted DNA fragments are separated based on their size.
3. Separated DNA fragments are stained with ethidium bromide, which forms a complex with DNA and produces fluoresces under UV light. The agarose gel is then photographed and size of each DNA fragment is determined by calculating the distance travelled by them and comparing it with a calibration curve.
Restriction Enzyme Analysis of Genes in the Genome:
1. Knowing the restriction sites present in the gene of interest is useful in analyzing introns organization or cloning some part of the gene into a vector.
2. Restriction site present in the gene of interest can be studied using cDNA or by studying same gene of interest from a related species. This is done as follows:
a. Genomic DNA is digested with different restriction enzymes.
b. Each sample of genomic DNA digest is electrophoresed in agarose gel, and using ethidium bromide these fragments are stained.
c. DNA fragments are denatured and by using southern blotting technique DNA is transferred into membrane filter.
d. Labelled probes are added to the membrane filter, probes will hybridize with complementary DNA fragments that were present on the original agarose gel. Bound DNA is visualized using appropriate technique for the label type.
e. Hybridization signal are compared with marker bands, this helps in determine the size and also construction of map.
Analysis of Gene Transcripts:
1. Northern blotting technique is used to analyze the gene transcripts or RNA, using following steps:
a. RNA is extracted from the cell, is electrophoresed and is also bound to a membrane filter.
b. Labelled probe is added to the filter, hence hybridization occur between cellular RNA and complementary probe. Further analysis is done to determine the size of the RNA.
2. Northern blotting technique is used to determine the size of mRNA encoded by a gene. Using these technique scientists deduced that same gene is capable of giving rise to different types of mRNA, by alternate mRNA splicing or differential use of promoter and terminator region of gene.
3. Northern blotting technique is also used to study RNA sampling from a variety of sampling. This is used to study development, specialization of tissue and also the cells response to different types of physiological stimuli.
These are some of the methods used for analyzing the gene and also its transcript or mRNA produced in a cell or tissue.
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