Initially, it was observed that gDNA was always read and transcribed into mRNA, which guided protein formation and then was disposed. Calling it that challenged scientists to find exceptions to this rule. Virologists eventually did find one such exception. It should be noted that prokaryotes are not capable of splicing out introns. Exons are a necessary part of the coding system, being retained after introns are spliced out. This is displayed in Figure 3.
Exitrons are introns that are not spliced out, despite containing no coding sequences. When scientists use viral enzymes to make cDNA from RNA isolated from the cells and tissues that they are studying, it does not contain introns due to being spliced out in mRNA. As a result, cDNA will only contain genes that are actively being used by a specific cell or tissue at a point in time.
Once isolated, gDNA can be used to make genomic libraries for DNA sequencing, fingerprinting, differentiation and other applications with both clinical and research fields. This way a protein expressed in a eukaryotic organism can be introduced into a prokaryote. Then, using a reverse transcriptase enzyme, cDNA can be made. Libraries also provide proportional insights into the abundance of RNA produced in a given cell or tissue because the more an mRNA is expressed, the more cDNA will be produced and vice versa.
This is extremely useful when using prokaryotic organisms for cloning since they do not have splicing capabilities. Just as a traditional library might have a book of interest, a cDNA library will hold copies of a gene of interest, and researchers need a way of identifying that gene.
There are many colonies on a master plate of a cDNA library since the library holds the mRNA representation of a given tissue or cell.
This is where library screening comes into play. In this screening method, a nylon filter paper is used to replicate a master plate containing colonies each colony contains a homogenous population of identical closed plasmid by pressing it onto the master plate thereby transferring cells from the colonies from the master plate onto the nylon transfer paper. The filter paper is treated with an alkaline solution to lyse the cells and denature the DNA. Then, radio labeled probes comprising complementary oligos of the target sequence are added.
The probes hybridize with DNA from the lysed cells. Then, the filter paper is exposed to X-ray which will once developed, will allow visualization of the target, and enable us to make a comparison between the labeled nylon paper and the master plate to find the colonies containing our DNA of interest. The selected colonies are then picked and grown on nutrient medium. Reverse transcription is the key to obtaining the initial DNA cDNA which can then be used in a number of applications to further study a gene.
The one step process can also have reduced sensitivity. Synthesized cDNA also allows researchers to perform protein purification and expression, and gene expression profiling. Bhagavan, N. Essentials of medical biochemistry: With clinical cases.
Amsterdam: Academic Press. Biology, S. Bremgartner, M. Coffin, J. Overview of Reverse Transcription. Liu, C. Construction of cDNA library and preliminary analysis of expressed sequence tags from Siberian tiger. Eberwine, J. Gonzales, M. Goodsell, D. Hu, P. Construction and characterization of a cDNA expression library from the endangered Hu sheep. Genetics and Molecular Research,13 4 , Isolation and use of cDNA clones.
McClean, P. Clone Library Screening. Namuth-Covert, D. Perbal, B. Avian myeoloblastosis virus AMV : Only one side of the coin. Retrovirology,5 1 , Below is a list of tissues that are part of different systems that could be used for isolating your target:.
If there are too many tissues to try at once, pool the cDNAs into one reaction. The purpose of pooling cDNA is to reduce the number of reactions that needs to be performed. Rather, add as much of each cDNA in the pooled reaction as you would in a single reaction. This ensures the target in abundant tissues is not diluted, which compromises the success of the reaction.
In the absence of expression data or poor success with other cDNAs, potential sources of a gene can also be determined by the function of the gene. For example, if a gene is a receptor known to be activated by glutamate a neurotransmitter , then that gene is most likely going to be found in the brain.
Likewise, if a mutation in a gene has been associated with increased risk of diabetes, then the pancreas or adipose tissue may be good tissues to try. If none of the above works, then you may have exhausted your options with regards to cDNA. The next step would probably be to look into buying a commercial clone. Has this helped you? Then please share with your network.
Usually we end up with the cDNA clone without a hitch. You must be logged in to post a comment.
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