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Isolation of the Gene Sequence of Interest from the DNA Libraries
#1
Knowledge on the complete genome of different organisms paves way for many developments and inventions in the field of molecular biology contributing to the advancement in various fields like medicine, Bio technology, Scientific research in the development of new medicines, new biotechnology products, understanding the genetic diseases and finding a cure for it through gene therapy, production of beneficial enzymes in large scale and so on. But there is always procedural difficulty to study the DNA of interest of any organism each and every time when required for the research. Hence molecular biologists found a solution to this problem by constructing gene library which holds the collection of DNA sequences derived from various organisms. They constructed two libraries, a genomic library and cDNA library.

A genomic library is created by extracting all the DNA from the nucleus of the selected organism, fragmenting the extracted DNA into suitable sizes using restriction enzyme, ligating the fragmented DNA into suitable vectors and introducing the vector with the inserted DNA sequence into suitable host bacteria. The group of transformed bacterial cells holds all the DNA sequences of the selected organism and is stored. A cDNA library is constructed in the similar way except that instead of the whole genome of the organism here the mRNA of the organism is turned into cDNA with the help of the reverse transcriptase enzyme which is then subjected to fragmentation and ligated into the vector and then introduced into the host bacterium. A genomic library holds the entire DNA sequences of an organism whereas the cDNA library holds the expression gene sequence of the organism as it is generated from the mRNA of the organism.

In this way molecular biologists construct the libraries and keep it ready so that as and when they want they can retrieve the gene of interest from the library for their research use. But extracting that particular DNA sequence or gene of interest from the whole lot of DNA sequences present in the library is quite a challenging task which is overcome by the development and application of various screening procedures and screening a genomic library and cDNA library have specific protocols. Hybridization technique, use of specific antibodies, hybrid arrest- hybrid release method, chromosome walking are some of the procedures applied to screen a gene library or cDNA library in order to isolate the gene sequence of interest.

Hybridization method employs the use of nucleic acid probes which are DNA sequences synthesized complementary to the gene sequence of interest to be extracted from the gene bank. The transformed bacteria carrying the vectors with inserted DNA sequence will either form colonies or plaques based on the type of vector used and the hybridization technique is also named accordingly as colony hybridization or plaque hybridization. Except for the plating procedure for the colony formation or the plaque formation rest of the procedure is similar. The group of colonies of transformed bacteria on a plate is imposed on a nitro cellulose membrane by placing the membrane on the plate. The membrane with copies of the colonies on the plate is then removed and the colonies on the plate are allowed to grow. The nitrocellulose membrane is then treated with sodium dodecyl sulfate and the protease enzyme inorder to lyse the bacterial colonies so that the DNA sequence gets exposed and adheres to the nitro cellulose membrane. The adhered DNA is denatured by treating with alkali like sodium hydroxide and the membrane is dipped in radioactive labeled nucleic acid probe containing solution. The nucleic acid probe attaches to the complementary DNA sequence on the membrane (hybridization) and the excess probe on the membrane is removed by washing. The areas of hybridization are seen by exposing the membrane to X-rays which is observed as dark spots on the X-ray film. The unique colony holding the gene sequence of interest is identified by comparing the main plate with bacterial colonies, the nitro cellulose membrane and the X-ray film. The identified colony carrying gene sequence of interest can then be extracted from the plate and grown as a step to amplify the gene of interest.

Using expression vectors in creating cDNA library makes the screening of the cDNA library much easier which is done with the help of specific antibodies. The ligation of cDNA fragments with special expression vectors enhances the transcription and translation of the cDNA sequence inside the host bacteria which in turn produces the protein coded by the cDNA. The screening procedure is similar to hybridization method except that here the nitrocellulose membrane holds the protein which is identified by using specific antibody which in turn helps identifying the particular bacterial colony producing the same protein of interest.

Thus screening procedures are the best index to find the gene sequence of interest from the constructed gene libraries.
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#2
Isolation of the Gene Sequence of Interest from the DNA Libraries' is quite an interesting topic to read. Please also post a sum up or a brief for your thread.
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#3
Screening based on properties of protein products of target genes have to be based on expression libraries, i.e., libraries constructed using expression vectors (expression cloning). All expression libraries of higher eukaryotes are, of necessity, cDNA libraries, but those of bacteria and many yeast are usually genomic libraries since few, if any, of their genes have introns, and there is very little spacer DNA between genes.

Generally, a random primer method is used to obtain cDNA; as a result, 5-end sequences are likely to be represented in a higher frequency. In addition, some cDNAs may be abundant and others rare due to the tissue used for library construction. A potential problem of genomic expression libraries of bacteria and yeast is that clones corresponding to a gene may carry termination sequences from the gene located immediately upstream; this can prevent efficient expression of the gen. therefore, DNA fragments used for cloning are smaller in size than the target gene.

In addition, a large enough number of recombinant clones are produced so that there is a reasonable chance for the DNA fragments representing each gene to be cloned in all six possible reading frames; three reading frames in each of the two orientations of the fragments with respect to the promoter (present in the vector).

Given below is a list of possible approaches for expression based screening:

1. Hybrid Arrested Translation: HART is an indirect approach for identification of clones having the desired DNA insert and resulting polypeptide.

2. Hybrid Selection: Recombinant DNAs are fixed to solid support like nitrocellulose filter discs which hybridizes to complementary mRNA which is used for in vitro translation and therefore identification of the DNA.

3. Antibodies Specific to the Protein Product: This is used if the protein lacks a recognizable and measurable function.

4. Colony / Plaque Screening with Antibodies: Antibodies are bound to solid support and brought in contact with agar containing colonies to be screened, proteins produced by the colonies bind to the antibodies and can therefore be identified.

5. Fluorescence Activated Cell Sorter (FACS): This rapid screening method is used for cells whose products are expressed on the cell surface and bind to specific antibodies.

6. Unique Gene Products (Gain of Function): cells can be identified by a function not performed by the proteins of non transformed host cells like enzyme activities or hormone effects.

7. Marker Rescue Approach: Cells are identified by using mutant E.coli strains that produce compounds essential for survival/multiplication of the cells.

8. South-Western Screening: Performed using transcription factors for identification of clones encoding proteins
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Isolation of the Gene Sequence of Interest from the DNA Libraries00