INSERTION OF FOREIGN GENES AND VECTORS

SOIL MICROBIOLOGY

BIOL/CSES 4684



This webpage was created by Jennifer Schuchert



1. OVERVIEW OF METHODOLOGY FOR INSERTION OF GENES AND VECTORS

The process of insertion of  foreign genes and vectors is used to create recombinant DNA which can  be inserted into a host for cloning. This would create multiple copies of the gene of interest.  This method is a relatively new procedure which allows analysis of proteins and other parts of most organisms which had previously been very difficult to study due to the scarcity of the gene of interest.  A genome is very large and the gene of interest is usually found only one or two times per cell.

This new method allows the introduction of almost any type of genetic information into an organism.  For example, a fragment of genome which contains the gene involved in the production of a toxin, can be removed from the bacteria from which it was found using restriction enzymes.  The gene can be inserted into a cloning vector, forming recombinant DNA.  The newly formed recombinant DNA can be inserted into the appropriate host, most likely E. coli, by the process of Transformation.  The cells produced will contain the recombinant DNA  which can be analyzed for possible use in a vaccine. (All figures are from www.blc.arizona.edu/marty/181/181Lectures98/Lecture18_98.html )

FIGURE 1. Illustration of the procedure to form recombinant DNA.
 
 

FIGURE 2. Illustration of Transformation procedure.



2. OVERVIEW OF INSERTION OF GENE AND VECTOR PROCEDURES

The procedure for insertion of foreign genes and vectors can be grouped into four categories based on the construction and insertion of recombinant DNA: DNA source, selected vector, restriction enzymes, transformation into host .

The source of DNA used to construct the recombinant DNA, is usually a small part of a genome that contains the gene or sequence of interest.  The gene or sequence of interest is usually of significant importance due to its unique function , whether it be the production of a protein, or capsule, or  regulate gene expression. Once the DNA source is chosen, it must be isolated.  This can be accomplished with the use of restriction endonucleases. The isolated DNA is then inserted into a vector.

Vectors are used as a vehicle by which foreign DNA can be inserted into cells , so the recombinant DNA may be replicated. The most common type of vector is a plasmid.  Plasmids are a small circular piece of extrachromosomal DNA found in bacterial cells, which are capable of replication independent of the host.  Plasmids usually carry a few genes which give them a selective advantage, such as genes for antibiotic resistance.

FIGURE 3. Common cloning vector which contains restriction sites and genes for antibiotic resistance.

Restriction endonucleases are used to cut the DNA and vector so they can be joined. Restriction endonucleases are bacterial enzymes that cleave DNA into fragments based on the recognition of specific nucleotide sequences. The enzymes are named after the bacterial species from which they were isolated. The DNA and vector to be joined are cut with the same restriction enzymes which can produce "sticky ends". DNA ligase is used to join the ends of the vector and DNA pieces.

The newly formed recombinant DNA is inserted into a host, usually E.coli., by the process of transformation.  As the cell divides, more cells are produced, each containing plasmid DNA which also exhibits antibiotic resistance.  This process of making copies of foreign DNA is called molecular cloning.  The cloned cells can be screened for by using antibiotic resistance markers.



3. PROS AND CONS

Problems that may arise in the process of creating Recombinant DNA include; difficulty in location and isolation of the sequence of interest,  finding the appropriate vector and successful transformation of the recombinant DNA into the host cell.  The recombinant DNA must be made competent by treatment with a calcium chloride solution, but this does not insure that the procedure will work.
One important concern with using this technique to produce recombinant DNA and inevitably cloned cells, is the use of the genetically engineered cells for biological or environmental purposes.  Transgenic plants and animals could be created but the uncertainty of their impact on society may not permit their use until further research is conducted.

This method however, has enabled the analysis of many proteins, enzymes and genes which previously had not been possible.  this method has a wide variety of applications.  It can be used in the medical field to study specific genes that might be linked to genetic disorders.  It can be used in the creation of recombiant vaccines.  This method can be applied in the agricultural field by creating transgenic plants which exhibit antibiotic resistance.



4. ADDITIONAL REFERENCES

Stansfield, Wiliam D.,  Schaum's Outline of Theory and Problems of Molecular and Cell Biology,  McGraw-Hill, NY, 1996. pp.183-200.

Turner, P.C., McLennan, A.G., Bates, A.D., and White, M.R.H.,  Instant notes in Molecular Biology, School of Biological Sciences, University of Liverpool, Liverpool, UK 1997. pp. 91-109.

www.blc.arizona.edu/marty/181/181Lectures98/Lecture18_98.html

www.mc.edu/~rhamilto/111/Lect_28.html
 



5. LINKS

www.hort.wisc.edu/faculty/faculty/Vierstra/hort550/strategies-lec11.htm

www.baysys.com/~jkimball/BiologyPages/T/TransgenicAnimals.html


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