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BTEC3301
Recombinant DNA technology procedures by which DNA from different species can be isolated, cut and spliced together -new "recombinant " molecules are then multiplied in quantity in populations of rapidly dividing cells (e.g. bacteria, yeast).
The term gene cloning, recombinant DNA technology and genetic engineering may seems similar, however they are different techniques in Biotechnology and they are interrelated
Human gene therapy, geneticallyengineered crop plants and transgenic mice have become possible because of the powerful techniques developed to manipulate nucleic acids and proteins.
In the early 1970s it became possible to isolate a specific piece of DNA out of the millions of base pairs in a typical genome.
Currently it is relatively easy to cut out a specific piece of DNA, produce a large number of copies , determine its nucleotide sequence, slightly alter it and then as a final step transfer it back into cell in.
Recombinant DNA technology is based on a number of important things: Bacteria contain extrachromosomal molecules of DNA called plasmids which are circular.
Bacteria also produce enzymes called restriction endonucleases that cut DNA molecules at specific places into many smaller fragments called restriction fragments.
A restriction enzyme cuts only doublehelical segments that contain a particular sequence, and it makes its incisions only within that sequence--known as a "recognition sequence".
Sticky end and blunt end are the two possible configurations resulting from the breaking of double-stranded DNA
If two complementary strands of DNA are of equal length, then they will terminate in a blunt end, as in the following example:
5'-CpTpGpApTpCpTpGpApCpTpGpApTpGpCpGpTpApTpGpCpTpApGpT-3' 3'-GpApCpTpApGpApCpTpGpApCpTpApCpGpCpApTpApCpGpApTpCpA-5'
However, if one strand extends beyond the complementary region, then the DNA is said to possess an overhang:
5'-ApTpCpTpGpApCpT-3' 3'-TpApGpApCpTpGpApCpTpApCpG-5'
If another DNA fragment exists with a complementary overhang, then these two overhangs will tend to associate with each other and each strand is said to possess a sticky end:
5'-ApTpCpTpGpApCpT
pGpApTpGpCpGpTpApTpGpCpT-3' CpApTpApCpGpA-5'
3'-TpApGpApCpTpGpApCpTpApCpGp
Becomes
5'-ApTpCpTpGpApCpT pGpApTpGpCpGpTpApTpGpCpT-3'
3'-TpApGpApCpTpGpApCpTpApCpGp CpApTpApCpGpA-5'
The first Recombinant DNA molecules were made by Paul Berg at Stanford University in 1972.
In 1973 Herbert Boyer and Stanley Cohen created the first recombinant DNA organisms.
Recombinant DNA technology requires DNA extraction, purification, and fragmentation. Fragmentation of DNA is done by specific 'restriction' enzymes and is followed by sorting and isolation of fragments containing a particular gene.
This portion of the DNA is then coupled to a carrier molecule. The hybrid DNA is introduced into a chosen cell for reproduction and synthesis.
Transformation is the genetic alteration of a cell resulting from the introduction, uptake and expression of foreign DNA.
Plasmids were discovered in the late sixties, and it was quickly realized that they could be used to amplify a gene of interest. A plasmid containing resistance to an antibiotic (usually ampicillin) or Tetracycline, is used as a vector.
The gene of interest (resistant to Ampicillin) is inserted into the vector plasmid and this newly constructed plasmid is then put into E. coli that is sensitive to ampicillin.( Text bk:Pg 58) The bacteria are then spread over a plate that contains ampicillin.
As long as the bacteria grow in ampicillin, it will need the plasmid to survive and it will continually replicate it, along with the gene of interest that has been inserted to the plasmid .
Once inside a bacterium, the plasmid containing the human cDNA can multiply to yield several dozen replicas.
Isolation of two kinds of DNA Treatment of plasmid and foreign DNA with the same restriction enzyme Mixture of foreign DNA with plasmids
Introduction of recombinant plasmid into bacterial cells Production of multiple gene copies by gene cloning
This segment is "glued" into place using an enzyme called DNA ligase. The result is an edited, or recombinant, DNA molecule.
When this recombinant plasmid DNA is inserted into E. coli, the cell will be able to process the instructions to assemble the amino acids for insulin production.