Recombinant DNA Technology

• 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.

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Recombinant DNA Technology

• Human gene therapy, genetically-engineered 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.

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Recombinant DNA Technology

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.

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Recombinant DNA Technology

Restriction Enzymes and plasmid

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• There are many different kinds of restriction endonucleases
• Each nuclei cuts DNA at a specific site defined by a sequence of bases in the DNA called a recognition site
• A restriction enzyme cuts only double-helical segments that contain a particular sequence, and it makes its incisions only within that sequence--known as a "recognition sequence".

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Recombinant DNA Technology

Restriction Enzymes and plasmid

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• 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:

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• 5'-CpTpGpApTpCpTpGpApCpTpGpApTpGpCpGpTpApTpGpCpTpApGpT-3'

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• 3'-GpApCpTpApGpApCpTpGpApCpTpApCpGpCpApTpApCpGpApTpCpA-5'

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Recombinant DNA Technology

Restriction Enzymes and plasmid

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• However, if one strand extends beyond the complementary region, then the DNA is said to possess an overhang:

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• 5'-ApTpCpTpGpApCpT-3'
• 3'-TpApGpApCpTpGpApCpTpApCpG-5'

Recombinant DNA Technology

Restriction Enzymes and plasmid

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• 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'

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• 3'-TpApGpApCpTpGpApCpTpApCpGp CpApTpApCpGpA-5'

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Becomes

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• 5'-ApTpCpTpGpApCpT pGpApTpGpCpGpTpApTpGpCpT-3'

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• 3'-TpApGpApCpTpGpApCpTpApCpGp CpApTpApCpGpA-5'

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Recombinant DNA Technology

Restriction Enzymes and plasmid

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• Restriction Enzymes are primarily found in bacteria and are given abbreviations based on genus and species of the bacteria.
• One of the first restriction enzymes to be isolated was from EcoRI
• EcoRI is so named because it was isolated from Escherichia coli strain called RY13.

Recombinant DNA Technology

Digestion of DNA by EcoRI to produce cohesive ends ( Fig. 3.1):

Recombinant DNA Technology

Creating recombinant DNA :

• The first Recombinant DNA molecules were made by Paul Berg at Stanford University in 1972.

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• In 1973 Herbert Boyer and Stanley Cohen created the first recombinant DNA organisms.

Recombinant DNA Technology

Creating Recombinant DNA (Fig 3.2):

Recombinant DNA Technology

Reading materials :Summary of Recombinant DNA technology process:

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• Recombinant DNA technology requires DNA extraction, purification, and fragmentation.

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• Fragmentation of DNA is done by specific 'restriction' enzymes and is followed by sorting and isolation of fragments containing a particular gene.

Recombinant DNA Technology

Summary of Recombinant DNA technology process:

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• This portion of the DNA is then coupled to a carrier molecule.

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• The hybrid DNA is introduced into a chosen cell for reproduction and synthesis.

Recombinant DNA Technology

Transformation and Antibiotic Selection

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• Transformation is the genetic alteration of a cell resulting from the introduction, uptake and expression of foreign DNA.

Recombinant DNA Technology

Transformation and Antibiotic Selection

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• There are more aggressive techniques for inserting foreign DNA into eukaryotic cells. For example, through electroporation.
• Electroporation involves applying a brief (milliseconds) pulse high voltage electricity to create tiny holes in the bacterial cell wall that allows DNA to enter.

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Recombinant DNA Technology

Plasmids and Antibiotic resistance

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• Plasmids were discovered in the late sixties, and it was quickly realized that they could be used to amplify a gene of interest.

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• A plasmid containing resistance to an antibiotic (usually ampicillin) or Tetracycline, is used as a vector.

Recombinant DNA Technology

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• 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)

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• The bacteria are then spread over a plate that contains ampicillin.

Recombinant DNA Technology

Plasmids and Antibiotic resistance

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• The ampicillin provides a selective pressure because only bacteria that have acquired the plasmid can grow on the plate.
• Those bacteria which do not acquire the plasmid with the inserted gene of interest will die.

Recombinant DNA Technology

Plasmids and Antibiotic resistance

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• 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 .

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Recombinant DNA Technology

Fig 3.3 (a).

Selecting a Gene in a plasmid and Antibiotic selection.

Recombinant DNA Technology

Human Gene cloning

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• Once inside a bacterium, the plasmid containing the human cDNA can multiply to yield several dozen replicas.

Recombinant DNA Technology

Recombinant DNA Technology

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• Addition of DNA ligase

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• Introduction of recombinant plasmid into bacterial cells

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• Production of multiple gene copies by gene cloning

Recombinant DNA Technology

Summary of Recombinant DNA and Cloning (Fig.):

Recombinant DNA Technology

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• This segment is "glued" into place using an enzyme called DNA ligase.

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• The result is an edited, or recombinant, DNA molecule.

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• 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.
• More importantly, the new instructions are passed along to the next generation of E. coli cells in the process known as gene cloning.

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Recombinant DNA Technology

Fig: Inserting a DNA sample into a Plasmid