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Preview of AQA A2 BIOLOGY UNIT 5: DNA Technology

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Techniques have been developed to isolate genes, clone them and transfer them into microorganisms.
These microorganisms are then grown for the continuous production of a desired protein.
The DNA of two different organisms that have been combined in this way is called recombinant DNA.
The resulting organism is known as a genetically modified organism.
The process of making a protein using the DNA technology of gene transfer and cloning involves a
number of stages:
1. Isolation of the DNA fragments that have the gene for the desired protein
2. Insertion of the DNA fragment into a vector
3. Transformation, that is the transfer of DNA into suitable host cells
4. Identification of the host cells that have successfully taken up the gene by the use of gene markers
5. Growth/cloning of the population of host cells
Before a gene can be transplanted, it must be identified and isolated from the rest of the DNA.
Two of the methods employed to use enzymes that have important roles in microorganisms:
· reverse transcriptase
· restriction endonuclease
Using Reverse Transcriptase
· A cell that readily produces the desired protein is selected
· These cells have large quantities of the relevant mRNA, which is therefore extracted
· Reverse transcriptase is then used to make DNA from RNA
This DNA is known as complementary DNA ­ cDNA because it is made up of the other nucleotides
that are complementary to the mRNA
· To make the other strand of DNA, the enzyme DNA polymerase is used to build up the
complementary nucleotides on the cDNA template
This double strand of DNA is the required gene
· since the cDNA doesn't have introns cDNA is therefore an artificial gene
Reverse transcriptase has several uses in biotechnology:
· It makes genes without introns.
Eukaryotic genes with many introns are often too big to be incorporated into a bacterial plasmid, and bacteria
are unable to splice out the introns anyway.
The artificial cDNA gene is made from mRNA that already has the introns spliced out of it, so it can be
expressed in bacteria.
· It makes a stable copy of a gene, since DNA is less readily broken down by enzymes than RNA.
· It makes genes easier to find.
However a given cell only expresses a few genes, so only makes a few different kinds of mRNA molecule.
For example the b cells of the pancreas make insulin, so make lots of mRNA molecules coding for insulin.
This mRNA can be isolated from these cells and used to make cDNA of the insulin gene.

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Using Restriction Endonuclease
All organisms use defensive measures against invaders.
Bacteria are frequently invaded by viruses that inject their DNA into them in order to take over the cell.
Some bacteria defend themselves by producing enzymes that cut up the viral DNA.
These enzymes are called restriction endonucleases.
There are many types of restriction endonucleases.
Each one cuts a DNA double strand at a specific sequence of bases called a recognition sequence.
Sometimes this cut occurs between two opposite base pair.…read more

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DNA Ligase
This enzyme repairs broken DNA by joining two
nucleotides in a DNA strand.
Ligase is therefore a bit like DNA polymerase.
It is commonly used in genetic engineering to do the
reverse of a restriction enzyme, i.e. to join together
complementary restriction fragments.
Two restriction fragments can anneal if they have
complementary sticky ends, but only by weak
hydrogen bonds, which can quite easily be broken,
say by gentle heating.
The backbone is still incomplete.…read more

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Introduction of DNA into host cells
Once the DNA has been incorporated into at least some of the plasmids, they must then be reintroduced
into bacterial cells.
This process is called transformation and involves the plasmids and bacterial cells being mixed together
in a medium containing calcium ions.
The calcium ions, and changes in temperature, make the bacteria permeable, allowing the plasmids, to
pass through the cell membrane into the cytoplasm.
However, not all the bacterial cells will possess the DNA fragments.…read more

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There are a number of different ways of using gene markers to identify whether a gene has been taken
up by bacterial cells.
They all involve using a second, separate gene on the plasmid.…read more

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Polymerase Chain Reaction
The polymerase chain reaction is a method of copying fragments of DNA.…read more

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In vivo In Vitro
Particularly useful where we want to Extremely rapid
introduce a gene into another organism Particularly valuable where only a minute
amount of DNA is available
Involves almost no risk of contamination Does not require living cells
This is because a gene that has been cut All that is required is a base sequence of
by the same restriction endonuclease can DNA that needs amplification
match the sticky ends of the opened up No complex culturing techniques required
Contaminant DNA will…read more

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Genetic engineering is known as recombinant DNA technology.
The genetic makeup of organisms can now be altered by transferring genes between the individuals of
the same species of between organisms of different species.
Organisms that have had their DNA altered by genetic engineering are called transformed organisms.
These organisms have recombinant DNA ­ DNA formed by joining together DNA from different sources.…read more

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Genetically Modified Plants…read more

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Genetically Modified Animals
An example of genetically modified animals is the transfer of genes from an animal that has natural
resistance to a disease into a totally different animal.
This second animal is then made resistant to that disease.
In this way, domesticated animals can be more economic to rear and hence help to reduce the price of
food production.…read more


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