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Gene Technology

Recombinant DNA Technology by In Vivo Cloning
1. Isolation of DNA Fragments
Using Restriction Endonucleases
DNA Gel Electrophoresis
Gene Identification and Gene Probes
Southern Blotting
Reverse Transcriptase
2. Insertion of the DNA fragment into the Vector
3. Transformation
4. Identification of Host Cells Using Genetic Markers
Uses of…

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Recombinant DNA Technology by In Vivo Cloning
This specialist technology allows genes to be manipulated, altered and transferred between
organisms helping scientists to understand how organisms work as well as assisting
industrial processes and medicine.

Techniques have been developed to isolate genes, clone them and
transfer them to microorganisms such…

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1. Isolation of DNA Fragments
Before transplantation, one must find the gene amongst billions of DNA nucleotides and then
isolate it from the rest of DNA there are two ways of doing this:

Using Restriction Endonucleases

A restriction endonuclease is an enzyme that recognises and cuts DNA at a specific…

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For example, BamHI is a restriction endonuclease that cuts at the sequence GGATCC,
whilst HindIII cuts at AAGCTT.




DNA Gel Electrophoresis

This is a form of chromatography that separates DNA fragments by their length. It can be
then used to separate restriction fragments, enabling scientists to locate the DNA fragment…

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The smaller the length of DNA,
the further down the gel it will move in a given time.




DNA cannot be seen on the gel so there are two options:
DNA can be stained with a coloured chemical that stains DNA bands blue, or use a
fluorescent molecule such as…

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Now the DNA fragments have been separated, they are put into alkali to allow the two
strands to break apart. The single strands are transferred on to a nylon membrane by a
technique called southern blotting. The single strands stick to the sheet in the same
positions as on the…

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and hence must turn their RNA into DNA so the host cell can read it for this they use the
enzyme reverse transcriptase.




Reverse transcriptase binds to the mRNA and begins to make a complementary
DNA strand known as cDNA.

The strands then separate and DNA polymerase builds a complementary…

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Reverse transcription has a few advantages over using endonucleases:
cDNA does not contain introns.
mRNA is in high concentrations in certain cells so it is easier to isolate than a single
DNA copy. (e.g. mRNA that codes for insulin is plentiful in the islet cells in the
pancreas so it…

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3. Transformation
Because of the insertion of the gene the plasmids are said to be recombinant, and when the
plasmids are inserted into bacteria, the bacterial cells are said to be transformed.

The bacterial cells and plasmids are mixed together in a solution containing calcium ions.
Calcium ions are used…

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Only a few bacterial cells become transformed. 99% do not take up a plasmid and others
may take up a nonrecombinant plasmid or a loop of DNA that has doubled back on itself.




4. Identification of Host Cells Using Genetic Markers
Because only a small number of bacteria take up…

Comments

Swallowtail

What a great contribution to the documents on this topic. These notes are well written, accurate and detailed. The annotated colour diagrams make learning easier. This would be a useful resource for any student needing to study biotechnology as part of their A level studies.

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