gene technologyng

8/7/2019 Gene TechnologyNG

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Procedures involving the manipulation of

DNA

Includes

taking genes from one organism andplacing them into another

genetic fingerprintingAlso called genetic engineering or

recombinant DNA technology


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Many techniques involved in gene

technology can be demonstrated using

the example of human insulin productionby genetically modified bacteria.


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This method involves cutting the geneout from a complete chromosome.

Electrophoresis and DNA probes areused to identify the gene in donor DNA.

The gene is cut using restrictionendonucleases.

This method has limitations due todifferent regulatory genes in prokaryotesand eukaryotes and the presence ofintrons.


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Molecular scissors

Found naturally in bacteria

Use to destroy viral DNA

Over 400 types now been obtained

Recognise a specific base sequence

Cut DNA unevenly to produce stickyends or make straight cuts to form blunt

ends


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mRNA is obtained from cells producinginsulin

Using the enzyme reverse transcriptase

A DNA sequence is copied from mRNA Making single stranded copy DNA (cDNA)

DNA polymerase uses free nucleotides tomake the complementary strand

This method is preferable as the cell hasmany mRNA copies versus one gene in DNAand the mRNA strand is the same length as

the gene and doesnt have to be cut


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Vectors are molecules of DNA which areused to carry the foreign gene into thehost DNA

Examples include bacterial plasmids andphage viruses


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Plasmids are circular pieces of double

stranded DNA found in bacteria in

addition to the main DNA. May contain useful gene such as

antibiotic resistance.

Can enter bacteria and plasmid DNAcan replicate when bacteria undergo

binary fission


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Both the DNA (if using method A) and

the plasmid are cut using the same

restriction endonuclease The sticky ends of the plasmid and the

gene are complementary and areattracted to each other

Hydrogen bonds form between

complementary bases

DNA ligase is used to glue the cut ends


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Plasmids are removed from the bacterialhost cells.

Recombinant plasmids are incubated withthe host bacteria

Calcium ions, temperature and electricalshocks can be used to make the bacterialcell walls more permeable and take up theplasmid

Some bacteria take up the recombinantplasmids to become genetically modified


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Only some of the bacteria have been

modified and need to be separated out

DNA probes orgenetic markers can be

used

The human insulin gene in inserted into the

middle of a gene for antibiotic resistance

Modified bacteria are not antibioticresistant and can be identified by replica

plating


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Modified bacteria are grown in industrial

fermenters on a large scale

Bacteria are allowed to reproduceasexually and produce clones

Plasmids also replicate each time the

cell divides The bacterial cells express the human

insulin gene


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Insulin made by the bacteria is excreted

into the culture medium

The insulin then needs to be extractedand purified


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Over 2 million people inject insulin daily

Previously used cow or pig insulin from

dead animals Different chemically

Immune response destroyed this insulin

Fermentation produces large quantitiescheaply

No ethical/religious/moral issues

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