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TECHNIQUES IN BIOTECHNOLOGY

TECHNIQUES IN BIOTECHNOLOGY

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TECHNIQUES IN BIOTECHNOLOGY. DNA EXTRACTION. http://learn.genetics.utah.edu/content/labs/extraction/. Purpose: to purify a DNA sample Uses genetic testing crime scene investigation gene studies. Steps: . collect cells remove DNA from cell purify it use it!. Ex. Insert for plasmid. - PowerPoint PPT Presentation

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Page 1: TECHNIQUES IN BIOTECHNOLOGY

TECHNIQUES IN BIOTECHNOLOGY

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DNA EXTRACTION

• http://learn.genetics.utah.edu/content/labs/extraction/

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• Purpose: to purify a DNA sample

• Uses– genetic testing– crime scene investigation– gene studies

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Steps:

• collect cells• remove DNA from cell• purify it• use it!

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Ex. Insert for plasmid

• From someone else’s DNA – ex. fish gene in strawberries, – jellyfish gene in plants

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• In order to do these things, we need a way to make many copies of the genes we want

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Making more DNA: PCR

• purpose: to make many copies of a specific piece of DNA

Steps:- denature DNA using temperature- add in the ingredients (ATGC, enzymes,

primers) to copy it- repeat

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Making an insert: Polymerase Chain Reaction

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Uses

• make the desired gene/piece of DNA to use it to make proteins, study it, etc.

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To Make a Recombinant Plasmid:

1. Cut the plasmid and the insert with the same restriction endonuclease to make complementary sticky ends.

Insert

2. Combine the sticky ends using ligase.ligase: enzyme used to join DNA together

3. Introduce the recombinant plasmid into bacteria.

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Making a Recombinant Plasmid

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Bacterial Transformation

- - - - - -

- - - -- - - -

- - - - - -

- - -

+ + + + +- ++ -++ - - ++ - ++

+ + + + +

+ - +

phospholipid bilayer

plasmid

Ca2+ ions

• introduction of foreign DNA into a bacterial cell• plasmid is used as a vector, a vehicle by which DNA can be introduced into host cell

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Following transformation bacteria are grown in medium with antibiotic…

Only the bacteria that have the plasmid (and therefore the antibiotic resistance) will survive.

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Example plasmid:

Origin of Replication:

• the specific sequence MUST NOT be cut by restriction endonucleases or it won’t be able to replicate

• where the plasmid starts to duplicate itself

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• easy to grow • no ethical issues• small genome • easy to

manipulate

Using Bacteria as Production Factories

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DNA SEQUENCING

• purpose: find out the order of DNA, the “fingerprint”

• uses: forensics, genetic testing, studying mutations

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Steps

• like PCR, put your template (DNA you want to “read”) with the nucleotides, primers, enzymes, and use heat to make “more”

• some of the nucleotides have fluorescent labels and stop the chain from being made

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• results in a soup of different-length fragments

• separate them by size, and “read”

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Gel Electrophoresis• used to “see” the DNA

• used in sequencing and to determine the origin of DNA

ex. Tsunami baby

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Sequencing

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Ex. RFLP: Restriction Fragment Length Polymorphism

Comparison of different lengths of DNA fragments produced by restriction enzymes to determine genetic differences between individuals

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Steps

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Therapeutic cloning

• used to produce tissue that is identical to the donor, to prevent rejection

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Reproductive Cloning

• creates an organism with the same genetic material (DNA) as the original organism – an EXACT COPY of the donor

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Dolly the Sheep

• the first cloned sheep

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Common uses of biotechnology:1. Making "stuff”

• proteins, enzymes, medication, etc. can be produced by engineered bacteria!

• Food can be altered to have new traits• Cloning (therapeutic and reproductive)

2. Genetic screening• crime cases, relationship, genetic screening, etc.

3. Gene Therapy

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Gene therapy - desired gene is inserted into cell's nucleus using a retrovirus as a carrier- defective gene replaced by functional gene

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Ex. ADA deficiency

- adenosine deaminase deficiency- little immunity with low chances of

recovery

- the T-cells of a four-year-old were removed, modified and re-inserted to fix her immune system