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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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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
• From someone else’s DNA – ex. fish gene in strawberries, – jellyfish gene in plants
• In order to do these things, we need a way to make many copies of the genes we want
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
Making an insert: Polymerase Chain Reaction
Uses
• make the desired gene/piece of DNA to use it to make proteins, study it, etc.
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.
Making a Recombinant Plasmid
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
Following transformation bacteria are grown in medium with antibiotic…
Only the bacteria that have the plasmid (and therefore the antibiotic resistance) will survive.
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
• easy to grow • no ethical issues• small genome • easy to
manipulate
Using Bacteria as Production Factories
DNA SEQUENCING
• purpose: find out the order of DNA, the “fingerprint”
• uses: forensics, genetic testing, studying mutations
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
• results in a soup of different-length fragments
• separate them by size, and “read”
Gel Electrophoresis• used to “see” the DNA
• used in sequencing and to determine the origin of DNA
ex. Tsunami baby
Sequencing
Ex. RFLP: Restriction Fragment Length Polymorphism
Comparison of different lengths of DNA fragments produced by restriction enzymes to determine genetic differences between individuals
Steps
CLONING
http://learn.genetics.utah.edu/content/tech/cloning/
Therapeutic cloning
• used to produce tissue that is identical to the donor, to prevent rejection
Reproductive Cloning
• creates an organism with the same genetic material (DNA) as the original organism – an EXACT COPY of the donor
Dolly the Sheep
• the first cloned sheep
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
Gene therapy - desired gene is inserted into cell's nucleus using a retrovirus as a carrier- defective gene replaced by functional gene
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