TECHNIQUES IN BIOTECHNOLOGY

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

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- - - -- - - -

- - - - - -

- - -

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

+ + + + +

+ - +

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