Biotechnology

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Combining Life Science and Technology

What is Genetic Engineering?

• Process of manipulating genes for a practical purpose. Making recombinant DNA is a process used for this.
• Examples of Genetic engineering:
• Making proteins/vaccines to treat diseases

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• Genetically modified plants-- that are created to be more drought resistant or grow bigger.

What is Recombinant DNA?

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• DNA from 2 or more different species combined.
• Why combine DNA?
• In this way, human genes can be inserted into other organism.. and that organism can then make human proteins for medical uses,
• This is called a transgenic organism.Example...
• Bacteria with the insulin gene can make human insulin for treatment of diabetes

What is a Vector?

• Anything that is used to transfer DNA into a host cell.
• What are three types of vectors?
• Plasmid, Virus, Yeast
• What is a Plasmid?
• A circular piece of DNA from bacteria that can replicate independently.

Give the steps to making Recombinant DNA.

• 1. Identify the gene of interest-- a gene sequence that codes for a protein for example.
• 2. Cut the DNA with...
• Restriction enzymes
• 3. Cut vector DNA with same restriction enzymes
• 4. Insert gene of interest into vector DNA
• 5. Sticky ends allow gene to attach with help of enzyme DNA ligase
• 6. Insert recombinant DNA into a host cell

How do Restriction Enzymes cut DNA?

• They recognize a pattern in the genetic code (order of nitrogen bases). This pattern is..
• A Palindrome sequence. It is the same...
• Backwards as forwards
• ex. R-A-C-E-C-A-R
• Restriction enzymes always cut at the same specific base sequence and leave sticky ends

What are sticky ends?

• The unpaired bases left in the specific sequence
• They will attract their complementary bases and allow the gene of interest to combine with the plasmid.

What is PCR?

• Polymerase Chain Reaction
• What is it used for?
• It is used to copy a DNA sample so that there is more of it for testing.
• What natural process does it mimic?
• DNA Replication
• Example: Crime scene may only yield a small amount of skin cells or a drop of blood. PCR replicates the sample so that they can complete many tests on the sample.

What the Steps to PCR?

• Denaturing- Raise the temperature (~95° C) to break hydrogen bonds of DNA, separating the strands to reveal unpaired bases.
• Annealing- Lower the temperature (~54° C) to add primers (short segments of DNA).
• Extension- Raise the temperature (~72° C) to an optimum range for Taq polymerase to add free nucleotides to the original strand.
• Result is two copies of the original DNA
• DNA is doubled every 2 minutes. In a few hours millions of copies can be made.

DNA Fingerprinting

• Identification method using DNA
• Is not the same as taking a print from the fingers.
• What does DNA Fingerprinting produce?
• It produces a pattern of dark bands that is unique for each individual.
• What is DNA Fingerprinting used for?
• 1. Criminal cases-- identifying crime scene DNA
• 2. Paternity cases-- finding out who the parents of a child are
• 3. sorting Recombinant vs. Nonrecombinant DNA

What are the steps to making �a DNA fingerprint?

• 1. Obtain a biological sample from..
• Blood, semen, saliva, skin cells
• 2. Make several copies of the DNA sample using PCR
• 3. Cut the DNA into segments using...
• Restriction enzymes
• 4. Use gel electrophoresis to sort the sections of DNA by size

What is gel electrophoresis?

• A technique for measuring DNA segments using a gel and electricity to separate DNA according to size.

Explain how �gel electrophoresis works.

• DNA samples are loaded into wells in the gel using a...
• Micropipette
• Electrical current is turned on, allowing the DNA to move through the gel toward the..
• Positive end of the gel because…
• DNA has a negative charge.

How are the bands created?

• DNA segments travel different distances depending on their...
• Size (length)
• Describe how long vs. short pieces move.
• Longer segments move shorter distances because they move more slowly through the gel
• Shorter segments move fastest and go... the greatest distance away from the start.

What is responsible for the different sized DNA fragments?

• Non-coding repeated base sequences between genes are responsible for the different sized DNA fragments (RFLP’s)

What is the gel made of?

• Agarose powder and buffer solution
• What does the gel have that makes it good for separating the DNA?
• There are holes (spaces) in the gel that the segments have to go through (like a filter)
• Why is buffer used?
• To conduct electricity through the gel

What is a clone?

• An organism that is an exact copy (genetically identical) of another organism
• What is a method used to transfer one organism's DNA into an enucleated host cell to create a clone?
• Somatic Cell Nuclear Transfer

List the steps to:�Somatic Cell Nuclear Transfer

• 1. Remove nucleus from somatic cell
• 2. Remove and discard nucleus from an egg cell..
• 3. Put somatic cell nucleus into enucleated (empty) egg cell.
• 4. Allow time for DNA to adjust and reprogram
• 5. Stimulate cell division electrically or chemically
• 6. Put divided cell embryo into a surrogate mother.

When the new organism is born, what will it be a clone of?

• The donor organism. The one that gave the somatic cell nucleus.

What is therapeutic cloning?

• Creating tissue from a patient's DNA that is an exact copy of the patient in order to grow organs that can be transplanted in the patient that his body will not reject.
• Example.. take liver cells and stimulate them to grow in the lab in order to make a new liver to replace the patient's damaged one.

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Define Stem Cells.

• Stem cells are cells that haven't been programmed yet-- they do not have jobs in the body, but could become many different kinds of cells.
• There are 3 kinds:
• Totipotent, Pluripotent, Multipotent
• If you remember TPM “The Perfect Man” it will help you remember the order of Best to Least useful.

Differentiate between the �types of stem cells

• Totipotent—Can become any kind of cell They are found in …
• early embryos
• Pluripotent—They can become almost any type of cell and are found in..
• Late embryos and early fetuses
• Multipotent—can only become certain kinds of cells and are found in...
• Adult blood, bone marrow and in umbilical cords

What are the medical uses �of stem cells?

• Treat paralysis
• Treat brain injuries and strokes
• Treat leukemia (bone marrow cells)
• Treat diabetes (pancreas cells so that pancreas will begin producing insulin)