Application: PCR & Electrophoresis

What do organisms use enzymes for?

to rapidly grow their DNA

What is PCR?

• an in vitro method for copying DNA

• “DNA replication in a tube”

What are the 5 ingredients of PCR?

1. Template DNA

2. Nucleotides (dNTPs)

3. Short DNA primers

4. DNA polymerase

5. Magnesium Ions (buffer)

What are the 3 steps?

1. Template DNA is denatured by heating to 95 degrees Celsius

2. Primers are annealed to teh DNA by cooling to 60 degrees Celsius

3. New DNA is synthesized by heating to 72 degrees Celsius

All new strands serve as ___ ____

futre templates (chain reactions)

How many times is the PCR process repeated? How does the amount of DNA copies grow in each cycle?

30+ cycles. grow exponentially.

Who invented PCR?

Kary Mullis in 1983

What is Gel Electrophoresis?

A method to separate and visualize DNA

What happens first in Gel Electrophoresis?

• DNA is added to wells inside a porous agarose gel.

  • the gel is suspended within a salt solution

  • To make sure the DNA sinks within this solution, it is mixed with loading dye that increases its density

  • The gel also contains a fluorescent dye that can bind DNA

What is the purpose of loading dye?

to make sure the DNA sinks within the solution, it increases the DNA’s density

What cant he fluorescent dye do?

it can bind DNA and is within the gel

What is the second step of Gel Electrophoresis?

• An electrical current is applied to the gel

• The negative cathode is located near the wells at the top, while the positive anode is located near the bottom

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• An electrical current is applied to the DNA within the gel

• The negative cathode pushes the DNA away from the top, while the positive electrode pulls the DNA towards the bottom

• “Run to Red”

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• As the DNA travels through the gel, it must squeeze through a network of pores of varying sizes

• Small DNA fragments have an easier time squeezing through, while larger fragments tend to get stuck

• Small DNA fragments end up at the bottom of the gel, while large DNA fragments remain near the top

Why is DNA repelled by the negative cathode?

Because DNA has its own negative charge from phosphates.

What is the final step of Gel Electrophoresis?

• The DNA fragments are visualized with UV light

• Ethidium bromide intercalates DNA that travels through the gel

• When exposed to UV light, ethidium bromide fluoresces, allowing us to visualize the DNA within the gel