PCR and Gel Electrophoresis

PCR (polymerase chain reaction)

Enzymatic reaction that links nucleotides together to make more DNA.

• Used to amplify a specific sequence or sequences of DNA

Primers

Short pieces of DNA (single-stranded) that target unique sequences of target DNA

• Flank the sequence you want to amplify

• Forward and Reverse should anneal to different strand

• Cause condensation reaction; provide free 3’ OH for polymerase to attach incoming dNTPs

Template

What you are amplifying

• one of the main ingredients of a PCR

dNTPs

monomers that become part of the polymer

• one of the main ingredients of a PCR

thermally stable DNA polymerase

Needed to withstand protein denaturing during PCR process

• Acquired from Thermus aquaticus

• One of the main ingredients of PCR

Buffer

Maintains pH and provided suitable environment for DNA polymerase activity

• one of the main ingredients of PCR

Mg2+

Cofactor for DNA polymerase

• neutralize negative charges on DNA phosphate backbone

• Facilitate formation of complex between primers and DNA template

• Too much concentration can produce non specific PCR products

• One of the main ingredients of PCR

Initial denaturation

Heat up to about 95^oC

• 1st step of PCR

• done in a thermal cycler

Denaturation

Heat sample again to about 95^oC

• complimentary strands split

• 2nd step of PCR

Annealing

Let sample cool down so primers can bind to DNA

• temperature depended on primer sequences

• 3rd step of PCR

Extension

Let sample reach room temperature (68-72^oC)

• For optimal DNA polymerase function

• 4th step of PCR

Final incubation

finishes any partial fragments

• 5th step of PCR

soak 4^oC

sixth step of PCR

How does electrophoresis take advantage of DNA’s charge?

Negatively charged DNA will travel through the gel towards the positive electrode

• smaller molecules move faster than larger ones

Ethidium bromide

“stains” the DNA as it passes through the gell

Ficoll, glycerol, or sucrose

One loading buffer component

• Increases density so sample sinks in the well

Dyes

One loading buffer component

• Visualizes sample for loading

• Tracks progress through gel

pH stabilizer (Tris)

One loading buffer component

• Keeps DNA from being affected by pH

EDTA

One loading buffer component

• chelating agent that separates Mg2+ ions so DNases are inactive

• prevents DNA degradation

SDS

one loading buffer component

• Eliminates DNA-protein interactions

Running buffer

a buffer that neutralizes the charge of water and protects DNA

• under electrical current, water molecule dissociates into H+ and OH-

• H+ ions can react with phosphate of DNA and neutralize it, interfering with its movement through gel