Cell Bio Lab 7: PCR and Agarose Gel Electrophoresis

PCR (Polymerase Chain Reaction) Defined

A repeated series of sequential reactions that results in the amplification of minute quantities of DNA

3 Steps of PCR

1. Denaturing the DNA template

2. Annealing of oligonucleotide primers to the DNA template

3. Elongation of a new DNA strand

Note: As this process is repeated, the newly formed DNA strands become part of the DNA template population → results in exponential amplification of the original DNA template

In theory, how many copies of the original DNA template is needed for amplification?

Only one copy

Historical Background of PCR + Applications

• By Kary Mullis, 1983 → he earned Noble Prize in Chem

• Applications:

  • Technique revolutionized forensic science

  • Used in diagnosing genetic problems

  • Can detect AIDS even before antibodies develop

  • May yield an early warning system for cancer

  • Etc

What two innovations made PCR an automated and routine procedure?

• 1) Development of programmable thermal cyclers

  • Instruments that cycle automatically through the diff temps required for each of the rxns in the sequence

  • Remove need for rxn tubes to be transferred manually from one incubation temp to another

• 2) Isolation of thermally stable DNA polymerases (most commonly used is Taq DNA polymerase, initially isolated from Thermus aquatiticus; there are others also available)

  • Aren’t denatured at high temps required for DNA denaturation step → their use eliminates need to replenish DNA polymerase after each denaturation step

    • Means that components of rxn can be mixed together, placed in a thermal cycler, & left unattended until the completion of the rxn

Why must target DNA be denatured?

• DNA requires a template for replication, since template contains info that directs sequence for synthesis of its complementary strand

• Note: target DNA is double-stranded

Step 1: Denaturation

• Sample is heated to 94°C → this high temp will disrupt the hydrogen bonds that hold the two strands together

• (45 sec - 1 min)

• Usually all components of PCR are mixed together before beginning cycling

• Sometimes, “HOT START” PCR is used

  • Target DNA is heated to 94°C - 98°C for several minutes before adding the Taq polymerase, adding to the duration of the enzyme’s activity

DNA polymerases (the enzymes that synthesize DNA) require …

• A primer → means they can’t copy a single-strand of DNA and must start w/ a double-stranded region

• In PCR, primers provide these double-stranded regions

What are primers?

Synthetic oligonucleotides (15-30 bases) that will bind by hydrogen-bonding to the complementary sequence on the target DNA

Step 2: Annealing

• Primers bind (hybridize) to their complementary sequences on the single-stranded target DNA template

• Then, primers provide the free 3’ hydroxyl required by DNA polymerase for addition of new nucleotides

• Investigator chooses primers that will anneal on either side of the part of the DNA of interest → Result: amplification of the DNA between these two primers

• 45 sec - 1 min

Factors to consider when selecting primers

• Sequence of the primers will influence the Tm (melting temp aka the temp at which the strands will separate)

• Possibility of primer hairpins (the primers complement within themselves)

• 3’ complementarity (primer-dimers; the two primers have complementary sequences and bind to each other preferentially to the target DNA

Between different PCR programs, what factor will be the most variable?

• The annealing temperature

  • Is chosen based on primer length and GC content

• Both primers should have ~same Tm

  • If not, use the lower Tm of the two primers and subtract 5°C

• Typical annealing temp might be 60°C for primers w/ high degree of match w/ template DNA sequence

• Much lower temps would be used if the sequences were less similar

Two reasons why primers anneal instead of the target DNA simply re-hybridizing to its complementary strand

1. The primers are present in much higher numbers than complementary strand

2. They are much shorter and hence will hybridize more quickly

Step 3: Extension

• Polymerase adds nucleotides to the 3’ end of the primer → this process is called extension

• Optimal extension temp usually 72-75°C by Taq polymerase

• Length of this step is usually 1-1.5 mins per kb of PCR product

• Note: Primers will also become part of product & as the cycles continue, the most common DNA in the rxn tube will be the sequence that’s defined by the two primers

Contents of a PCR Tube

1. Target DNA (this is what will be amplified)

2. Primers

3. PCR buffer (consisting of 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2)

4. dNTPs (the four diff deoxytrinucleotide phosphates, present in conc of 200 nM each)

5. Taq polymerase (a thermostable DNA polymerase)

6. Silicone oil (prevents tube contents from evaporating

   1. Not necessary if have thermal cycler w/ a heated lid