BTN315: Practical Prep (3, part 2)

PCR – CONTINUED

The major steps of PCR:

1) Denaturation
• DNA target sample is heated (remember that DNA denatured when heated)
• DNA is denatured – DNA unwinds, hydrogen bonds are broken, DNA strand separate
• Result: ssDNA(single-stranded DNA) is formed.

2) Annealing
• Temperature that the solution is held in is lowered
• Primers are added
• Primers anneal to ssDNA – providing a starting point for DNA replication.

3) Extension
Taq polymerase attaches to template DNA (specifically potion marked by primers)
Taw polymerase moves along the template and synthesizes DNA strands
complementary to the template.

Question: WHY IS PCR EFFICIENT?

PCR exponentially multiplies the target DNA. We usually make use of +25 cycles. In a cycle, the
newly synthesized ssDNA can serve as templates to make new DNA strands.

Question: WHY IS TAQ POLYMERASE ADDED LAST?

It minimizes any side-reactions that may occur during the PCR thus improving efficiency of the
process.

Factors to optimize results using PCR

1) Annealing temperature
• Usually 3-5 degree Celsius below melting temperature(Tm) [Tm=4(G+C)+2(A=T)]
• Usually ranges ~50-60 degree Celsius (best to start at lowest temperature)
• Temperature too low: primers likely to make more mistakes(result=when sample is run
on a gel the will be many bands)
• Temperature too high: primers won’t bind to DNA(result= none)

2) Concentration of magnesium
• [Mg2+] must be between 1.5-3mM
• [] too high: polymerase makes more mistakes
 3) Design of primer
• All primers used should have the same Tm
• Having the same Tm means all primers used will bind to the DNA at the same
temperature.
• To get good hydridization, 2/3 bases on 3 prime end should be a C or G
• Avoid primer dimers as all costs!

Questions 1: Why should the 3 prime end be a C or G?

Cytosine and Guanine both have 3 hydrogen bonds – thus ensuring better polymerization.

Question 2: When do primer dimers occur?

Primer dimers occur when the end of the primers bind to one another.

Question 3: If there is primer dimers then what will happen?

No product will be produced.

4) [DNA] and Taq polymerase

By increasing Taq polymerase you increase the amount of nonspecific products (that’s
produced during PCR)
If [DNA] is too high = PCR reactions won’t WORK!!!

QUESTION: EXPLAIN THE PROCESS OF ANNEALING IN DETAIL BY LISTING IMPORTANT FACTS

• The annealing step will specify what region of the DNA will be amplified.
• The solution is cooled and primers are added.
• Primers attach to the template DNA using hydrogen bongs
• Forward primer: matches the sequence of a ssDNA at the beginning regions that we wish to
amplify.
• Reverse primer: match the end of the region of the other side of the ssDNA.

QUESTION: WHY IS PCR REACTIONS NOT ALWAYS 100% SUFFICIENT?

1. Some reagents are in excess and become depleted

2. Not every DNA duplex will denature in every cycle
3. Taq polymerase may not copy the entire amplified region of the DNA in each cycle
4. Taq polymerase and primers won’t bind to every DNA molecule in every cycle