There was a time when to amplify DNA,
You had to grow tons and tons of tiny cells.
(Oooh) Then along came a guy named Dr. Kary Mullis,
Said you can amplify in vitro just as well.
Just mix your template with a buffer and some primers,
Nucleotides and polymerases too.
Denaturing, annealing, and extending,
Well it’s amazing what heating and cooling and heating will do.
[Chorus]
PCR when you need to detect mutation (detect mutation)
PCR when you need to recombine (recombine)
PCR when you need to find out who the daddy is (who’s your daddy?)
PCR when you need to solve a crime (solve a crime)
[x2]
POLYMERASE CHAIN REACTION (PCR)
1.
Before Briefly explain the steps involved in normal DNA replication in a cell.
2.
What is the function of each of the following in a PCR reaction?
HEAT:
PRIMER:
taq POLYMERASE:
STUDENT SHEET
Bio Rad PCR Song Lyrics
3.
Briefly describe what the thermocycler (PCR machine) does and what happens in each
of the following three steps of Polymerase Chain Reaction:
DENATURING:
ANNEALING:
ELONGATION/EXTENSION:
4.
Suppose you start with one double-stranded molecule of DNA and you want to
amplify this one DNA molecule using PCR. You add an excess of primers, each of
which will anneal to the DNA molecule in only one place, copying the segment of DNA
between them. Draw representations of the DNA and primers during the three steps
of PCR during one cycle.
5.
Illustrate the exponential growth of the DNA from question 4 through 3 cycles of PCR.
6.
How many molecules of double-stranded DNA will you have after three cycles? After
five cycles? After 30 cycles?
In a cell, the double-stranded DNA helix unwinds and unzips at the replication
origin. DNA polymerase makes a new copy of the template strand by adding
complimentary bases, producing a replication fork.
2. What is the function of each of the following in a PCR reaction?



Heat: turning up the temperature causes the double stranded DNA to separate or
denature
Primer: these are the hooks that provide the starting point for the polymerase.
There are two primers, a forward and a reverse which flank the target sequence.
Taq polymerase: heat-stable DNA polymerase
3. Briefly describe what the thermocycler (PCR machine) does and what happens in each of
the following three steps of Polymerase Chain Reaction:



Denaturing: 94˚C, raising the temp separates the DNA strands from doublestranded to single-stranded
Annealing: 50-65˚C, dropping the temperature allows the primers to anneal to their
complimentary sequences
Extension: 72˚C, bring the temperature back up allows the Taq polymerase to
extend the template strands by adding complimentary base pairs to the singlestranded DNA molecules
4. Suppose you start with one double-stranded molecule of DNA and you want to amplify
this one DNA molecule using PCR. You add an excess of primers, each of which will anneal to
the DNA molecule in only one place, copying the segment of DNA between them. Draw
representations of the DNA and primers during the three steps of PCR during one cycle.
Student drawings will vary.
5. Illustrate the exponential growth of the DNA from question 4 through 3 cycles of PCR.
Student drawings will vary.
6. How many molecules of double-stranded DNA will you have after three cycles? After five
cycles? After 30 cycles?
3 cycles = 8 ds DNA (23); 5 cycles = 32 ds DNA (25); 30 cycles = 1,073,741,824 (230)
ANSWER SHEET
STUDENT WORKSHEET – ANSWER SHEET
1. Briefly explain the steps involved in normal DNA replication in a cell.
Advantages of Real-Time PCR vs. Traditional PCR
OVERHEAD
Types of PCR:
Traditional vs. Real Time PCR
Overview
Quantitative?
Applications
Summary
Real-time PCR
Measures PCR amplification as it
occurs.
Yes, because data is collected
during the exponential growth
(log) phase of PCR when the
quantity of the PCR product is
directly proportional to the
amount of template nucleic acid.

Quantitation of Gene
Expression
 Microarray Verification
 Quality Control and
Assay Validation
 Pathogen detection
 SNP Genotyping
 Copy Number Variation
 MicroRNA Analysis
 Viral Quantitation
 siRNA/RNAi experiments
Advantages of Real-Time PCR
 Increased dynamic range
of detection
 No post-PCR processing
 Detection is capable
down to a 2-fold change
 Collects data in the
exponential growth
phase of PCR
 An increase in reporter
fluorescent signal is
directly proportional to
the number of amplicons
generated
 The cleaved probe
provides a permanent
record amplification of
an amplicon
Traditional PCR
Measures the amount of
accumulated PCR product at the
end of the PCR cycles.
No, though comparing the
intensity of the amplified band
on a gel to standards of a known
concentration can give you
'semi-quantitative' results.
Amplification of DNA for:
 Sequencing
 Genotyping
 Cloning
Disadvantages of Traditional
PCR
 Poor Precision
 Low sensitivity
 Short dynamic range < 2
logs
 Low resolution
 Non-Automated
 Size-based
discrimination only
 Results are not
expressed as numbers
 Ethidium bromide for
staining is not very
quantitative
 Post-PCR processing
OVERHEAD
PLANS
LESSON
Types of PCR:
Traditional vs. Real Time PCR