The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2−ΔΔCT method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2−ΔΔCT method. In addition, we present the derivation and applications of two variations of the 2−ΔΔCT method that may be useful in the analysis of real-time, quantitative PCR data.
6. Primer
3’
5’
5’
3’
Primer Anneals &
DNA Polymerase Adds
Deoxynucleoside triphosphates
37°C
Extension
New DNA strand
is created
Sequencing is performed by DNA replication
8. What if DNA extension could be terminated
at a known nucleotide using a mixture of
normal bases and termination bases
TACGTACGTACGTGT
ATGCATGCATGC????????????????????????????????????
3’ 5’
5’
Primer
DNA
Pol.
A
TACGTACGTACGTGT CG
ATGCATGCATGC????????????????????????????????????
3’ 5’
5’
Primer
A
DNA
Pol.
A
Normal base gets
incorporated
By probability termination will occur
at every “A”
16. Deoxynucleoside triphosphates
Deoxy adenosine triphosphate (dATP)
Deoxy guanosine triphosphate (dGTP)
Deoxy thymidine triphosphate (dTTP)
Deoxy cytidine triphosphate (dCTP)
Chain
Termination
3’
5’
DNA
Polymerase
Lacks a 3’ hydroxyl group.
Acts as a terminator because,
once incorporated, no other
nucelotide can be added.
X3’
5’
DNA
Polymerase
Dideoxynucleoside triphosphates
Dideoxy adenosine triphosphate (ddATP)
Dideoxy guanosine triphosphate (ddGTP)
Dideoxy thymidine triphosphate (ddTTP)
Dideoxy cytidine triphosphate (ddCTP)
Chain
Extension
17. PhD 1943
Cambridge University
Nobel Prize In Chemistry 1958
Amino acid sequence of insulin
Nobel Prize In Chemistry 1980
Sequenced the first genome, phage Φ-X174, by hand
using a method that he developed.Frederick Sanger
The Sanger Dideoxy sequencing method
was the foundation for the discovery of
PCR.
21. Why not do Sanger
sequencing at a single
base pair?
Kary B. Mullis
5’-TACGTACGTACGA
*GGAGTCCGGAATG-3’
CCTCAGGCCTTAC-5’+
ddTTP ddCTPddGTPddATP
22. First step to a Nobel Prize:
As you think, ignore obvious
problems.
23. Kary wanted to use total genomic DNA, but he
forgot the primer would likely mis-pair and ruin
his experiment. In “misguided puttering”, Kary
kept thinking!
CCTCAGGCCTTAC-5’
CCTCAGGCCTTAC-5’
CCTCAGGCCTTAC-5’
CCTCAGGCCTTAC-5’
25. What if I use two
primers for
confirmation?
+
ddTTP ddCTPddGTPddATP
3’-ATGCATGCATGCT
*CCTCAGGCCTTAC-5’
5’-GAATTCTACGTACGTACGAF-long
5’-TACGTACGTACGA
*GGAGTCCGGAATG-3’
CCTCAGGCCTTAC-5’
R-short
27. What about stray
nucleotide
triphosphates?
+
ddTTP ddCTPddGTPddATP
3’-ATGCATGCATGCT
*CCTCAGGCCTTAC-5’
5’-GAATTCTACGTACGTACGAF-long
5’-TACGTACGTACGA
*GGAGTCCGGAATG-3’
CCTCAGGCCTTAC-5’
R-short
dNTP
dNTP
28. I can destroy stray
dNTPs with alkaline
phosphatase!
But, bacterial alkaline phosphatase
will remain because it cannot
be heat killed. It will destroy the
ddNTP’s (not true).
29. Second step to a Nobel Prize:
Make up problems that
do not exist and try
to solve them.
30. I can deplete nucleotides
by adding polymerase
first without ddNTP’s
3’-ATGCATGCATGCT
*CCTCAGGCCTTAC-5’
5’-GAATTCTACGTACGTACGAF-long
5’-TACGTACGTACGA
*GGAGTCCGGAATG-3’
CCTCAGGCCTTAC-5’
R-short
dNTP
dNTP
31. Denature and anneal primers
Polymerase extension
DNA replicated!
Anderson Valley
Third step to a Nobel Prize.
Recognize PCR when
you find it.
1 Copy
2 Copies!
33. Final step to a Nobel Prize:
Try to get someone
to listen to you.
34. “…no one was particularly enthusiastic about it.”
1984 annual Cetus Scientific Meeting…..”nobody
seemed to be interested in my poster….” “People would
glance at it and keep walking.”
At first, people did not get it.
Then Joshua Lederberg (also a Nobel
Laureate) said:
“Why didn’t I think of that?”
38. Loci for Type 2 Diabetes and Triglyceride Levels
GCAGCTCACCTCCAGCTTTAGTTTTC[C/T]CATGACAGTAAGTCTATTACCCTCC
Risk allele
39. First, you need to select primers for PCR
• No, you do not need a computer program to select
primers.
• I prefer 24 bp long and end on G or C
• Others prefer 20 bp long and end on A or T
• Try to have at least 50% G’s +C’s to ensure
reasonable annealing temperature
That’s about it. Do not waste too much time selecting
primers.
48. Old School “Perkin Elmer 2400” PCR Thermocycler
Hot bonnet prevents condensation.
49. Mineral Oil
Sample
If your thermocycler does not have a hot bonnet
or if you will need to open the hot bonnet to
remove or modify a reaction, use mineral
oil.
50. Runs on antifreeze and refrigerant
Paper towels to adsorb leaking
orange-colored antifreeze.
54. New Thermocyclers = Peltier Cooling & Gradient Blocks
Peltier effect
It occurs when a current is passed through two
dissimilar metals or semiconductors (n-type and p-
type) that are connected to each other at two
junctions (Peltier junctions). The current drives a
transfer of heat from one junction to the other: one
junction cools off while the other heats up; as a
result, the effect is often used for thermoelectric
cooling. This effect was observed in 1834 by
Jean Peltier.
58. RT-PCR is like any other PCR except
it uses cDNA as a template.
59. How do you make cDNA?
cDNA can be created
from RNA using
RNA-dependent DNA
polymerase
(reverse transcriptase)
60. How do you make cDNA?
mRNA AAAAAAAAAAAA5’- -3’
TTTTTTTTTTTT-5’
mRNA AAAAAAAAAAAA5’- -3’
TTTTTTTTTTTT-5’cDNA
Reverse transcriptase Oligo (dT) Primer
Template For PCR
3’-
3’-
61. RT-PCR measures the presence
of cDNA corresponding to
its respective RNA.
RT-PCR is, therefore, used to indirectly
estimate RNA abundance which
MAY indicate the level of gene expression.
62. Major Points
• Sequencing and PCR both use DNA polymerase and
replicate DNA (PCR uses TAQ DNA polymerase).
• Kary Mullis discovered PCR while thinking about
a possible dideoxy sequencing experiment.
• Half of a Nobel discovery is finding it, the other half is
realizing what you have found.
• RT-PCR is used to estimate gene expression