PCR (polymerase chain reaction) was invented by Kary Mullis in 1987 and allows for the specific amplification of a single DNA sequence. It takes advantage of basic DNA replication requirements by using DNA polymerase, primers, nucleotides, and thermal cycling to exponentially amplify the target sequence. Key aspects of PCR include using specific primers, thermocycling between denaturation, annealing, and extension steps, and the use of Taq polymerase which functions at high temperatures. PCR has many applications including cloning, detection of genetic diseases, forensics, and quantification of DNA or RNA.
4. “I was working for Cetus, making oligonucleotides.
They were heady times. Biotechnology was in flower
and one spring night while the California buckeyes
were also in flower I came across the polymerase
chain reaction. I was driving with Jennifer Barnett to
a cabin I had been building in northern California.
She and I had worked and lived together for two
years. She was an inspiration to me during that time
as only a woman with brains, in the bloom of her
womanhood, can be. That morning she had no idea
what had just happened. I had an inkling. It was the
first day of the rest of my life.”
- from Karry Mullis’s autobiography at the Nobel e-Museum
5. PCR
Specifically targets and amplifies a
SINGLE sequence from within a complex
mixture of DNA.
How is this different from cloning?
6. Takes advantage of basic
requirements of replication
A DNA template
Nucleotides
Primers
polymerase
PCR is DNA replication in a test tube
7. Primers
Must have some information about
sequence flanking your target
Primers provide specificity
8. 5’ 3’
3’ 5’
Complementary to opposite strands with 3’
ends pointing towards each other
Should have similar melting temperatures
Be in vast excess
9. Melting temperature
TmoC = 2(A/T) + 4(G/C)
TmoC Temperature at which
half possible H bonds are
formed
16. Problems with Taq
Does not have proof readng ability
Error rate 1 in 2 X 104 bases
Seems rare but can be recovered in
cloning a single molecule
Newer polymerases have high fidelity
17. Termplates for PCR
Small amount of template
In theory a single molecule
Do not need to isolate sequence of
interest
DNA template need not be highly
purified
DNA is stable in absence of nucleases
27. Nested primers
PCR primers are not always an exact match!
Degeneracy
Lower annealing temperatures increase chances of
amplifying something!
Could be wrong thing!
30. Real Time PCR
Detection and quantitation of fluorescent reporter the
signal of which increases in direct proportion to the
amount of PCR product in a reaction
Does not measure the amount of end product but its
production in real time
31. SYBR
green
Also binds
primer dimers
Can
overestimate
product
35. Other application of PCR
Detection of mutations
screen for inherited disorders
Detection of HIV
Not standard test given
Detect tuberculosis without culturing
Prenatal sex determination
DZY1 = Y specific sequence present in
5000 copies
36. Other application of PCR
Preimplantation diagnosis of genetic
diseases
Forensics
Paternity testing
37. Forensics
STR
Short Tandem Repeats
2 to 7 base pairs repeated 7-40 times
Replaced VNTRs in forensic analysis
13 Highly polymorphic loci have been
selected by FBI
Population match probabilities 0.1 - 0.28
Probability One in 5.7 X 10-15
Combined DNA Index System (CODIS)