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PCR

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Cassie Stines

on 12 July 2013

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Transcript of PCR

PCR
Kary Mullis
Awarded Nobel Prize in Chemistry for his invention of Polymerase Chain Reaction (PCR) method in 1993
Contributions
Arthur Kornberg 1959
Began studying the mechanism of DNA replication and identified DNA polymerase
H. Gobind Khorana 1968
Pioneered techniques needed to make and use synthetic DNA oligonucleotides
Discovered the double helix structure of DNA
Thomas D. Brock 1969
Found new species of bacteria Thermus aquaticus able to withstand higher temperatures than E. coli
Klenow Fragment 1970
Modified version of DNA polymerase I from E. coli
1976
DNA polymerase isolated from T. aquaticus and found to retain at activity at temperatures above 75°C
Frederick Sanger 1977
Reported a method for determining the sequence of DNA
Stage is set for PCR!
Applications
Genetic Testing
Tissue Typing
Detecting infectious diseases
Genetic Fingerprinting for Forensic Science
Parental Testing
Before
Experiments
Method
olymerase
Before PCR, if you wanted to examine a specific sequence in DNA you had to clone it.
Chop up DNA into several thousand base pairs
Isolate each of them by growing them in a bacteria colony
Pick the best one, pick it off the plate, and grow it up
Problems:
Labor intensive, slow, and it was subject to inaccuracies
DNA is analyzed for genetic disease mutations
Prospective parents can be tested for being carriers of genetic disease mutations
PCR SONG
Denaturing
High temperatures is applied to double stranded DNA molecule to separate the strands from one another
Heat sample to 94-98°C for 20-30 seconds
Annealing
Extending
Repeat
hain
eaction
20 to 30 times
Cooled to 50-65°C for 20-40 seconds
Oligonucleotide primers anneal to the ssDNA template
DNA
How does DNA replicate?
What is DNA?
Heated to approximately 72°C
Taq Polymerase used to replicate DNA strands
Synthesizes new double stranded DNA molecules
Organ Transplantation
Detects Hepatitis B and C, Human Papillomavirus, Chlamydia trachomatis, Neisseria gonorrhoeae, Mycobacterium tuberculosis, HIV, etc.
Genetic analysis can be used to detect antibiotic resistance
Can detect as little as one viral genome among the DNA of over 50,000 host cells
Infections can be detected earlier
Donated blood can be immediately screened
Newborns can be immediately tested
Laser Pointer
Cetus 1979
Deoxyribonucleic Acid
Helicase attaches and breaks apart hydrogen bonds between the bases of the DNA strands
Primase attaches to each strand and assembles a foundation at which replication can begin
DNA Polymerase wraps around the strand and attaches new nucleotides to the exposed nitrogen bases
The new strand is complete containing the same sequence as the original strand
Hired to synthesize DNA
Components
DNA Template
DNA Polymerase
Primers
Nucleotides
Buffer
First Experiment
DEAR THOR!
2 oligonucleotides, DNA polymerase, and four nucleosidetriphosphates
Functionally Sober
eptember,
1983
Added human DNA and nerve growth factor primers to a test tube
Boiled for about 10 minutes, cooled, and added 10 units of DNA polymerase, then allowed it to sit at 37°C overnight
Next morning, nothing had happened
Meant he needed to consider thermal cycling
December
Point Genetic Mutations
DNA oligonucleotides
Taq
Machine invented to create oligonucleotides
pBR322, a purified plasmid
December 16,
First successful experiment!
Questions?
Full transcript