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Transcript of PCR
Sometimes called "molecular photocopying" PCR enables scientists to produce millions of copies of a specific DNA sequence in approximately two hours.
PCR is also valuable in numerous laboratory and clinical techniques
Detection of bacteria or viruses (particularly AIDS)
Diagnosis of Genetic Disorders What is PCR used for? History The invention of PCR is credited to Kary Mullis.
He developed this technique in 1983 when he worked in Emeryville, California
Worked for Cetus Corporation, one of the first biotechnology companies
Came up with the idea while on the Pacific Coast Highway in his car. He was playing in his mind with a new way of analyzing DNA mutations. In doing this, he realized that he had instead created a new method of amplifying any DNA region through repeated cycles of duplication facilitated by DNA polymerase. Why use PCR? This automated process bypasses the need to use bacteria for amplifying DNA
Involves the use of a DNA polymerase able to withstand the high temperatures of >90°C required for the separation of the two DNA strands in the DNA double helix after each replication cycle
Studies of isolated pieces of DNA are nearly impossible without PCR amplification. Steps Step 1 Known as the "Initiation Step"
Contains heating the reaction to a temperature of 94-96°C which is held for 1-9 minutes
***The reaction is heated to 98 if extremely thermostable polymerases are used***
Only required for DNA polymerases that require heat activation by hot-start Animation http://www.dnalc.org/resources/animations/pcr.html Consists of a series of 20-40 repeated temperature changes, called cycles, with each cycle commonly consisting of 2-3 discrete temperature steps, usually three
Cycling is preceded by a single temperature step (called hold) at a high temperature (>90°C), and followed by one hold at the end for final product extension or brief storage
Temperatures used and the length of time they are applied in each cycle depend on a variety of parameters
The enzyme used for DNA synthesis
The melting temperature (Tm) of the primers Step 2 Also known as the annealing step
Reaction temperature is lowered to 50-65 °C for 20-40 seconds
Allows for the annealing of the primers to the single-stranded DNA template
Usually the annealing temperature is about 3-5 degrees Celsius below the melting temperatures of the primers used
Stable DNA-DNA hydrogen bonds are formed when the primer sequence matches the template sequence very closely
The polymerase then binds to the primer-template hybrid and begins DNA formation Step 3 Also known as the extension/elongation step
The temperature depends on the DNA polymerase used
DNA Polymerase synthesizes a new DNA strand complementary to the DNA template strand
Time span depends on both the DNA polymerase used and on the length of the DNA fragment to be amplified
At its' optimum temperature, the DNA polymerase will polymerize at a thousand bases per minute
Under optimum conditions (no limitations due to limiting substrates), the DNA target is doubled
Leads to exponential (geometric) amplification of the specific DNA fragment Step 4 Also known as final elongation
Time Span: 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended. Stages of PCR 1. Exponential Amplification
At every cycle, the amount of product is doubled (assuming 100% reaction efficiency)
2. Leveling off stage
Reaction slows as the DNA polymerase loses activity and its consumption of reagents and primers causes them to become limiting
3. Plateau stage
No more product accumulates due to exhaustion of reagents and enzyme Applications of PCR Selective DNA Isolation Allows for the isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA
This use of PCR augments many methods:
Generating hybridization probes for southern or northern hybridization
PCR supplies these techniques with great amounts of pure DNA
Enables analysis of DNA samples even from very small amounts of starting material
Example: Bacterial colonies (E.coli) can be rapidly screened by PCR for correct DNA vector constricts Example: Can also be used for fingerprinting
A forensic technique used to identify a person or organism
Some PCR fingerprint methods have high discriminative power and can be used to identify genetic relationships between individuals, such as parent-child or between siblings
Used in paternity testing
Can also be used to determine evolutionary relationships among organisms Since it amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of a sample
Critical for forensic analysis when only a trace amount of DNA is available for evidence
Also can be used in the analysis of ancient DNA that is tens of thousands of years old
These PCR techniques have been successfully used on animals, such as a forty-thousand-year-old mammoth, and also on human DNA
Example: analysis of Egyptian mummies and the identification of a Russian tsar
This type of PCR allows for the estimation of the amount of a given sequence present in a sample
Applied to quantitatively determining levels of gene expression Amplification and quantification of DNA PCR in diagnosis or diseases PCR permits early diagnosis of malignant diseases such as leukemia and lymphoma which is currently the highest developed in cancer research and is already being used routinely
Permits the identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses from tissue culture assays and animal models
Viral DNA can be detected by PCR
The primers used must be specific to the targeted sequences in the DNA of a virus
The PCR can be used for diagnostic analysis or DNA sequencing of the viral genome
The high sensitivity of PCR permits virus detection soon after infection
This early detection gives physicians significant lead time in treatment
The amount of virus ("viral load") in a patient can also be quantified by PCR-based DNA quantification techniques References "Biology Animation Library." DNALC Blogs. N.p., n.d. Web. 06 Feb. 2013.
"Polymerase Chain Reaction." Wikipedia. Wikimedia Foundation, n.d. Web. 06 Feb. 2013. <http://en.wikipedia.org/wiki/Polymerase_chain_reaction>.
"PCR." NCBI. U.S. National Library of Medicine, n.d. Web. 06 Feb. 2013. <http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechPCR.shtml>.