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The polymerase chain reaction, generally known as PCR, is used in almost every application of gene technology. It is a method for rapid production of a very large number of copies of a particular fragment of DNA. Virtually unlimited quantities of a length of DNA can be produced
The polymerase chain reaction (PCR) is an artificial method of replicating DNA under laboratory conditions
- The PCR technique is used to amplify large quantities of a specific sequence of DNA from an initial minute sample
- Each reaction doubles the amount of DNA – a standard PCR sequence of 30 cycles creates over 1 billion copies (230)
The reaction occurs in a thermal cycler and uses variations in temperature to control the replication process via three steps:
- Denaturation – DNA sample is heated (~90ºC) to separate the two strands
- Annealing – Sample is cooled (~55ºC) to allow primers to anneal (primers designate sequence to be copied)
- Elongation – Sample is heated to the optimal temperature for a heat-tolerant polymerase (Taq) to function (~75ºC)
Taq polymerase is an enzyme isolated from the thermophilic bacterium Thermus aquaticus (remember this guy?)
- As this enzyme’s optimal temperature is ~75ºC, it is able to function at the high temperatures used in PCR without denaturing
- Taq polymerase extends the nucleotide chain from the primers – therefore primers are used to select the sequence to be copied
Watch Me - video shown in class
Further detail about the process including buffers and dNTPs
Great animations to show the exact process -
very dramatic music :)
Virtual Simulation - you can learn about every part of the process. You are the scientist
Mr. Dayaram's Students doing an actual Polymerase Chain Reaction Experiment
Mr. Dayaram's Students doing an actual Polymerase Chain Reaction Experiment
What goes into the PCR Reaction?
What goes into the PCR Reaction?
Primers are short pieces of DNA that are made in a laboratory. Since they're custom built, primers can have any sequence of nucleotides you'd like.
In a PCR experiment, two primers are designed to match to the segment of DNA you want to copy. Through complementary base pairing, one primer attaches to the top strand at one end of your segment of interest, and the other primer attaches to the bottom strand at the other end. In most cases, 2 primers that are 20 or so nucleotides long will target just one place in the entire genome.
Primers are also necessary because DNA polymerase can't attach at just any old place and start copying away. It can only add onto an existing piece of DNA.
DNA Polymerase is a naturally occurring complex of proteins whose function is to copy a cell's DNA before it divides in two. When a DNA polymerase molecule bumps into a primer that's base-paired with a longer piece of DNA, it attaches itself near the end of the primer and starts adding nucleotides. (In nature, these primers are made by an enzyme called primase).
The DNA polymerase in our bodies breaks down at temperatures well below 95 °C (203 °F), the temperature necessary to separate two complementary strands of DNA in a test tube. The DNA polymerase that's most often used in PCR comes from a strain of bacteria calledThermus aquaticus that live in the hot springs of Yellowstone National Park. It can survive near boiling temperatures and works quite well at 72 °C (162 °F).
Nucleotides are the building blocks that DNA molecules are made of. You add a mixture of four types of nucleotides to your PCR reactionA's, C's, G's and T's. DNA polymerase grabs nucleotides that are floating in the liquid around it and attaches them to the end of a primer.
The Role of the Thermocycler
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Polymerase Chain Reaction Presentation -->
A LOT OF DETAIL!
(un-necessary for A Level)
Good for the applications of the PCR process and detailed steps
Applications of PCR
Applications of PCR
PCR analysis in Influenza
PCR analysis in Influenza
Influenza is caused by an RNA virus that can rapidly mutate to exist as distinctive serotypes (i.e. different strains exist)
- These strains will have different glycoprotein antigens and hence engender different antibody responses
- Identifying the specific strain of influenza virus in an infected individual will help in the selection of optimal drug treatments
Different strains of influenza can be detected using a modified PCR protocol
- Amplification of the virus requires the conversion of its RNA sequences into cDNA (reverse transcription via RT-PCR)
- Because several different strains of the influenza virus exist, appropriate primers need to be selected to reflect this range
Specific strains of influenza virus can be detected using molecular beacons that target sequences unique to a given strain
- A molecular beacon is a hairpin-shaped nucleotide sequence with an internally quenched fluorophore molecule
- When the beacon anneals to a complementary target sequence, the hairpin unfolds and fluorescence is restored
- Hence, different strains of influenza virus can be detected following RT-PCR amplification according to fluorescence
Test for Pathogens
Test for Pathogens
The polymerase chain reaction (PCR) is a widely used technique in molecular biology, microbiology, forensics, and other disciplines. With PCR, it is possible to amplify a single piece of DNA and generate millions of copies of the original DNA molecule.
By using PCR in diagnostic tests, researchers can determine whether a particular pathogen is present in a sample.
The test is set up so that the reaction mixture contains a pair of oligonucleotide primers that match up with a region of a pathogen's DNA. If the pathogen is present in the sample, a fragment of its DNA will be amplified by the PCR technique, producing a positive result. Primers can be created to identify any pathogen with a known DNA sequence. Because PCR requires very little starting material, a small test sample is all that is required to set up many reactions, each using a primer pair corresponding to a different pathogen.
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