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PCR

PCR. Polymerase Chain Reaction By: Sarah D ^2. What is PCR?. PCR stands for ‘polymerase chain reaction’. PCR is the amplification of DNA sequence by repeated cycles of strand separation and replication. It is intended to replicate many copies of one small section of DNA very quickly.

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PCR

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  1. PCR Polymerase Chain Reaction By: Sarah D ^2

  2. What is PCR? • PCR stands for ‘polymerase chain reaction’. • PCR is the amplification of DNA sequence by repeated cycles of strand separation and replication. • It is intended to replicate many copies of one small section of DNA very quickly.

  3. How does PCR work? • PCR is a simple process which replicates DNA by following a cycle of increasing and reducing heat.

  4. Watch Animation WOW CHECK OUT THIS ANIMATION THAT MAKES THE WORLD OF PCR MAKE SENSE!!!!

  5. Step 1: Denature • Increase heat to 92 o C • This denatures the DNA, and allows the hydrogen bonds to break, splitting the DNA into its 2 strands

  6. Step 2: Anneal • Heat is decreased to about 60oC • This allows the DNA to ‘anneal’ to DNA primers. • ANNEAL: matching up of the nitrogenous bases

  7. Step 3: Synthesize • Temperature is raised again, this time to 70 oC • Enzyme ‘Taq Polymerase’ acts upon DNA strands, adding nucleotides and replicating the DNA segment (similar to the process performed by DNA Polymerase III).

  8. Step 4…ish • This process is repeated many times over. • Some pieces of DNA extend past the targeted area and some do not. These create ‘variable strand lengths’ • The more cycles that occur the more targeted DNA only strands are created, and the fewer you have of the wasteful DNA strands

  9. What the strands might look like: Phase 1 5’ 3’ CONSTANT LENGTH STRANDS 3’ 5’ Phase 2 5’ 3’ 3’ 5’ Phase 3 3’ 5’ 5’ 3’ VARIABLE LENGTH STRANDS 5’ 3’ 3’ 5’

  10. Differences Between PCR and DNA Replication DNA Replication PCR • Replicates an entire strand of DNA • Use RNA primers • More accurate for checking for mistakes • Occurs in the body all the time • Targeted at replicating a segment of DNA • Use DNA primers • Quicker to amplify DNA amounts (growth is exponential) • Requires heating and cooling not available from the human body

  11. Game • Purpose: Read text to distinguish between DNA replication and PCR, and to distinguish between DNA and DNA fragments. • Two teams: Team DNA Rep. and Team PCR • One person starts at each and must read a sentence, as soon as one of them is done reading, the next person in line, joins their team and reads a sentence as well. • Compare who finishes first. What was unfair about this game?

  12. Why did we do this? • DNA Rep. read your text. It is the longest. This represents DNA having to replicate an entire strand. • PCR first person, read your text. Slightly shorter than DNA Rep. This is the first replication in PCR; stretching from a fragment tip to the end of the DNA strand. • Another PCR, read your text. It is short, representing only a DNA fragment. There are many more of these as the game goes on, making PCR go much faster than DNA.

  13. Uses of PCR • Genetic Testing (i.e. Forensics) • Diagnosis of diseases (i.e. Leukemia, AIDS, etc. as well as bacterial, and virus driven diseases) • Determining ancestry (both of animals and humans)

  14. Benefits & Drawbacks of PCR • Can take small amounts of DNA and replicate them to a size quantifiable in other tests (good for forensics). • It is very accurate, especially for determining various diseases, leading to better diagnoses and earlier treatment. • The technique is very sensitive, and poor attention to detail or sloppy technique can lead to contamination by DNA from a source other than the patient's sample. • There may be difficulties replicating the targeted area if it is too large or too small.

  15. Textbook Terms: • DNA primers: short sequences of DNA nucleotides that are complimentary to the opposing 3’ to 5’ ends of the DNA target sequence that is to be replicated • TaqPolymerase: DNA polymerase extracted from Thermusaquaticus (bacteria found in hot springs) that is able to withstand high temperatures • Variable-length strands: mixture of strands of DNA that have been replicated and are of unequal length • Constant-length strands: mixture of strands if DNA that have been replicated and are of equal length

  16. References • Giuseppe, M. (2003). Nelson Biology 12 . Toronto: Nelson Thomson Learning. • http://www.nhlcyberfamily.org/tests/pcr.htm • http://www.dnalc.org/resources/animations/pcr.html • http://www.infolizer.com/5emb2r1a3c5e3ci81all5en7g5ea1n5et/Comparison-between-pcr-and-human-dna-replication.html • http://www.sumanasinc.com/webcontent/animations/content/pcr.html

  17. Some Good places to look… • For the animation shown here you can go back to the slide for it and click the link. • There is a diagram in your textbook on page 297, if you would like to look at that. It’s better than most of the internet ones. • Consult the textbook at any point for definitions and additional information if you would like.

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