heat-tolerant bacterium thesis

heat-tolerant bacterium thesis

Week 4

Methods and the Human Genome Project

What is PCR?

    • Polymerase chain reaction (PCR) is a common lab technique used to make millions or billions of copies of a particular region of DNA
    • This region can be anything the experimenter wants to know more about – it could be a gene with a function a researcher wants to understand
    • It is an in vitro practice

 

 

 

  • PCR requires a DNA polymerase enzyme that makes new strands of DNA, using the existing strands as templates – just like in DNA replication in an organism. In PCR, this DNA polymerase is usually Taq polymerase

 

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  • Taq is named after the heat-tolerant bacterium it was isolated from (thermus aquaticus)
  • Interestingly, Taq was first discovered at Yellowstone National Park
  • Thermus aquaticus is a thermophile bacterium. is stable at elevated temperatures.
  • DNA using Taq is achieved at the optimal temperature of 70°C and is used during PCR to denature the template DNA, or separate its strands
    • Taq polymerase can only make DNA if it’s given a primer – a short sequence of nucleotides that provides a starting point for DNA synthesis
    • PCR amplification is achieved by using oligonucleotide primers for flanking regions with a known base sequence.

 

 

 

 

 

 

  • PCR primers are short pieces of single-stranded DNA, usually around 20 nucleotides in length
  • Two primers are used per each PCR reaction. They flank the target region to be copied – aka, they are given sequences that cause them to bind to opposite strands of the template DNA.

 

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Image source: Khan Academy

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    • Taq polymerase can only make DNA if it’s given a primer – a short sequence of nucleotides that provides a starting point for DNA synthesis
    • PCR amplification is achieved by using oligonucleotide primers for flanking regions with a known base sequence.

 

 

 

 

 

 

  • PCR primers are short pieces of single-stranded DNA, usually around 20 nucleotides in length
  • Two primers are used per each PCR reaction. They flank the target region to be copied – aka, they are given sequences that cause them to bind to opposite strands of the template DNA.

 

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  • Major components
  • Taq polymerase
  • Primers
  • Template DNA
  • Nucleotides
  • Assemble these components in a tube and put through repeated cycles of heating/cooling to allow for DNA synthesis to occur
  • Basic steps:

Denaturation: Heating the reaction will separate (denature) the DNA strands to provide the single-stranded template for step 2.

Annealing: Cooling the reaction so primers can bind to their complementary sequences on the single-stranded template DNA.