Medication design

Week 4

Methods and the Human Genome Project

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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.

Extension: Raising the reaction temperature so Taq polymerase can extend the primers, causing the synthesis of new DNA strands.

Repeat this cycle 25-35 times, ~2-4 hours

Can make BILLIONS of copies

Steps of PCR:

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Electrophoresis

  • We can use gel electrophoresis to see the results of PCR
  • DNA fragments of the same length form a “band” on the gel that we can actually visually detect with the naked eye

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The Human Genome Project

  • A 13-year-long project initiated in 1990 to determine the DNA sequence of the entire human genome within 15 years
  • Met with skepticism early on – would the huge cost of the project outweigh any potential benefits?
  • Initial goals/principles:
  • HGP would be an all-inclusive effort to have collaboration from any nation
  • All human genome sequence info would be freely and publicly available within 24 hours of its assembly
  • First sequence yeast and worm genomes as a test run for the human genome

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Impact of HGP

  • HGP has revealed there are ~20,500 human genes
  • There are 3 billion chemical base pairs
  • Can help us with:
  • Genotyping viruses for treatment
  • Identifying mutations linked to cancer
  • Medication design
  • Advances in forensic science
  • Biofuels and energy
  • Agriculture advances
  • The sequence of the DNA is stored in databases available to anyone on the internet

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