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Polymerase Chain Reaction (PCR) & DNA SEQUENCING

Polymerase Chain Reaction (PCR) & DNA SEQUENCING. Polymerase Chain Reaction. Allows for the production of many copies of a desired gene fragment Process is closely related to DNA replication.

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Polymerase Chain Reaction (PCR) & DNA SEQUENCING

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  1. Polymerase Chain Reaction (PCR)&DNA SEQUENCING

  2. Polymerase Chain Reaction Allows for the production of many copies of a desired gene fragment • Process is closely related to DNA replication

  3. Denaturation. The DNA is subjected to heat (94-96°C) which causes the H bonds between the complimentary bases to separate; the two single strands are used as templates to build complimentary strands • Annealing. Temperature is brought down to 50-65°C. DNA primers (complimentary to the opposing 3’-5’ ends of the DNA target sequence to be replicated) anneal with the template DNA

  4. Extension(Elongation). Temperature is raised slightly to 72°C. Taq polymerase (a DNA polymerase) builds complimentary strands by adding nucleotides in the 5’ to 3’ direction. • Solution is reheated and cycle repeats itself • Taq polymerase is heat resistant so no need to add new enzymes • Can quickly generate billions of copies of DNA sequences

  5. Gene Sequencing • Used to determine the exact sequence of base pairs in a DNA strand • The Human Genome Project uses computer technology to read the human DNA sequence using laboratory based techniques • The Sanger dideoxy method is the most widely used because it is easily automated • Uses a variant of each nucleotide DNA known as a dideoxynucleotide (ddNTP) • ddNTPs resemble regular DNA except lack the 3’ hydroxyl group

  6. How it works: • 4 separate suspensions are prepared • Each contains nucleotides and polymerase used in the standard PCR process and a small quantity of ONE of the four variant ddNTP • As PCR proceeds, there is a high probability that a regular nucleotide will be added to the growing chain • Occasionally, the polymerase will bind a ddNTP and the reaction will terminate • Also called the chain termination technique

  7. Thus, in the suspension containing billions of elongating fragments, there is a series of fragments ending with the ddNTP • The fragments are separated with gel electrophoresis and are read with an automated DNA sequencer which speeds up the reading process • The chain termination reaction can be used to sequence DNA up to about 1000 base pairs in a single reaction

  8. Homework • Read 8.2 • Start reviewing your notes for test next week

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