Polymerase Chain Reaction (PCR) & DNA SEQUENCING

Polymerase Chain Reaction PCR is DNA replication of a specific region of the genome, outside of the nucleus, in a laboratory setting, without the need of a host organism Allows for the production of many copies of a desired gene fragment Process is closely related to DNA replication

PCR Steps: 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

Extension(Elongation). Temperature is raised slightly to 72°C 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 it does not denature at high temps. Can quickly generate billions of copies of DNA sequences

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

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

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

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