BME100 s2015:Group8 12pmL4
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LAB 4 WRITE-UP
have the same forward primer and reverse primer.
samples will be cross-contaminated.
Research and Development
PCR - The Underlying Technology
PCR consists of five steps and a holding phase, three of the steps are cycled 35 times. A PCR solution will require four main
ingredients. The first ingredient that it will require is a template DNA source. The source nor the organism that it came from
matters but purer DNA yields higher results. Primers which are short single-strand segments of DNA are necessary to bind to the
complementary segment of the single-stranded DNA and for Taq polymerase to extend upon, thus replicating and amplifying the sought
after DNA segment. The final component deoxyribonucleotides or dNTP's are needed for the polymerase to base pair the template strand
with.Taq Polymerase which has been derived from a thermophile is used instead of regular DNA polymerase because it works optimally
at 72 degrees Celsius which is the optimal temperature for the extension step. Most PCR solutions will also contain a reaction
buffer in order to maintain optimal pH which will also maximize yields.
During the first initial step, the PCR mix will be held at 95 degrees Celsius for 3 minutes. The high heat breaks the hydrogen
bond that exist between the two stands of DNA leaving them as two single strands of DNA. This step is only repeated once. The next
three steps are each 30 seconds and are cycled 35 times, starting with the denature step at 95 degrees Celsius which seems
repetitive the first time but will serve to separate the primers from the single strand DNA so that the the next two steps may once
again occur. In the anneal step the mix is held at 57 degrees Celsius so that the primers may bond to their complementary DNA
segment. Two primers are made so that first, oriented in the forward direction and the second, in the reverse direction will contain
the segment meant for amplification in between. The final cycled step is the extension step which is at 72 degrees Celsius, in this
step Taq polymerase binds to the two primers and starts extending them. This extension is done by Taq polymerase grabbing a free
floating dNTP that complements the base immediately following the primer and bonding it with the template strand. This is repeated
until it meets the Taq polymerase coming in the opposite direction. The final extension step which is once again 72 degrees Celsius
but for three minutes lets any partially replicated strands finish. It is held at 4 degrees Celsius until retrieval time.
Taq polymerase follows very strict restrictions for base pairing. In other words it doesn't match randomn nucleotides. Purines
are always matched with a pyrimidine. In specific the purine adenine bonds with the pyrimidine thymine and the purine guanine base
pairs with the pyrimidine cytosine.