Class notes
Biol 2200 PCR Cycle Notes
- Course
- BIOL 2200
- Institution
- University Of Virginia
Comprehensive and detailed PCR Cycle Notes for Biol 2200. *Essential!! *At a price that's fair enough!!
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Exam 5 learning objectivesPCR
• Explain what’s occurring in each step of a PCR cycle, noting the purpose and fate of
the primers and why some DNA products are longer than intended (or desired)
- PCR is based on three simple steps required for any DNA synthesis reaction: (1)
denaturation of the template into single strands; (2) annealing of primers to each
original strand for new strand synthesis; and (3) extension of the new DNA
strands from the primers. These reactions may be carried out with any DNA
polymerase and result in the synthesis of defined portions of the original DNA
sequence. However, in order to achieve more than one round of synthesis, the
templates must again be denatured, which requires temperatures well above
those that inactivate most enzymes. Therefore, initial attempts at cyclic DNA
synthesis were carried out by adding fresh polymerase after each denaturation
step (1,2). The cost of such a protocol becomes rapidly prohibitive.
• Given a target sequence, recognize primers that could be used for amplification
- PCR primers are synthetic DNA oligonucleotides of approximately 15–30 bases.
PCR primers are designed to bind (via sequence complementarity) to sequences
that flank the region of interest in the template DNA. During PCR, DNA
polymerase extends the primers from their 3′ ends.
• Predict the length of PCR products given primer and target sequence information
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• Compare and contrast DNA replication in your cells with DNA replication in PCR
- The main difference between PCR and DNA replication is that PCR is an in vitro
process which synthesizes DNA, while DNA replication is the in vivo process of
DNA synthesis.
Dideoxy DNA (Sanger) sequencing
• Explain what a dideoxy nucleotide is and effect of its presence during DNA synthesis
- Dideoxynucleotides are used to terminate growing DNA chains and create the
subsets of truncated fragments in a sequencing reaction.
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