MBIO 3410 Lecture Notes - Lecture 3: Thermus Aquaticus, Taq Polymerase, Low Fidelity
24 Dec 2015
School
Department
Course
Professor
Document Summary
Taq dna polymerase i from thermus aquaticus: thermophilic, single subunit, low fidelity (no proof-reading, adds 3"a overhang. Requirements: sequence information, dna template (single-stranded, primers with 3"oh group (aka dna primers, dna polymerase, dntps, buffer, mg2, pcr machine , denaturation of dna at 93 degrees, anneal dna primer to dna at 55 degrees. Depends on proportion of at to gc. Two primers: forward (for 5" end) and reverse (complement; for 3" end) Forward primer easier to make; goes as written: elongation. Taq dna polymerase works best at 72 degrees: repeat many times, new product becomes template for the next reaction (cid:1) (cid:1) (cid:1) amplification. Use of restriction enzymes to determine differences between samples of homologous dna. Resulting dna fragments are run on agarose gel. Pcr requires prior knowledge of the dna sequence to be amplified. The site of mutation must be known before pcr analysis can be attempted (cid:1) to make a diagnosis (cid:1)
