ZOL 341 Lecture 4 Outline of Last Lecture I Base stacking II DNA replication Outline of Current Lecture II Proofreading III Telomeres IV PCR V Sanger sequencing Current Lecture DNA proofreading has high fidelity due to instability of mismatched pairs configuration of the DNA polymerase active site proofreading function of DNA polymerase telomeres repetitive sequence at the end of linear chromosomes telomerase lengthens end of telomere prevents shortening of telomeres with multiple rounds of replication like the little piece of plastic on the end of a shoelace only in some cells telomere length is important for chromosome stability cell longevity and reproductive success telomerase is active in germ line cells and some stem cells in eukaryotes other cells have limited life spans 30 50 divisions polymerase chain reaction PCR an automated version of DNA replication that produces millions of copies of a short target DNA segment requires These notes represent a detailed interpretation of the professor s lecture GradeBuddy is best used as a supplement to your own notes not as a substitute a double stranded DNA template containing the target sequence to be amplified a supply of the 4 DNA nucleotides heat stable DNA polymerase ie Taq polymerase 2 different single stranded DNA primers buffer solution steps of PCR 1 denaturation heat to 95 C 2 primer annealing reduce temperature to 45 68 C to allow primers to hybridize to their complementary sequences in the target DNA 3 primer extension raise temperature to 72 C to allow polymerase to synthesize DNA limitations of PCR some knowledge of the target DNA sequence is required in order to determine primer sequences amplification products longer than 10 to 15 kb are difficult to produce sanger sequencing dideoxynucleotide DNA sequencing dideoxy sequencing uses DNA polymerase to replicate new DNA from a single stranded template 4 standard deoxynucleotide bases are present in large amounts each of 4 reactions contains a small amount of one ddNTP
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