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UT Knoxville BIOL 140 - 19. PCR and DNA sequescing

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Tell us what you think of the course!10 point Assignment – Due April 28th Interesting Facts about genetic engineering and cloning50 Assignment points19. PCR and gene Sequencing: Key Concepts1. DNA - Amplification (of fossil DNA!)Slide 6The Polymerase Chain ReactionRequirements of PCRSlide 9The Steps of Polymerase Chain ReactionSlide 11Slide 12Selected DNA sequences can be amplified by PCR:PCR in Action: Studying Fossil DNASlide 15Slide 16By comparing the DNA sequences among organisms, scientists can determine:Slide 182. Dideoxy DNA SequencingDideoxy DNA SequencingSlide 21Slide 22Agarose Gel electrophoresisSlide 24Slide 25© 2011 Pearson Education, Inc.Tell us what you think of the course!• Please fill out the course evaluations online!!•Thanks…….© 2011 Pearson Education, Inc.10 point Assignment – Due April 28th Interesting Facts about genetic engineering and cloning•Genetically modified Rice  Golden Rice•abc news clip  genetically modified salmonSept 2010•NY Times article  cloning RedwoodsApril 2011© 2011 Pearson Education, Inc.50 Assignment points•20 points on written assignment on 3 of the 5 concepts from your chapter•20 points from chapter 16 - as we discussed, this will not be included in MB•10 points from genetic engineering and cloning- this will not be counted for MB© 2011 Pearson Education, Inc.19. PCR and gene Sequencing: Key ConceptsBiologists can obtain many identical copies of a gene by (1) inserting it into a bacterial cell that copies the gene each time the cell divides or (2) by conducting a polymerase chain reaction (PCR).The sequence of bases in a gene can be determined by dideoxy sequencing method.© 2011 Pearson Education, Inc.1. DNA - Amplification (of fossil DNA!)Copies of DNA sequences can be made by the polymerase chain reaction (PCR) technique in vitro DNA synthesis reactionPCR is a cyclical process: (DNA AMPLIFICATION)•DNA fragments are denatured by heating.•A primer, plus nucleotides and DNA polymerase are added  primer annealing•New DNA strands are synthesized  elongation© 2011 Pearson Education, Inc.•Eliminates the use of restriction enzymes, vectors, host cells…….© 2011 Pearson Education, Inc.The Polymerase Chain Reaction•The polymerase chain reaction (PCR) is an in vitro DNA synthesis reaction in which a specific DNA sequence is replicated over and over again.•This technique generates many identical copies of a particular DNA sequence.© 2011 Pearson Education, Inc.Requirements of PCR•PCR is possible only when DNA sequence information surrounding the gene of interest is available, because PCR requires primers that match sequences on either side of the gene.•One primer is complementary to a sequence on one strand upstream of the target DNA and the other primer is complementary to a sequence on the other strand downstream of the target.•The primers will bind to single-stranded target DNA.© 2011 Pearson Education, Inc.© 2011 Pearson Education, Inc.The Steps of Polymerase Chain Reaction1. A reaction mix containing dNTPs, a DNA template, copies of the two primers, and Taq polymerase (heat stable -DNA Polymerase)2. Denaturation – heating the mixture to 95°C separates the two strands of the DNA.3. Primer annealing – cooling the mixture allows the primers to bond, or anneal, to complementary sections of single-stranded target DNA.4. Extension – heating the mixture to 72°C causes the Taq polymerase to synthesize the complementary DNA strand from the dNTPs, starting at the primer.5. Steps 2–4 are continually repeated to yield the necessary number of copies.© 2011 Pearson Education, Inc. YellowstoneTemperature = 160°F (70°C)Thermus aquaticus makes PCR practicalThermus aquaticus can live (and replicate DNA) in hot springs!© 2011 Pearson Education, Inc.© 2011 Pearson Education, Inc.Selected DNA sequences can be amplified by PCR:Strand separation (~950C), Hybridization of primers (~520C), and DNA synthesis (~720C).Powerful technique in medical diagnostics, forensics, and molecular evolution.Ancient DNA© 2011 Pearson Education, Inc.PCR in Action: Studying Fossil DNA•Svante Pääbo and colleagues used PCR to compare DNA sequences from a 30,000-year-old Homo neanderthalensis fossil with modern Homo sapiens DNA to analyze how similar the two species are.•These sequences proved to be highly distinct and so support the hypothesis that Neanderthals never interbred with modern humans.•Because the complete genomes of a wide array of organisms have now been sequenced, researchers can find appropriate primer sequences to use in cloning almost any target gene by PCR.© 2011 Pearson Education, Inc.•Bacterial genomes have between 1 and 6 million base pairs (Mb). Most plants and animals have about 100 Mb. Humans have approximately 2900 Mb. •As a result, individual chromosomes may contain millions of base pairs. It is difficult to work with DNA sequences this large. As a result, the DNA is broken into smaller pieces (approximately 500 to 1000 bp each). •These pieces are sequenced and then the sequenced pieces are examined and aligned based on overlapping sequence homology at their ends.© 2011 Pearson Education, Inc.© 2011 Pearson Education, Inc.By comparing the DNA sequences among organisms, scientists can determine:•what parts of the genomes are most similar among organisms and therefore likely evolved earliest,•what key changes exist in the genomes that may account for differences among related species,•what changes within species exist that may account for development of specific types of disease.© 2011 Pearson Education, Inc.•In 1980, Frederick Sanger was awarded the Nobel Prize for inventing the dideoxy method (or Sanger method) of DNA sequencing.© 2011 Pearson Education, Inc.2. Dideoxy DNA Sequencing•Determining a cloned gene’s base sequence is useful for understanding more about the gene’s function.•Fredrick Sanger developed dideoxy sequencing as a method for determining DNA sequence. Sanger had to link three important insights to make his sequencing strategy work.•The method is based on an in vitro DNA synthesis reaction.© 2011 Pearson Education, Inc.Dideoxy DNA Sequencing•Dideoxy sequencing is carried out by adding both dideoxynucleotide triphosphates (ddNTPs) and deoxyribonucleotide triphosphates (dNTPs) to the synthesis reactions. •ddNTPs are identical to dNTPs except that they lack the 3' hydroxyl group.•Because of this


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UT Knoxville BIOL 140 - 19. PCR and DNA sequescing

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