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Lecture 16

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Biology 1202B
Brenda Murphy

1 DNA Technologies: PCR, Sequencing (Sanger & Genome) (Chapter 15) The Polymerase Chain Reaction Amplifies DNA In Vitro  Polymerase Chain Reaction (PCR) o Purpose – To amplify—produce large numbers of copies of—a target DNA sequence in the test tube without cloning o A rapid process to produce an extremely large number of copies of a specific DNA sequence from a DNA mixture without having to clone the sequence in a host organism  By cycling 20-30 times through a series of priming and replication steps, PCR amplifies the target sequence, producing millions of copies o Process is called amplification as it increases the amount of DNA to the point where it can be analyzed or manipulated easily  Usually, no further purification of the amplified sample is needed o PCR is essentially a special case of DNA replication in which a DNA polymerase replicates just a portion of a DNA molecule rather than the whole molecule o PCR takes advantage of characteristics common to all DNA polymerases: these enzymes add nucleotides only to the 3’ end of an existing chain called the primer  For replication to begin, a primer must be available, base-paired to the template chain o Process:  Cycle 1 – Produces 2 molecules  Denaturation – Heat DNA containing target sequences to 95*C to denature it to single strands  Annealing – Cool the mixture to 55-65*C (depending on primers) to allow the two primers to anneal their complimentary sequences at the two ends of the target sequence  Extension – Heat o 72*C, the optimal temperature for DNA polymerase to extend the primers, using the four nucleoside triphosphate precursors to make complimentary copies of the two template strands  Cycle 2 – Produces 4 molecules  Repeat the same steps of denaturation, annealing of primers, and extension in cycle 2, producing a total of 4 molecules  Cycle 3 – Produces 8 molecules  Repeat the same steps, producing a total of 8 molecules  Two of the eight match the exact length of the target DNA sequence o Note:  The primers are made up of DNA rather than RNA like natural DNA replication 2  The left primer binds to one strand while the right primer binds to the opposite strand of the original DNA  Of all the DNA sequences put in the PCR reaction tube, only the target sequence, the sequence between the primers, is amplified  The DNA polymerase is reading the template 3’ to 5’ o The characteristics of PCR allow extremely small DNA samples to be amplified to concentrations high enough for analysis o Can use root of human hair, small amount of blood or semen or saliva, skeletal muscles or ancient remains, fossils etc. Dideoxy (Sanger) Method for Sequencing DNA  Purpose – To obtain the sequence of a piece of DNA, such as in gene sequencing or genome sequencing  Based on the properties of nucleotides known as dideoxyribonucleotides; therefore, the method is also called Dideoxy sequencing o Dideoxyribonucleotides have a single –H bound to the 3’ carbon of the deoxyribose sugar instead of the –OH normally at this position in deoxyribonucleotides o DNA polymerases (replication enzymes) recognize the
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