DNA Sequencing PCR REPLICATION AND ITS APPLICATIONS

DNA REPLICATION IS SEMICONSERVATIVE  Confirmed by Messelson- Stahl:  hill.com/sites/ /student_view0/chapter14/ animations.html# hill.com/sites/ /student_view0/chapter14/ animations.html#

 Begins at specific sites called origins of replication  DNA helicase unwinds double helix by breaking H-bonds – forms replication forks  Single-stranded binding proteins hold strands open STEPS IN DNA REPLICATION

 DNA polymerases add nucleotides to 3’-end of growing DNA strand  Synthesis is always in 5’  3’ direction  Requires a RNA primer to build off of  DNA primase synthesizes a RNA primer

 Strands run in opposite directions  Therefore, only 1 strand can replicate toward the replication fork: leading strand  Strand replicating away from the fork is called the lagging strand  Can only synthesize short pieces at a time  Okazaki fragments DNA REPLICATION OCCURS ON BOTH STRANDS AT THE SAME TIME

 Synthesis on leading strand is continuous  Synthesis on lagging strand requires multiple primers  When RNA primer of previous Okazaki fragment is reached, DNA polymerase breaks it down  DNA ligase seals Okazaki fragments together   hill.com/sites/ /student_view0/cha pter14/animations.html# hill.com/sites/ /student_view0/cha pter14/animations.html#

 DNA polymerase proofreads new nucleotides against template  If mistake made, DNA polymerase repairs mistake  If mistake not corrected, mutation has occurred PROOFREADING

PRO VS. EUKARYOTES  Prokaryotes have 1 origin of replication  Eukaryotic chromosomes have multiple origins of replication  Replication bubbles eventually meet and merge  Speeds up process

 End of each chromosome is left short, unreplicated strands of DNA  These ends are repeating, non-coding sections called telomeres  Help to regulate the # of times of cell can divide  Cancer cells possess telomerase, which adds repeating sequences to ends of chromosomes TELOMERES

USING REPLICATION IN BIOTECHNOLOGY

 Amplifies small amounts of DNA  Without cloning  Uses nucleotides, primers to replicate DNA sequence   hill.com/sites/ /student_view0/chapter 16/animations.html# hill.com/sites/ /student_view0/chapter 16/animations.html# POLYMERASE CHAIN REACTION

 Determining the order of bases that make up the genome  Requires dideoxynucleotides (ddnt)  Stop addition of new nucleotides DNA SEQUENCING