DNA SequencingPCR

REPLICATION AND ITS APPLICATIONS

DNA REPLICATION IS SEMICONSERVATIVE

Confirmed by Messelson-Stahl:

http://highered.mcgraw-hill.com/sites/0072437316/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 strandSynthesis is always in 5’3’ directionRequires a RNA primer to build off ofDNA 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 strandCan 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

http://207.207.4.198/pub/flash/24/menu.swf

http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/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 replicationReplication 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 telomeresHelp 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 DNAWithout cloning

Uses nucleotides, primers to replicate DNA sequence

http://learn.genetics.utah.edu/content/labs/pcr/

http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/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