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Replication

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					Replication
Qu i ckTi me ™ an d a GIF d ec om pres s or are n ee de d to s ee th i s p ic ture .
    CH3              H
              O   H N
                             N
        U N H
        T
                    N    A       N
    N
R                        N           R
          O

              H
              N H   O
                             N
        C N       H N    G       N
    N
                         N           R
R         O       H N
                    H
                       DeoxyAdenine (dA)       H
DeoxyAdenineTriphosphate (dATP)                 N H
                                    N
           O O O
                              N            A    N
         O P O P O P OH
           O O O CH2 O      OH             N
               Ribose
            DeoxyRibose
            DiDeoxyRibose
                        OH OH
 5’P
       3’OH
               5’P 3’OH




3’OH          3’OH 5’P
       5’P
3’   3’   3’
Just adding
polymerase
  clamp
  and
  polymerase




clamp loader,
clamp, and
polymerase
clamp loader,
clamp, and
polymerase




primase,
clamp loader,
clamp, and
polymerase
 helicase




helicase,
clamp loader,
clamp, and
polymerase
How much is 1010?
Typical single space typewritten page 3000 characters/sheet



1 sheet/3000 characters * 1010 characters = 3,333,333 sheets
1 ream/500 sheets * 3,333,333 sheets = 6666 reams
1 box/10 reams * 6666 reams = 666 boxes

666 boxes of single-spaced typed sheets would fill the front of
this room up to the ceiling with only a single spelling error.
 How fast is the fork going?
E. coli can replicate in about 20 minutes under optimal conditions.
E. coli genome contains 4.5*106 basepairs

4.5*106 basepairs/2 replication forks/1200 s =
1875 basepairs/replication fork/s
By comparison….
1 deck of cards/26 pairs
1875 pairs * 1 deck of cards/26 pairs = 72 decks of cards

To move as fast as a replication fork you would have to be able to
sort 72 decks of shuffled cards….
pairing every club with a spade and every heart with a diamond…
each second.
  How many mistakes are made each time the cell replicates?


   E. coli genome 4.5*106 basepairs
   (1 genome/4.5*106 basepairs) * (1*1010 basepairs/1 error)=
   2222 genomes/error
   H. sapiens genome 3.1*109 basepairs
   (1 genome/ 3.1*109 basepairs) * (1*1010 basepairs/1 error)=
    3 genomes/error


What would happen in E. coli if mismatch repair did not occur?

What would happen in humans if mismatch repair did not occur?
Sorting it out
And keeping things
straight
2) Supercoiling helps
to condense and pack
the DNA, keeping it
organized so that it
can fit in the cell.
1) Partitioning
functions help to
keep things
sorted/separated as
replication occurs so
that things aren’t too
badly tangled when
they finish.
Tools for examining / rearranging / changing the DNA
           Nucleic Acid Hybridization
• DNA denaturation: Two DNA strands can be separated by heat
  without breaking phosphodiester bonds

• DNA renaturation = hybridization: Two single strands that are
  complementary or nearly complementary in sequence can come
  together to form a different double helix

• Single strands of DNA can also hybridize complementary
  sequences of RNA




                                                           34
Fig. 6.24
                Restriction Enzymes
• Restriction enzymes cleave duplex DNA at particular
  nucleotide sequences

• The nucleotide sequence recognized for cleavage by a
  restriction enzyme is called the restriction site of the enzyme

• In virtually all cases, the restriction site of a restriction
  enzyme reads the same on both strands A DNA sequence
  with this type of symmetry is called a palindrome




                                                                  36
Fig. 6.26
                 Southern Blot Analysis
• DNA fragments on a gel can often be visualized by staining with ethidium
  bromide, a dye which binds DNA
• Particular DNA fragments can be isolated by cutting out the small region of
  the gel that contains the fragment and removing the DNA from the gel.
• Specific DNA fragments are identified by hybridization with a probe = a
  radioactive fragment of DNA or RNA
• Southern blot analysis is used to detect very small amounts of DNA or to
  identify a particular DNA band by DNA-DNA or DNA-RNA hybridization




                                                                         39
            Southern Blot Analysis




Fig. 6.27
          Polymerase Chain Reaction
• Polymerase Chain Reaction (PCR) makes possible the
  amplification of a particular DNA fragment

• Oligonucleotide primers that are complementary to the ends
  of the target sequence are used in repeated round of
  denaturation, annealing, and DNA replication

• The number of copies of the target sequence doubles in each
  round of replication, eventually overwhelming any other
  sequences that may be present



                                                            41
        Polymerase Chain Reaction


• Special DNA polymerase is used in PCR = Taq
  polymerase isolated from bacterial thermophiles which
  can withstand high temperature used in procedure

• PCR accomplishes the rapid      production of large
  amounts of target DNA which can then be identified and
  analyzed




                                                           42
Polymerase
chain
                                 Allows you to
reaction
                                 amplify
(PCR)                     Heat   (generate a ton
1)   Needs only the              of) any gene or
     smallest amount of
     DNA                         sequence that
2) Short DNA primers             you need
     (that you can
     synthesize)
                          Cool




                  Polymerize
      DNA Sequence Analysis
•   DNA sequence analysis
    determines the order of bases in
    DNA

•   The dideoxy sequencing method
    employs DNA synthesis in the
    presence of small amounts of
    fluorescently labeled nucleotides
    that contain the sugar
    dideoxyribose instead of
    deoxyribose




                                        Fig. 6.29   45
         DNA Sequencing:
         Dideoxy Method
•   Modified sugars cause chain termination because
    it lacks the 3’-OH group, which is essential for
    attachment of the next nucleotide in a growing
    DNA strand

•   The products of DNA synthesis are then
    separated by electrophoresis. In principle, the
    sequence can be read directly from the gel
     DNA Sequencing: Dideoxy Method
•   Each band on the gel is one base longer than the previous band
•   Each didyoxynucleotide is labeled by different fluorescent dye
•   G, black; A, green; T, red; C, purple
•   As each band comes off the bottom of the gel, the fluorescent dye that it
    contains is excited by laser light, and the color of the fluorescence is
    read automatically by a photocell and recorded in a computer




                                                                           47
Fig. 6.31

				
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posted:8/5/2012
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