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DNA Structure and Replication
Chapter Nine
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2005We know:• DNA is the hereditary material• DNA has a double helix structure
– Made of four bases; A,T,C,G– Sugar-Phosphate backbone
• DNA replication is semi-conservative• How to do PCRHow did we gain all this knowledge?
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Timeline of Experiments1871 – Miescher – identifies Nucleic Acid1928 – Griffith – “transformation” of bacteria1944 – Avery, MacLeod and McCarty –
DNase expt1950 – Chargaff – G&C, A&T1952 – Hershey and Chase – Blender expt1953 – Franklin – picture of DNA1953 – Watson and Crick – Double Helix
structure and base pairing
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Friedrich Miescher1871• Isolates a type of acid• From white blood cells of bandages• Coins the term Nucleic Acid – because it
was found in the Nucleus of the cell
• Most people thought proteins were hereditary material, so no one cared.
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Proteins?• Scientists had noted that chromosomes
moved and divided as cells divided• Chromosomes had something to do with
heredity• Chromosomes are made of both Nucleic
Acids and Proteins• Choice of either 4 bases or 20 amino acids• Code of all life? – 20 amino acids
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Frederick Griffith1928• Transforming factor in bacteria
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Avery, MacLeod, McCarty1944• DNase vs. Protease
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Hershey and Chase1952• Blender Experiment
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By 1950’s• DNA was the hereditary molecule
• Protein was not
• DNA had transforming ability
• Scientists began examining DNA’s structure
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Erwin Chargaff1950• Found that strands were always same
distance apart• Also, that the amount of A always the
same as amount of T• Amount of G always same as amount of CTherefore: Purines must always bind
PyrimidinesA binds to T; G binds to C
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Rosalind Franklin1953• Crystallized DNA and X-ray diffraction
• From picture it was clear that DNA was in a helix
• With symmetrically organized bases in center
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Watson and CrickApril 25th, 1953• Seen Franklin’s picture:
– Assumed Sugar-Phosphate backbone• Knew Chargaff’s rule:
– A&T, G&C must bind each other• Determined turning radius of beta-Helix• Realized it must be two complementary
strands because of base pairing• Determined it was a double Helix
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Watson and Crick
James WatsonAmerican
Francis CrickEnglish
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Summary of DNA• String of Nucleotides• deoxyribose Sugar-Phosphate backbone• 4 Bases:
– A, G are Pyrimidines– T, C are Purines– A = T– G = C
• Two complementary strands (double helix)
=
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What is DNA?• Deoxyribonucleic Acid:
– String of nucleotides• Nucleotides made up of three parts:
deoxyribose(a sugar)
OH
OH
HO-CH2
+ P –
–=
–
O
O
O
O -
-
-
phosphate
+
cyclic amine(base)
N
N
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NucleotideOH
O-CH2P –
–
=
–O
O
O
-
-
N
N
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DNA
O-CH2P –
–=
–
O
O
O
-
N
N
O
O-CH2P ––
=
–
O
O
O
-
N
N
O-CH2P –
–
=–
O
O
O
-
-
N
N
Sugar-Phosphate Backbone
(negatively charged)
Specific Bases
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The Five Bases• A = Adenine• T = Thymine• G = Guanine• C = Cytosine
• RNA only:– U = Uracil (replaces T)
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Structures of Bases
N
N
Pyrimidines
N
N
N
N
Purines N
N
N
N
CH3O O
O O O
O
NH2
NH2
N
N
N
N
NH2
G
T U C
A
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DNA
O-CH2P –
–=
–
O
O
O
-
O
O-CH2P ––
=
–
O
O
O
-
O-CH2P –
–
=–
O
O
O
-
- T
A
C
Sequence of DNAis order of the basesattached to backbone
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Double Helix• Sugar-Phosphate backbone is on outside• Bases are inside - Hydrogen-bonding to
opposing base on opposite strand• Forming Base Pairs
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Base Pairing1. A Purine must always be base paired
to a Pyrimidine
2. A = T – with two Hydrogen Bonds
3. G = C – with three Hydrogen BondsTherefore:Strands must be complementary
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Complementary StrandsDouble Helix has two strands:• Complementary – means when you read
the message on one strand, you automatically know the message on other strand
• Not identical, because in reverse• “Antiparallel” strands• Exact same message on both strands
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Antiparallel Strands• DNA strands match
up in opposite directions
• DNA always “read”5’ to 3’ direction
• In the end, both strands have the exact same message
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DNA ReplicationMakes logical sense how DNA replication
happens:1. DNA has two strands with identical
information2. Must open up3. Exposing unpaired bases4. Bases are matched perfectly
(compliment)5. Forming two double helixes from one
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Three Theories:1. Semi-conservative
– The one Watson and Crick just suggested– Strands split – New strand forms from reading other
2. Conservative– Makes copy of double strand, from double
strand (without splitting)3. Dispersive
– Double helix breaking and reforming at random
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Meselson and Stahl1957• Density Shift Experiments• Labeled DNA with “heavy” Nitrogen• Follow label:Does it split equally = Semi-conservativeDoes it stay together = ConservativeDoes it split randomly = Dispersive
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Meselson and Stahl• Insert figure 9.13
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Parental DNA strands
Daughter DNA strands
Each of the parental strands serves as atemplate for a daughter strand
Semi-conservative
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DNA Replication• DNA from cell to cell, has to be replicated
with fidelity• Mistakes – mutations, cancer, etc.• Only one mistake in a billion nucleotides is
made by the replication machinery• DNA replication is a result of the
coordination of > 10 protein complexes and enzymes
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Enzymes• Helicases – unwind the DNA• Primase – attracts complimentary bases to
form a “primer” sequence• DNA Polymerase – add bases to the
primer strand by reading the code– Therefore, extends the new strand– According to the original strand’s sequence
• Ligase – seals the sugar-phosphate backbone back together
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Synthesis• Two strands• Opposite directions• One strand can be “read” directly because
it is 5’ to 3’– Leading Strand
• Other strand is 3’ to 5’– Lagging Strand– Discontinuous synthesis of this strand
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DNA Replication
Notes:
1.Enzymes and their functions
2.Direction of replication- Always 5’ to 3’
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DNA Replication
Notes con’t:
3. Difference between leadingand lagging strands
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Error CorrectionDNA Polymerase – can also “proofread” the
newly formed strand:1. Excise bases that are “mismatched”
2. Replace with the correct base
3. Then move forward to next base
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Summary of DNA Replication• DNA is a double helix• Bases in middle make up sequence• Semi-conservative replication
1. Strands separate2. Each one is copied3. One “lagging” and one “leading”4. Forming two double helixes from one5. Proteins do all the copying6. Incredibly accurate
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DNA Highly Condensed• DNA is a very long molecule• To fit into the nucleus it must be
condensed• Double helix coils around Histones• Forms “beads on a string”• Beads = Nucleosomes• Nucleosomes coil into Chromatin• Chromatin is condensed into Chromosomes
Chromosomes are BOTH – DNA and Protein!
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DNA Highly Condensed
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PCR1977• PCR – Polymerase Chain Reaction• Kary Mullis• Idea just came to him while he was
thinking about DNA Replication (on LSD)• Won Noble Prize• Brought three women to the ceremony:His wife, his mistress and the hooker from
the night before
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PCR1. Separate the Double Helix2. Bind primers (2) to sequence you want to
replicate3. DNA Polymerase copies between two
primers4. Rinse and Repeat5. Copies DNA between two primers
exponentially
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PCR1. Separate the Double Helix
2. Bind primers (2) to sequence you want to replicate
Heat
Add
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PCR3. DNA Polymerase copies between two
primers
4. Repeat Many Times
AddDNA Pol.
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PCR Needs• You must know the sequence you are
trying to amplify• Primers:
– One for each side, exact sequence correct• Excess of four dNTPs:
– 4 nucleotides• Heat insensitive DNA Polymerase:
– So that reagents can be heated and cooled repeatedly
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PCR Uses• Forensics
– ID a body– ID a criminal– Free the innocent
• Genetic Tests– Testing for specific polymorphism/mutation
• Paternity Tests• Research
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Next Class:• Read Chapter Ten
• Homework – Chapter Nine Problems;– Review: 2, 4, 5, 6, 7, 8, 10– Applied: 2, 4, 6, 8
• Pedigrees Due October 11th