The Genetic Code of Genes and Genomes

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• The Genetic Code of Genes and Genomes

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DNA Structure Double Helix

• DNA backbone forms right-handed helix
• Each DNA strand has polarity directionality
• The paired strands are oriented in opposite
  directions antiparallel

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Central Dogma
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Defects in an enzymatic pathway Can produce in
phenotypes
Black urine diease (Alkaptonuria )
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Mutations

• Mutation refers to any heritable change in a gene
• The change may be substitution of one base pair
  in DNA for a different base pair deletion or
  addition of base pairs
• Any mutation that causes the insertion of an
  incorrect amino acid in a protein can impair its
  function

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Normal, wild type
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Chapter 6 DNA Structure, Replication, and
Manipulation
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DNA Molecule of Heredity

• Inherited traits are affected by genes that are
  transmitted from parents to offspring in
  reproduction
• Genes are composed of the chemical
  deoxyribonucleic acid DNA

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DNA Molecule of Heredity

• DNA was discovered by Friedrich Miescher in 1869
• In 1920s microscopic studies with special stains
  showed that DNA is present in chromosomes
• In 1944 Avery, McLeod and McCarty provided the
  first evidence that DNA is the genetic material

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Griffith's experiment 1928 demonstrating
bacterial transformation
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Avery, McLeod and McCarty identified DNA as the
chemical substance responsible for changing
rough, nonvirulent cells of S pneumoniae (R) into
smooth encapsulated infectious cells (S)
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Genome Size

• The genetic complement of a cell or virus
  constitutes its genome
• In eukaryotes, this term is commonly used to
  refer to one complete haploid set of chromosomes,
  such as that found in a sperm or egg
• The C-value the DNA content of the haploid
  genome
• The units of length of nucleic acids in which
  genome sizes are expressed
• kilobase (kb) 103 base pairs
• megabase (Mb) 106 base pairs

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Genome Size

• Viral genomes are typically in the range 1001000
  kb
• Bacteriophage MS2, one of the smallest viruses,
  has only four genes in a single stranded RNA
  molecule of about 4000 nucleotides (4kb)
• Bacterial genomes are larger, typically in the
  range 110 Mb
• The chromosome of Escherichia coli is a circular
  DNA molecule of 4600 kb.
• Eukaryotic genomes are typically in the range
  1001000 Mb
• The genome of a fruit fly, Drosophila
  melanogaster is 180 Mb
• Among eukaryotes, genome size often differs
  tremendously, even among closely related species

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The C-value Paradox

• Genome size among species of protozoa differ by
  5800-fold, among arthropods by 250-fold, fish
  350-fold, algae 5000-fold, and angiosperms
  1000-fold.
• The C-value paradox Among eukaryotes, there is
  no consistent relationship between the C-value
  and the metabolic, developmental, or behavioral
  complexity of the organism
• The reason for the discrepancy is that in higher
  organisms, much of the DNA has functions other
  than coding for the amino acid sequence of
  proteins

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DNA Chemical Composition

• DNA is a linear polymer of four
  deoxyribonucleotides
• Nucleotides composed of 2'- deoxyribose (a
  five-carbon sugar), phosphoric acid, and the four
  nitrogen-containing bases denoted A, T, G and C

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DNA Chemical Composition

• Two of the bases, A and G, have a double-ring
  structure these are called purines
• The other two bases, T and C, have a single-ring
  structure these are called pyrimidines

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DNA Structure

• The duplex molecule of DNA consists of two
  polynucleotide chains twisted around one another
  to form a right-handed helix in which the bases
  form hydrogen bonds
• Adenine pairs with thymine guanine with
  cytosine
• A hydrogen bond is a weak bond
• The stacking of the base pairs on top of one
  another also contribute to holding the strands
  together
• The paired bases are planar, parallel to one
  another, and perpendicular to the long axis of
  the double helix.

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DNA Structure

• The backbone of each polynucleotide strand
  consists of deoxyribose sugars alternating with
  phosphate groups that link 5 ' carbon of one
  sugar to the 3' carbon of the next sugar in line
• The two polynucleotide strands of the double
  helix run in opposite directions
• The paired strands are said to be antiparallel

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DNA Replication

• Watson-Crick model of DNA replication
• Hydrogen bonds between DNA bases break to allow
  strand separation
• Each DNA strand is a template for the synthesis
  of a new strand
• Template (parental) strand determines the
  sequence of bases in the new strand (daughter)
  complementary base pairing rules

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M. Meselson and F. Stahl
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Circular DNA Replication

• Autoradiogram of the intact replicating circular
  chromosome of E. coli shows that
• DNA synthesis is bidirectional
• Replication starts from a single site called
  origin of replication (OR)
• The region in which parental strands are
  separating and new strands are being synthesized
  is called a replication fork

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Replication of Linear DNA

• The linear DNA duplex in a eukaryotic chromosome
  also replicates bidirectionally
• Replication is initiated at many sites along the
  DNA
• Multiple initiation is a means of reducing the
  total replication time

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DNA Synthesis

• One strand of the newly made DNA is synthesized
  continuously leading strand
• The other, lagging strand is made in small
  precursor fragments Okazaki fragments
• The size of Okazaki fragments is 10002000 base
  pairs in prokaryotic cells and 100200 base pairs
  in eukaryotic cells.

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How fast is the fork going?
E. coli can replicate in about 20 minutes under
optimal conditions. E. coli genome contains
4.5106 basepairs
4.5106 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.
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The error rate of replication is 1 error/ 1010
bases
How much is 1010?
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.
Typical single space typewritten page 3000
characters/sheet
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How many mistakes are made each time the cell
replicates?
E. coli genome 4.5106 basepairs
(1 cell/1 genome)(1 genome/4.5106 basepairs)
(11010 basepairs/1 error)
2222 cells/1 error
H. sapiens genome 3.1109 basepairs
(1 cell /2 genomes)( 1 genome/ 3.1109
basepairs) (11010 basepairs/1 error)
1.6 cells/ 1 error
What would happen in E. coli if mismatch repair
did not occur?
What would happen in humans if mismatch repair
did not occur?
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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

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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

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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

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Southern Blot Analysis
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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

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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

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• Polymerase
• chain
• reaction
• (PCR)
• Needs only the smallest amount of DNA
• 2) Short DNA primers (that you can synthesize)

Heat
Allows you to amplify (generate a ton of) any
gene or sequence that you need
Cool
Polymerize
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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

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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

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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

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