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Nucleic Acids
Biochem 4511
Figures: Essentials of Biochemistry, 3rd Ed
...
, Nelson & Cox
Principles of Biochemistry 5th Ed
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Fundamentals of Biochemistry, Voet, Voet & Pratt

Nucleotides
• DNA and RNA are polymers of nucleotides
• Nucleotides are comprised of three main components
1) Base - Purine or Pyrimidine
2) Sugar molecule - Deoxyribose or Ribose
3) Phosphate

Nucleotides, Nucleosides, and Bases
• Base: Aromatic heterocyclic molecules that provide the
name and identity for different nucleotides
• Nucleosides: Bases plus a 5-membered sugar ring derived
from ribose
...

Adenine, A
Guanine, G
• Draw ring system
• Draw ring system
• Two more steps
• Three more steps
– Add amino group (C6)
– Add amino group (C2)
– Fill in bonds
– Add oxo group (C6)
– Fill in bonds

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Sons, Inc
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Learn how to draw pyrimidines
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reserved
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D
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• He identified nucleotides Adenine, Guanine,
Cytosine, Thymidine as the components of DNA
• Components were linked together in the order
phosphate-sugar-base to form units –
nucleotides
• DNA molecule consist of a string of nucleotides
linked by the phosphate groups, which are the
'backbone' of the molecule
...


Friedrich Miescher

Phoebius Levene

History: DNA carries genetic information
Griffith (1928) & Avery, MacLeod, McCarty (1944)

Pneumococcus

Transfer of (genetic) information from virulent to nonvirulent strains

History: DNA carries
genetic information
1952: Hershey-Chase Experiment
• Two batches of phage were
prepared containing 32P labeled
DNA or 35S labeled protein
•

32P

was found in infected cells
producing progeny phage and
32S was not

• DNA is the hereditary information

Chargaff rules
•In 1950s Erwin Chargaff discovered that DNA molecules
always contain A and T and C and G in equal amounts
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Erwin Chargaff

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Chargaff Base Content Rules
1) Base composition of DNA varies from one species to another
2) DNA specimens from different tissues of one species have the
same base composition
3) Base composition of DNA in a given species does not change
with age, nutritional state, or environment
4) In all cellular DNA, the percentage composition %A = %T and
%G = %C
5) The total number of purine bases equals the total number of
pyrimidine bases:
(%A + %G) = (%T + %C)

Watson-Crick’s DNA double helix
Nature, 1953 (find on Carmen)

1962 Nobel Prize in Physiology or Medicine

Watson-Crick’s DNA double helix

Francis Crick and James Watson

The theoretical structure prediction based on:
1) Known chemical composition of DNA
2) Chargaff’s rules
3) Maurice Wilkins & Rosalind Franklin X-ray data,
suggesting the helical nature of DNA
GENETIC ENHERITANCE

DNA Double Helix
• Two strands, wrapping around a
common axis with right-handed
twist, to form a double helix
• The two strands run in opposite
directions (antiparallel), but both
are right-handed
• Bases occupy the core of the helix
• Sugar-phosphate chains are on
the outside
• Each base has a “Watson-Crick”
pairing to generate a planar
hydrogen bonded pair
• RNA typically does not form
double strand helices

DNA Double Helix
Space-filling
representation

Ball-and-stick
model

What drives DNA to form
a double helix?

DNA Double Helix
DNA double helix is stabilized
by several forces:
• Base pairs form the core of
the double helix and
associate through H-bonding
• Phosphate backbone
forms the periphery
(significant charge repulsion)
• Double helix formation is
driven by base stacking
(hydrophobic effect)

Watson-Crick Base Pairing
A - T base pairs
form 2 H-bonds

G - C base pairs
form 3 H-bonds

Base pair width is similar
within a given conformation
of DNA

Drawing Nucleic Acids

The Hydrophobic Effect
• The Hydrophobic Effect is the phenomenon by which
nonpolar molecules aggregate to avoid contact with
hydrophilic molecules, particularly water
• Aggregating nonpolar molecules releases caged water
which is VERY entropically favorable

∆G = ∆H – T∆S
∆G <0 i
...
∆HUnfavorable

Preferred

Many caged water molecules
are ordered around the
nonpolar molecules

Fewer water molecules
are ordered around the
nonpolar molecules

DNA is Stabilized by Multiple Forces
• Hydrophobic Effect: the predominant force:
hydrophobicity, base stacking and entropy
• Hydrogen bonding (in base pairs)
• Ionic interactions
– Cations (e
...
Mg2+, Na+, K+)
– Polyamines

Denaturation
• Double-stranded nucleic acids are denatured (unfold
and dissociate) at high temperatures
• At lower temperatures, complementary polynucleotides
anneal to native state
• Annealing is determined
by base complementarity
• Tm = melting temperature
• G≡C base pair has three
H-bonds => stronger
• A=T base pair has two
H-bonds => weaker

Nucleic Acids Renature
• Annealing is determined
by base complementarity
• Tm = melting temperature
• G≡C base pair has three
H-bonds
• A=T base pair has two
H-bonds
• The ability to re-anneal is VERY important in nature and
in biochemical research

From Genes to Proteins
The biological information encoded by a sequence of DNA is:
• Transcribed to RNA
• Translated into the amino acid sequence of a protein
• This is commonly referred to as the Central Dogma of Biology

•
•
•
•
•

Genes are sequences of DNA
Replication: copying DNA
Transcription: converting DNA into RNA
Reverse transcription: converting RNA into DNA (viruses)
Translation: making proteins from an RNA template

All these reactions are catalysed by Enzymes –
proteins and protein-RNA complexes
• Replication: copying DNA by DNA polymerases
• Transcription: converting DNA into RNA by RNA
polymerases
• Reverse transcription: converting RNA into DNA (by
viruses e
...
HIV) by reverse transcriptases
• Translation: making proteins from an RNA template by
a ribosome (a Protein-RNA complex)

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DNA Replication
In vivo
• DNA must be copied in order to sustain life
• Excessive DNA replication can be indicative of cancer
In vitro
• Technology known as the “polymerase chain reaction” or
PCR has revolutionized researchers capability to study
nucleic acids, genes, and proteins
• Catalyzed by DNA polymerases
DNA replication is semi-conservative and requires high
fidelity (needs to be error-free to prevent mutations)

DNA replication
Meselson & Stahl,
1958:
• DNA replication is semiconservative
• Analysis by 15N labeling
and ultracentrifugation
• Density gradient
equilibrium
sedimentation carried
out in cesium chloride

Transcription

• RNA polymerase (not shown) unwinds and separates dsDNA
at the position where transcription occurs
• The RNA product is a “copy” of the coding and complimentary
to the noncoding (template) strand
• Cells cannot survive if transcription is shut down!

Central Dogma of Biology
• http://www
...
com/watch?v=9kOGO
Y7vthk&list=PL771LqkVTavQLbGniWkzQ
hNQ9LKHWY5bX

RNA
There are three primary classes of RNA:
1) Messenger RNA (mRNA): encodes protein sequences
2) Transfer RNA (tRNA): carries amino acids to ribosome
3) Ribosomal RNA (rRNA): aids in protein synthesis

Transcription in
Nucleus
Translation of mRNA
by ribosome aided
by tRNA
in Cytoplasm

tRNA is single-stranded and
forms unique conformations
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©

Genes

• The genomes vary in size and number of genes
• Genes have regulatory elements and can be identified by their
nucleotide sequences
Largest
genomes

Amoeba dubia
670,000,000,000 bp

Marbled lungfish
133,000,000,000 bp

Paris japonica
149,000,000,000 bp

KEY CONCEPTS
• A DNA molecule can be sequenced or amplified
by using DNA polymerase to make a copy of a
template strand
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• Gel-based separation is replaced by capillary
electrophoresis
• Up to 1000 nucleotides can be read in a single run

© 2014 John Wiley & Sons, Inc
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Modern DNA sequencing techniques:
SMRT- single molecule real-time sequencing
• Polymerase is covalently bound
to transparent (glass) surface
• Continues polymerization – NO
ddNTPs!!!
• Nucleotides are fluorescently
labeled at the phosphoryl groups
• Removal of fluorescent signal
upon nucleotides incorporation
results in low background
•

http://www
...
com/products/
smrt-technology/

© 2014 John Wiley & Sons, Inc
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Amplification of specific DNA sequences
The Polymerase Chain Reaction
(PCR) can be used to make billions
of copies of DNA efficiently and
accurately:
• PCR was developed by Kary
Mullis, who won the 1993 Nobel
Prize in Chemistry for his
discovery
• PCR requires:
1) Heat stable polymerase (Taq)
2) DNA template
3) DNA primers
4) Temperature regulation

DNA Amplification: PCR
1) Denaturation
92–95 ºC
• dsDNA denatures (and separates)
at high temp to form ssDNA
2) Annealing
~55 (50-60) ºC
• Primers base pair to ssDNA
• Actual temperature depends on
DNA template and primers
3) Extension
72 ºC
• Optimal temp for heat-stable DNA
polymerases to work (varies based
on which polymerase is used)
• New strand is synthesized
4) Repeat from Step 1 for 30-35
cycles…

DNA Processing
Restriction Enzymes are a class of proteins whose role
is to cleave dsDNA in a sequence specific manner:
• In nature, restriction enzymes defend bacteria from
infection by bacteriophages (viruses that infect
bacteria)
• Bacteria methylate their own DNA
• Bacteriophages have unmethylated DNA
• Bacterial restriction enzymes recognize and excise
unmethylated viral DNA
• DNA generated by PCR is unmethylated which allows
the use of restriction enzymes in processing of in vitro
generated DNA – Molecular Cloning

Restriction Enzymes
Restriction Enzymes:
•

Endonucleases that recognize a
specific nucleotide sequence
(target) in DNA and break the
DNA chain at or near the target

• A variety of these enzymes are
known, and they are extensively
used in genetic engineering
• Recognition sequences are
Overhangs are called “sticky ends”
palindromic in dsDNA
(complementary on both strands)
Example:
• EcoRI recognizes the dsDNA
sequence GAATTC and cleaves
both strands at GAATTC

“Sticky ends” from two different
DNA pieces can be connected by
Ligases (enzymes)

DNA Fragmentation
• DNA fragmented by enzymatic digestion can
be analyzed to give sequence information
• Gel electrophoresis is a method of separating
DNA fragments by size
• Gel is made of agarose
• DNA bands can be stained with chemicals
such as Ethidium bromide for fluorescent
detection
• Ethidium bromide intercalates
in between aromatic bases of
DNA

Plasmid DNA
Plasmid DNA is double-stranded, circular, bacterial DNA
• Covalently closed, circular
• Typical Size: 1–200 kbases
• Can be modified by restriction enzyme digestion and
easily incorporated into cells
• Dependent on host cell’s proteins for replication and
transcription machinery
• Useful for expressing proteins of interest

Site-Directed
Mutagenesis
• A method of introducing a
single (or multiple) amino
acid change in a protein as
a result of a mutation at a
specific site in the DNA
• A very powerful
biochemical technique that
allows the study of mutant
proteins
• Mutant proteins are
responsible for a variety of
disease and cancer

Forensic DNA
Genetic fingerprinting- used in parental testing and
criminal investigation (first reported in1984)
• 99
...

• Identified by restriction analysis or PCR
Variations of
VNTR allele
lengths in 6
individuals
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Title: Biochemistry -- Nucleic Acids
Description: Biochemistry Intro class -- Thorough Notes of Nucleic Acids.

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