Chapter Outline

• 3.1 Structure of DNA and RNA

• 3.2 Genomics: The Study of Genomes
• 3.3 Methods in Nucleic Acid Biochemistry
 3.1 Structure of DNA and RNA
• DNA and RNA are formed from nucleotides that are linked together
through a phosphodiester backbone in a linear direction.
Nitrogenous
Bases Found
in
Nucleotides
Nomenclature
• Based on the bases and sugars involved, the naming conventions are
different.

Copyright © 2017 W. W. Norton & Company

Structure of DNA

• Primary structure
• Found in all biological molecules
• Unique arrangement of deoxyribonucleotides or ribonucleotides arranged in
a single chain
• Usually depicted as single letters in a row
• Secondary structure
• Two complementary strands of DNA bind (anneal) together through
complementary base pairing in an antiparallel fashion
• Resembles a double helix

Copyright © 2017 W. W. Norton & Company

Secondary Structure of DNA

Copyright © 2017 W. W. Norton & Company

DNA Must Be Condensed to Fit within the Cell

Copyright © 2017 W. W. Norton & Company

 Complementary Base Pairing
• Based on Chargaff’s rule
• States that A-T means A binds to T and G-C means that G binds to C
• These are called Watson-Crick base pairs.

Copyright © 2017 W. W. Norton & Company

Base Stacking
• Bases are oriented so that hydrogen
bonding with another base requires
that they are arranged in a planar
fashion, parallel to the adjacent
bases on the same strand, and
located in the interior of the helix.
• The base pairs are stacked upon
each other within van der Waals
distance.
• This provides stability to the
molecule through the hydrophobic
effect and van der Waals
interactions.

Copyright © 2017 W. W. Norton & Company

Crystal
Structure
of DNA

Copyright © 2017 W. W. Norton & Company

A-, B-, and Z-DNA

• A – DNA
• Short and wide
• Right handed
• Dehydrated (cannot bind to water easily)

• B – DNA
• Most stable
• Right handed

• Z – DNA
• Most narrow
• Left handed

Copyright © 2017 W. W. Norton & Company

 DNA Denaturation and Renaturation
• Denaturation
• “Melting”
• Occurs under heating or addition of acid
or base
• Separation into two individual strands
• Causes a “hyperchromic shift”
• Renaturation
• “Annealing”
• Two DNA strands reform a helix.

Copyright © 2017 W. W. Norton & Company

Hyperchromic
Shift
• Difference in
absorbance
between double
stranded and
single stranded
DNA after
denaturation

Copyright © 2017 W. W. Norton & Company

DNA Supercoiling
• Supercoil
• Structure where the majority of the DNA molecules inside a cell fold upon
themselves
• The area where the double helix crosses itself
• Found in prokaryotes and eukaryotes

Copyright © 2017 W. W. Norton & Company

Supercoils

Copyright © 2017 W. W. Norton & Company

DNA Topology, Part 1

• Linking number (Lk)

• Number of times a strand of DNA winds in the right hand direction around
the helix in an imaginary plane
• Lk = Tw + Wr

• Twist number (Tw)

• Measures the winding DNA strands around each other

• Writhe (Wr)
• Measures crossing of the DNA strands

Copyright © 2017 W. W. Norton & Company

DNA
Topology,
Part 2

Copyright © 2017 W. W. Norton & Company

Nucleosome
Assembly
• Nucleosome: Circular DNA
is wrapped around histone
proteins.
• One turn must be
removed in order for this
wrapping to occur, which
produces a negative
supercoil, and which is
balanced by adding a
positive supercoil.

Copyright © 2017 W. W. Norton & Company

DNA-Protein Interaction through Histones

Copyright © 2017 W. W. Norton & Company

Topoisomerases

• Enzymes that relieve positive supercoils through cleavage and

reannealing of DNA

• Type I (Topoisomerase I)
• Cleave one strand of DNA
• Reduce supercoiled region by one turn

• Type II (Topoisomerase II)

• Cleave two strands of DNA
• Reduce supercoiled region by two turns

Copyright © 2017 W. W. Norton & Company

Topoisomera
se Activity

Copyright © 2017 W. W. Norton & Company

Topoiso
merase II

Copyright © 2017 W. W. Norton & Company

DNA vs. RNA
• DNA • RNA
• Deoxyribose sugar • Ribose sugar
• Thymine base • Uracil base
• Complex intrastrand structures
• Can form ribozymes (catalytic
molecules)

Copyright © 2017 W. W. Norton & Company

Autocleavage Can Occur in RNA

Copyright © 2017 W. W. Norton & Company

Spontaneous
Deamination of
Cytosine

Copyright © 2017 W. W. Norton & Company

 Ribozymes
• RNA molecules with catalytic activity
• Example: Ribonuclease P (RNAse P)
• Cleaves nucleic acids

Copyright © 2017 W. W. Norton & Company

Three Dimensional Structure of tRNA

Copyright © 2017 W. W. Norton & Company

Modified
Nucleotide
Bases in
RNA

Copyright © 2017 W. W. Norton & Company

Inosine Interactions

Copyright © 2017 W. W. Norton & Company

Unconventional Base Pairing in RNA and
DNA– 1
• Triplet interactions can occur between a single stranded region of
DNA, or RNA with an RNA, DNA, or RNA-DNA duplex.
• This can result in a triple helix (i.e., triplex).
• Quadraplexes can occur among guanine bases found in particular G-
rich DNA sequences.

Copyright © 2017 W. W. Norton & Company

Unconventional Base Pairing in RNA and
DNA– 2

Copyright © 2017 W. W. Norton & Company

 3.2 Genomics: The Study of Genomes
• The genome is the collection of genes.
• The number of genes increases the higher the organism is.

Copyright © 2017 W. W. Norton & Company

Condensation
of Eukaryotic
DNA

Copyright © 2017 W. W. Norton & Company

Further Condensation of DNA

Copyright © 2017 W. W. Norton & Company

Chromosome and Telomere Formation

Copyright © 2017 W. W. Norton & Company

Organization of a Gene

Copyright © 2017 W. W. Norton & Company

Prokaryotic Gene Structures

Copyright © 2017 W. W. Norton & Company

Eukaryotic
Gene
Structures

Copyright © 2017 W. W. Norton & Company

Exon Shuffling

Copyright © 2017 W. W. Norton & Company

Computational Methods in Genomics

Copyright © 2017 W. W. Norton & Company

Bioinformatics
• The field used in
biochemistry to
discover the function
of an unknown gene,
such as a human
disease gene, that can
be targeted with
pharmaceutical drugs
• NCBI BLAST searches are
used to analyze
information.

Copyright © 2017 W. W. Norton & Company

 Bioinformatics Breakthrough
• Identification of the ICMT mutation observed in Hutchinson-Gilford
progeria
Polymorphisms – 1
Mutations that occur within the genome
• Single nucleotide polymorphisms (SNP)
• One mutation
Polymorphisms – 2

• Short tandem repeats (STR)

• Deletions and insertions in the genome
• Variable number tandem repeats (VNTR)
• Repeats of a core sequence
• Can be used in forensic applications
Polymorphisms Used in DNA Analysis: Forensic Applications
Precision Medicine, Part 1

• A method used to “tailor” treatment of a disease to fit the genetic

material of individuals
• Can be used to increase effectiveness of treatment and minimize side
effects
Precision
Medicine,
Part 2
3.3 Methods in Nucleic Acid Biochemistry

• Chemical and physical properties of cell membranes

• Organization of prokaryotic and eukaryotic cell membranes
Plasmids

• Self-replicating pieces of circular DNA

• Can carry extra genetic information not contained in chromosomal
DNA
• Are found in both prokaryotic and eukaryotic organisms
• Contain an original of replication
• Can be cloned, conjugated, transformed, or transduced
Transfer of Genetic Material Using Plasmids
Plasmids Used as Cloning Vectors
Restriction Endonucleases

• Enzymes that cleave

• Type I
• Require ATP
• Type II
• Cleave DNA at specific recognition sequences
• Type III
• Require ATP
Type II
Restriction
Endonucleases
Recombinant
Technology
cDNA Libraries
• Complementary
DNA (cDNA):
Generated from
mRNA by reverse
transcriptase
• RNA fragments
serve as primers.
• DNA
polymerases and
DNA ligase aid in
the generation of
cDNA.
High-Throughput DNA Sequencing

• 1977 – Sanger sequencing developed

• Uses ddNTPs (dideoxynucleoside triphosphates)
• Generates chain termination DNA molecules
• Uses fluorescently labeled ddNTPs
• Followed by capillary gel electrophoresis
• 1980 – Nobel Prize awarded for DNA sequencing

Copyright © 2017 W. W. Norton & Company

Sanger
Dideoxy
Sequencing

Copyright © 2017 W. W. Norton & Company

Polymerase Chain Reaction (PCR)

• A method used to exponentially amplify a specific targeted DNA

segment
• Number of DNA molecules increases by 2n in each cycle.
• Three temperature phases:
• Phase I – DNA denaturation
• Phase II – Annealing
• Phase III – Primer extension and DNA synthesis

Copyright © 2017 W. W. Norton & Company

PCR Schematic

Copyright © 2017 W. W. Norton & Company

Polymeras
e Chain
Reaction

Copyright © 2017 W. W. Norton & Company

Transcriptome Analysis

• Gene expression microarrays

• Provide a readout of transcript abundance
using a predetermined cDNA attached to a
solid surface
• Next generation sequencing using RNA sequencing
• Provides readout of all transcripts from same
gene
• Permits identification of alternatively spliced
RNA products

Copyright © 2017 W. W. Norton & Company

GeneChi
p
technol
ogy
 Gene Expression Assays
Click to view the Gene Expression Assays Animation
https://digital.wwnorton.com/biochem
Next Generation Sequencing
Applications for Next Generation Sequencing