Nucleic Acid Biotechnology

Techniques

Chapter 13, Biochemistry, Campbell, Farrell, McDougal

1
Image Credit: Sangharsh Lohakare
Chapter Outline
Purification and detection of nucleic acids
Cloning
Genetic engineering
DNA libraries
The polymerase chain reaction (PCR)
Sequencing DNA
CRISPR
Genomics and proteomics

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Separation Technique for Nucleic Acids: Gel
Electrophoresis
• Method of separating molecules on the basis of
charge-to-size ratio using a gel as a support + sieving
material
• Based on the motion of charged particles in an
electric field

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Separation Technique for Nucleic Acids: Gel
Electrophoresis
• Sample is applied to a
supporting medium
• Supporting media -
Polymeric gels, such as
agarose +
polyacrylamide
• With the use of
electrodes, an electric
current is passed
through the medium to
achieve the desired
separation

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Figure 13.2 - The Experimental Setup for Gel
Electrophoresis

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Detection Methods for Nucleic Acids
• Fluorescence: Sensitive method for detection and
identification of substances that absorb and re-emit
light
• Ethidium bromide
• Slips between DNA bases
and has different
fluorescence characteristics
as opposed to when it is
free in solution
• Used as a stain for DNA on
gels
• Fluorescent dyes - SyBr Green
and SyBr Gold

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Recombinant DNA
• Can be constructed using sticky ends
• Sticky ends that anneal to one
another are from different sources in
some cases
• Occurs if samples of DNA from 2
different sources are digested with the
same RE + then mixed
• Nicks in the covalent structure can be
sealed → DNA ligases

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Cloning of DNA
• Introduction of a section of DNA into a genome in
which it can be reproduced many times
• Clone: Genetically identical population of organisms,
cells, viruses, DNA molecules

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Plasmids
• Small, circular DNA molecules, contain genes for antibiotic
resistance
• Selection: Process that allows transformed bacteria to be
identified/ isolated
• Each plasmid chosen for cloning has a selectable marker
that indicates that the growing bacterial colonies contain
the plasmid

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Figure 13.10 - Plasmid pBR322

pBR322:
1. Origin of replication (ori)
2. Antibiotic resistance
genes
3. Several unique RE sites
4. Small size (4.3kb)

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Plasmids (continued)
• Multiple cloning site (MCS)
• Region of a bacterial plasmid with many restriction
sites
• e.g. of a polylinker
• Popular cloning vector series is based on pUC
plasmids

• Ribosome Binding Site (RBS)

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Figure 13.12 - Cloning with pUC Plasmids

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Universal Cloning Plasmid
• pUC plasmids contain lacZ gene → basis for
blue/white screening
• Blue/white screening: Method for determining
whether bacterial cells have incorporated a plasmid
that includes a gene of interest
• lacZ gene → -subunit of -galactosidase, which
cleaves disaccharides

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Antibiotic
Selection

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Identification
of Empty
Plasmid Select for TETR

AMPS

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Use of a plasmid vector

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Genetic Engineering
• When an organism is intentionally altered at the
molecular level to exhibit different traits, it has been
genetically engineered
• Focuses on gene therapy
• Gene therapy: Cells of specific tissues in a living
person are altered in a way that alleviates the effects
of a disease

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DNA Recombination
• Has the ability to:
• Change specific genes and
specific DNA sequences within
those genes
• e.g.
• Bacteria Thermus aquaticus
• Plants → to produce greater crop
yields or be given increased
resistance to pests

Image Source: https://upload.wikimedia.org/wikipedia/commons/3/38/Swiss_cheese.jpg

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DNA Recombination
• Has the ability to:
• Change specific genes and
specific DNA sequences within
those genes
• e.g.
• Bacteria Thermus aquaticus
• Plants → to produce greater crop
yields or be given increased
resistance to pests

Image Source: https://upload.wikimedia.org/wikipedia/commons/3/38/Swiss_cheese.jpg

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Bacteria as Protein Factories
• Reproductive power of
bacteria can be used to
express large quantities of
a mammalian protein of
interest
• Most mammalian proteins
are heavily processed
after the initial
transcription + translation

• Most rely on the a version of the lacZ

promoter. Protein expression is
activated by addition of IPTG

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Bacteria as Protein Factories
• Production of human
insulin by E. coli
• Eliminated the problems
related to harvesting
insulin from large # of
laboratory animals and
giving humans a peptide
from another species

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Figure 13.17 - Production of Recombinant
Human Insulin

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Cloning and Expression Vectors
• Cloning vectors pBR322 and pUC are used to insert
foreign DNA + amplify it

© 2018 Cengage Learning. All Rights Reserved. Fig. 8-40 Molecular Biology of the Cell (© Garland Science 2008)
Cloning and Expression Vectors
• Expression vectors: Plasmids that have the
machinery to direct the synthesis of a desired protein
• Must be able to be transcribed by the genetic
machinery of the bacteria where it is transformed
• Should have transcription termination sequence

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Figure 13.18 - pET Expression Vectors

• Common attributes
• Origin of replication
• MCS
• At least one selectable
marker

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DNA Library
• Collection of clones that
include the total genome
of an organism
• All the DNA of an
organism can be taken
and cloned in chunks of
reasonable size

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Polymerase Chain Reaction (PCR)

• Method of amplifying a small amount of DNA based on

the reaction of isolated enzymes
• Any chosen DNA can be
amplified, it does not need
to be separated from the
rest of the DNA in a
sample before the
procedure is applied
• Copies both
complementary strands of
the desired DNA
sequence
Credit: Carlos de Paz

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 95°C
~ 5°C < Tm for the duplex
Cycle 1
72°C
Typical PCR reaction:
- DNA template
- Oligonucleotides
- All 4 dNTPs
- Taq Polymerase

Cycle 2

Taq Polmerase:
thermostable DNA
polymerase named after
Cycle 3 the thermophilic
bacterium Thermus
aquaticus
Quantitative PCR (qPCR)
• Allows:
• Sensitive measurement of DNA samples
• PCR reaction to develop time-point data to help
discern how much DNA was in the cell originally

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Sequencing DNA
• Nature and order of monomer units determine the properties
of the whole molecule
• Sanger–Coulson method for determining base sequences of
nucleic acids depends on selective interruption of
oligonucleotide synthesis
• Single-stranded DNA fragment whose sequence is to be
determined is used as a template for the synthesis of a
complementary strand
• Synthesis is interrupted at every possible site in a
population of molecules depending on the presence of 2′,3′
-dideoxyribonucleoside triphosphates (ddNTPs)

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2′,3′ -dideoxyribonucleoside triphosphates (ddNTPs)

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• each ddNTP
has a different
fluorescent tag

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• each ddNTP
has a different
fluorescent tag Fragments of synthesized
DNA containing specific
labels

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Summary: Sequencing DNA (continued)
• Incorporation of ddNTP into the growing chain causes
termination at the point of incorporation
• Standard Sanger sequencing relies on the use of dideoxy (2’ and 3’ H) nucleotides.
These can be put into a polymerase reaction but act as chain terminators; the next
nucleotide has no 3’OH to be added to so the primer extension reaction stops

• DNA to be sequenced is mixed with a short oligonucleotide =

primer for synthesis
• Gel electrophoresis is performed on each reaction mixture,
and a band corresponding to each position of the chain
termination appears
• Sequence of the newly formed strand, complementary to that
of the template DNA, can then be read directly from the
sequencing gel

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 "Fur-ensic Evidence"
• Murder of Shirley Duguay (1994) by Douglas Beamish
• 1st time animal DNA = evidence during a murder trial

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Next-generation, deep or massively parallel
sequencing?
• Primer extensions are performed as before, but
instead of using ddNTPs, the added dNTPs are
fluorescent with different colours. The sequencing
reaction will glow differently based on the identity of
the nucleotide added during primer extension

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The CRISPR/Cas9 era of genome editing
• Bacteria fight phage infection by taking a sequence from the
phage DNA and placing it into the CRISPR (clustered regularly
interspaced palindromic repeats) locus (region of the
chromosome). The CRISPR DNA is transcribed into RNA and
the Cas9 endonuclease uses this to cleave complementary
(new infecting phage) DNA as a double strand break

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Genomics and Proteomics
• Knowing the full DNA sequence of the human genome allows
to address the causes of a disease
• Assignment of
sequences to the
chromosomes in
which they
belong remains a
challenge

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Genomics

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Genomics and Proteomics (continued)
• Proteomics: Study of interactions among all the
proteins in a cell
• Proteome
• Protein sequence → structure and how they interact
with each other
• Interactions determine how they behave in a living
organism

• Transcriptome: Sum of all genes being transcribed

into RNAs in the cell

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