Chapter 2

Methods in
Molecular Biology
and Genetic
Engineering
 2.1 Introduction

• restriction endonuclease – An enzyme that

recognizes specific short sequences of DNA and
cleaves the duplex (sometimes at the target site,
sometimes elsewhere, depending on type).
 2.1 Introduction

• cloning vector – DNA (often derived from a

plasmid or a bacteriophage genome) that can be
used to propagate an incorporated DNA
sequence in a host cell.
– Vectors contain selectable markers and
replication origins to allow identification and
maintenance of the vector in the host.
 2.2 Nucleases

• Nucleases hydrolyze an
ester bond within a
phosphodiester bond.
• Phosphatases hydrolyze
the ester bond in a
phosphomonoester bond.

Figure 2.1: The targets of a

phosphatase and a nuclease
 2.2 Nucleases

• endonuclease – Nuclease that cleaves

phosphoester bonds within a nucleic acid chain.
– It may be specific for RNA or for single-stranded
or double-stranded DNA.
• exonuclease – Nuclease that cleaves
phosphoester bonds one at a time from the end
of a polynucleotide chain.
– It may be specific for either the 5′ or 3′ end of
DNA or RNA.
2.2 Nucleases

• Restriction
endonucleases
can be used to
cleave DNA into
defined
fragments.

Figure 2.2: Recognition site

cleavage.
 2.2 Nucleases

• A map can be generated by using the overlaps

between the fragments generated by different
restriction enzymes.

Figure 2.3: A restriction map is a linear sequence of sites separated by defined distances
on DNA.
 2.3 Cloning

• Cloning a fragment of DNA requires a specially

engineered vector.
• recombinant DNA – A DNA molecule that has been
created by joining together two or more molecules
from different sources.
• subclone – The process of breaking a cloned
fragment into smaller fragments for further cloning.
• multiple cloning site (MCS) – A sequence of DNA
containing a series of tandem restriction
endonuclease sites used in cloning vectors for
creating recombinant molecules.
2.3 Cloning

Figure 2.4: (a) A plasmid together

with insert DNA (b) Restricted
insert fragments and vector will
be combined and (c) ligated
together.
 2.3 Cloning

• transformation – The
acquisition of new genetic
material by incorporation of
added exogenous, nonviral
DNA.
• Blue/white selection allows
the identification of bacteria
that contain the vector
plasmid and vector
plasmids that contain an Figure 2.5: The white colonies
insert. will be used to prepare DNA for
further analysis.
 2.4 Cloning Vectors Can Be
Specialized for Different Purposes

Table 2.1: Cloning vectors can be based on plasmids or phages or can mimic eukaryotic
chromosomes.
 2.4 Cloning Vectors Can Be
Specialized for Different Purposes

Figure 2.6: pYac2 is a shuttle vector

 2.4 Cloning Vectors Can Be
Specialized for Different Purposes
• Cloning vectors may be bacterial plasmids, phages,
cosmids, or yeast artificial chromosomes.
• Shuttle vectors can be propagated in more than
one type of host cell.
• Expression vectors contain promoters that allow
transcription of any cloned gene.
 2.4 Cloning Vectors Can Be
Specialized for Different Purposes
• Reporter genes can be used to measure promoter
activity or tissue-specific expression.
Courtesy of Joachim Goedhart, Molecular Cytology, SILS, University
of Amsterdam.
Stowers Institute for Medical Research
Photo courtesy of Robb Krumlauf,

Figure 2.9A: (a) Since the discovery of

GFP, derivatives that fluoresce in
Figure 2.8: Expression of a lacZ gene can different colors have been engineered.
be followed in the mouse by staining for
β-gal (in blue).
 2.4 Cloning Vectors Can
Be Specialized for
Different Purposes

• Numerous methods exist to

introduce DNA into different target
cells.

Figure 2.10: DNA can be released into target

cells by several methods.
 2.5 Nucleic Acid Detection

• Hybridization of a labeled nucleic acid to

complementary sequences can identify specific
nucleic acids.
• probe – A radioactive nucleic acid, DNA or RNA,
used to identify a complementary fragment.
 2.5 Nucleic Acid Detection

• autoradiography –
A method of
capturing an image
of radioactive
materials on film.

Figure 2.11: An autoradiogram of a gel prepared

from the colonies described in Figure 2.5.
 2.5 Nucleic Acid Detection

• in situ hybridization
– Hybridization of a
probe to intact tissue
to locate its
complementary
strand by
autoradiography.

Figure 2.12: Fluorescence in situ hybridization

(FISH).
Data from an illustration by Darryl Leja,
National Human Genome Research Institute
(www.genome.gov).
 2.6 DNA Separation
Techniques
• Gel electrophoresis
separates DNA
fragments by size,
using an electric
current to cause the
DNA to migrate toward
a positive charge.

Figure 2.13: DNA sizes can

be determined by gel
electrophoresis.

Data from an illustration by Michael

Blaber, Florida State University.
Reproduced from W. Keller, Proc. Natl. Acad. Sci.
USA 72 (1975): 2550-2554. Photo courtesy of
Walter Keller, University of Basel.

Figure 2.14: Supercoiled DNA molecules separated by agarose gel electrophoresis.

2.6 DNA Separation
Techniques
• DNA can also be
isolated using
density gradient
centrifugation.

Figure 2.15: Gradient

centrifugation separates
samples based on their density.
 2.7 DNA Sequencing

• Classical chain termination sequencing uses

dideoxynucleotides (ddNTPs) to terminate DNA
synthesis at particular nucleotides.
• Fluorescently tagged ddNTPs and capillary gel
electrophoresis allow automated, high-throughput
DNA sequencing.
• The next generations of sequencing techniques aim
to increase automation and decrease time and cost
of sequencing.
Figure 2.16: DideoxyNTP sequencing using fluorescent tags.
 2.8 PCR and RT-PCR

• Polymerase chain reaction

(PCR) permits the exponential
amplification of a desired
sequence, using primers that
anneal to the sequence of
interest.

Figure 2.17: Denaturation (a) and rapid cooling (b)

of a DNA template molecule in the presence of
excess primer.
 2.8 PCR
and RT-PCR
• RT-PCR uses
reverse
transcriptase to
convert RNA to
DNA for use in a
PCR reaction.

Figure 2.18: Exponential

production of the short,
primer-to-primer–defined
sequence (the “amplicon”).
 2.8 PCR and RT-PCR

• Real-time, or quantitative, PCR detects the

products of PCR amplification during their
synthesis, and is more sensitive and quantitative
than conventional PCR.
• PCR depends on the use of thermostable DNA
polymerases that can withstand multiple cycles of
template denaturation.
 2.8 PCR and RT-PCR

• fluorescence resonant energy transfer (FRET) –

A process whereby the emission from an excited
fluorophore is captured and reemitted at a longer
wavelength by a nearby second fluorophore whose
excitation spectrum matches the emission frequency
of the first fluorophore.
 2.9 Blotting Methods

• Southern blotting involves the transfer of DNA

from a gel to a membrane, followed by detection
of specific sequences by hybridization with a
labeled probe.
Figure 2.20: Southern blot.
 2.9 Blotting Methods

• Northern blotting is similar to Southern blotting, but

involves the transfer of RNA from a gel to a
membrane.
• Western blotting entails separation of proteins on a
sodium dodecyl sulfate (SDS) gel, transfer to a
nitrocellulose membrane, and detection of proteins
of interest using antibodies.
 Figure 2.23: In a Western blot, proteins are separated by size on an SDS gel,
transferred to a nitrocellulose membrane, and detected by using an antibody.
 2.9 Blotting Methods

• epitope tag – A short peptide sequence that

encodes a recognition site (“epitope”) for an
antibody, typically fused to a protein of interest for
detection or purification by the antibody.
 2.10 DNA
Microarrays
• DNA microarrays
comprise known
DNA sequences
spotted or
synthesized on a
small chip.

Figure 2.24: Gene expression

arrays are used to detect the
levels of all the expressed genes
in an experimental sample.
 2.10 DNA Microarrays

• Genome-wide transcription analysis is performed

using labeled cDNA from experimental samples
hybridized to a microarray containing sequences
from all ORFs of the organism being used.
• SNP arrays permit genome-wide genotyping of
single-nucleotide polymorphisms.
• Array comparative genome hybridization (array-
CGH) allows the detection of copy number changes
in any DNA sequence compared between two
samples.
 2.11 Chromatin Immunoprecipitation

• Chromatin immunoprecipitation (ChIP)

allows detection of specific protein–DNA
interactions in vivo.
• “ChIP on chip” or “ChIP-seq” allows mapping
of all the protein-binding sites for a given
protein across the entire genome.
Figure 2.25: Chromatin immunoprecipitation detects protein-DNA
interactions in the native chromatin context in vivo.
 2.12 Gene Knockouts, Transgenics,
and Genome Editing
• transgenics – Organisms created by introducing
DNA prepared in test tubes into the germline.
– The DNA may be inserted into the genome or exist in
an extrachromosomal structure.

Figure 2.26: Transfection

can introduce DNA directly
into the germline of
animals.
Whole animal experimental systems
 2.12 Gene Knockouts, Transgenics,
and Genome Editing
• Embryonic stem (ES) cells that are injected into
a mouse blastocyst generate descendant cells
that become part of a chimeric adult mouse.
– When the ES cells contribute to the germline, the next
generation of mice may be derived from the ES cell.
– Genes can be added to the mouse germline by
transfecting them into ES cells before the cells are
added to the blastocyst.
Figure 2.28: ES cells can be used to generate mouse chimeras.
Whole animal experimental systems
Whole animal experimental systems
Figure 2.0.27: Hypogonadism can be averted in the progeny of hpg mice by introducing a transgene that has
the wild-type sequence.
 2.12 Gene Knockouts, Transgenics,
and Genome Editing
• An endogenous gene can be replaced by a
transfected gene using homologous recombination.
• The occurrence of successful homologous
recombination can be detected by using two
selectable markers, one of which is incorporated
with the integrated gene, the other of which is lost
when recombination occurs.
 2.12 Gene Knockouts, Transgenics,
and Genome Editing
• The Cre/lox system is widely used to make inducible
knockouts and knock-ins.
– knockout – A process in which a gene function is
eliminated, usually by replacing most of the coding
sequence with a selectable marker in vitro and
transferring the altered gene to the genome by
homologous recombination.
– knock-in – A process similar to a knockout, but more
subtle mutations are made.
2.12 Gene Knockouts, Transgenics,
and Genome Editing

Figure 2.30: The Cre

recombinase catalyzes a site-
specific recombination
between two identical lox
sites, releasing the DNA
between them.
Figure 2.0.29: A transgene containing neo within an exon and TK downstream can be selected by resistance to
G418 and loss of TK activity.
 Figure 2.0.31: By placing the Cre recombinase under the control of a regulated promoter, it is possible to
activate the excision system only in specific cells. One mouse is created that has a promoter-cre construct, and
another that has a target sequence flanked by lox sites. The mice are crossed to generate progeny that have
both constructs. Then excision of the target sequence can be triggered by activating the promoter.
Whole animal experimental systems
Figure 2.0.32: An endogenous gene is replaced in the same way as when a knockout is made (see Figure 2.30),
but the neomycin gene is flanked by lox sites. After the gene replacement has been made using the selective
procedure, the neomycin gene can be removed by activating Cre, leaving an active insert.
Table 2.0.2: Basic features of endonuclease-based genome-editing systems.
CRISPR-Cas9