Chapter 3 Methods in Molecular Biology and Genetic Engineering

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Chapter 3


Methods in Molecular Biology
and Genetic Engineering

1953. Double helix structure, 1962. Noble Prize

for their discoveries concerning the
molecular structure of nucleic acids
and its significance for information
transfer in living material"
.

3.1 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).


Type II:
recognition site and cleavage site are the same

Type I:
cleavage site can be up to 1000 bp away from recognition site

Type III:
have closer cleavage sites, usually 20 to 30 bp

3.1 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.


3.2 Nucleases


nucleases

hydrolyze
an ester bond within a
phosphodiester bond.


phosphatases

hydrolyze the ester
bond in a
phosphomonoester
bond.


FIGURE 01: The target of a phosphatase (a) and a
nuclease (b). An endonuclease (c) and an exonuclease (d)

3.2 Nucleases


endonuclease


Nucleases that cleave
phosphoester bonds within a nucleic acid
chain.


They may be specific for RNA or for single
-
stranded or double
-
stranded DNA.


exonuclease


Nucleases that cleave
phosphoester bonds one at a time from
the end of a polynucleotide chain.


They may be specific for either the 5′ or 3′ end
of DNA or RNA.



3.2 Nucleases


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

FIGURE 02: Restriction endonuclease

1.
Recognize a specific
sequence


2.
Cut, or restrict, that
sequence

sticky end

blunt end

http://bioinfo.cs.technion.ac.il/projects/Engel
-
Freund/anzime.html

The Nobel Prize in Medicine 1978

Discovery of restriction enzymes and their application to problems of molecular
genetics

www.nobelprize.org

Biozentrum der
Universität, Basel,
Switzerland

Johns Hopkins
University School of
Medicine, Baltimore,
MD, USA

Johns Hopkins
University School of
Medicine, Baltimore,
MD, USA

3.2 Nucleases


A map can be generated by using the overlaps between
the fragments generated by different restriction
enzymes.


FIGURE 03: A restriction map is a linear sequence of sites separated by
defined distances on DNA

3.3 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.


ligating (or ligation)


The process of joining together
two DNA fragments.

3.3 Cloning



subclone


The process of breaking a cloned fragment
into smaller fragments for further cloning.


MCS (multiple cloning site)


A sequence of DNA
containing a series of tandem restriction endonuclease
sites used in cloning vectors for creating recombinant
molecules.

3.3 Cloning

FIGURE 04: Plasmid transformation

Three site in plasmid:

ori

amp
r

lacZ

with MCS

Action of DNA ligase

O

P

O

O

O

CH
2

Base

DNA ligase

Restriction Enzyme

O

Base

O

OH

P

O

O

O

CH
2

Base

O

Base

O

O

_

_

_

5


3


Enhance of the cloning efficiency:


Dephosphorylation of the 5

-
phophate in the vector by alkaline phosphatase

to prevent self
-
ligation

From Dr. Yu

3.3 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
insert
.


FIGURE 05: E. coli colonies on agar plates with
ampicillin, IPTG, and the color indicator X
-
gal

1.
LacZ

gene encodes β
-
galactoside (β
-
gal)

2.
β
-
gal
can cleave
X
-
gal
into blue compound

3.
IPTG

as β
-
gal inducer



3.4 Cloning Vectors Can Be Specialized for
Different Purposes

FIGURE 06: Several types of cloning vectors are available

3.4 Cloning Vectors Can Be Specialized for
Different Purposes


Shuttle vectors
can be propagated in more than one
type of host cell.



Expression vectors
contain promoters that allow
transcription of any cloned gene.

Shuttle Vector

FIGURE 07: A
vector can be used
in both yeast and
bacteria

(shuttle vector)

Expression vector

RBS:
Ribosome Binding Site

FIGURE 08: Luciferase graph/Firefly

Photo ©

Cathy Keifer/Dreamstime.com

Expression Vector

3.4 Cloning Vectors Can Be Specialized for
Different Purposes


Reporter genes
can be used to measure promoter
activity or tissue
-
specific expression.

Photo courtesy of Robb Krumlauf, Stowers Institute for Medical Research

FIGURE 09: A mouse promoter controls
tissue
-
specific expression of lacZ

FIGURE 10: Fluorescent proteins are powerful
research tools

Courtesy of Joachim Goedhart, Molecular
Cytology, SILS, University of Amsterdam.

Fluorescent proteins are powerful research tools

Reprinted from Vision Res., vol. 45, T. G. Wensel, et al., Rhodopsin
-
EGFP
knock
-
ins..., pp. 3445
-
3453. Copyright 2005, with permission from Elsevier
[http://www.sciencedirect.com/science/journal/00426989]. Photo courtesy
of Theodore G. Wensel, Baylor Col

GFP(Green), YFP(Yellow),
CFP(Cyan), BFP(Blue)

The Fly brain

GAL4/UAS

Binary

System

Targeted gene expression in fly

Brand & Perrimon
1993

Shih & Wu unpublished

GFP expression in subset of fly mushroom body

Wu et al., 2011

GFP express in single neuron in fly brain

The Nobel Prize in Chemistry 2008

For the discovery and development of the green fluorescent protein, GFP

www.nobelprize.org

Woods Hole & Boston
University Medical
School

Columbia University

Howard Hughes
Medical Institute

3.4 Cloning Vectors Can Be
Specialized for Different
Purposes


Numerous methods exist to
introduce DNA into different target
cells.

FIGURE 11: DNA can be introduced into
cells in several ways

3.5 Nucleic Acid Detection


DNA, RNA nucleic acids absorb light at 260 nm


Protein absorb light at 280 nm


260/280 ratios to quantify the amount of nucleic acid



DNA, RNA can be nonspecifically stained with
ethidium bromide(EtBr) or SYBR

3.5 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.

3.5 Nucleic Acid Detection


autoradiography


A method of capturing an image of
radioactive materials on film.

FIGURE 12: An
autoradiogram of a gel
prepared from the
colonies described in
Figure 3.5

3.5 Nucleic Acid Detection


in situ

hybridization


Hybridization of a probe to intact
tissue to locate its complementary strand by
autoradiography.

FIGURE 13: The
fluorescent in situ
hybridization (FISH)
technique

Adapted from an illustration by Darryl Leja,
National Human Genome Research
Institute (www.genome.gov).

In situ

Hybridization: Locating genes in chromosomes

FISH:

F
luorescence

i
n
s
itu

h
ybridization

From Dr. Yu

3.6 DNA Separation Techniques


Gel electrophoresis separates DNA fragments by size,
using an electric current to cause the DNA to migrate
toward a positive charge.

Adapted from an illustration by Michael Blaber, Florida State University.

FIGURE 14:
DNA sizes can
be determined
by gel
electrophoresis

FIGURE 15: Agarose gel electrophoresis pattern of SV40 DNA

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

Type I topoisomerase:

Relaxes negative supercoiled DNA to
relaxed or linear DNA

3.6 DNA Separation
Techniques


DNA can also be
isolated using density
gradient centrifugation.

DNA density dependents on
G
-
C content


An AT base pair has a lower molecular weight
than a GC base pair

3.7 DNA Sequencing


Chain termination sequencing uses
dideoxynucleotides

(ddNTPs) to terminate DNA synthesis at particular
nucleotides.



Primer
-

A single stranded nucleic acid molecule with a
3′

OH used to initiate DNA polymerase replication of a
paired template strand.

3.7 DNA Sequencing


Chain termination sequencing uses ddNTPs to terminate
DNA synthesis at particular nucleotide.



Fluorescently tagged ddNTPs and capillary gel
electrophoresis allow automated, high
-
throughput DNA
sequencing.

Inset photo courtesy of Jan Kieleczawa

FIGURE 17: DideoxyNTP sequencing using fluorescent tags

1.
Different fluorescents label for each ddNTP

2.
Hit with a laser and pass by an optical
sensor

3.
Glass capillary tube dissipate heat rapidly

T
hermus
aq
uaticus

Taq

DNA polymerase)

3.8 PCR and RT
-
PCR

3.8 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.

*
Kary

Mullis in the 1980s

*
Taq

polymerase from
Thermus

aquaticus

*Thermal cycler 95
o
C 40
o
C 72
o
C

20x

T
m
:
annealing temperature for primer/template pair

Exponential produce
the primer to primer
-
defined sequence

Press Release
13 October 1993

The Royal Swedish Academy of Sciences

has decided to award the 1993
Nobel Prize in Chemistry for
contributions to the development of methods
within DNA
-
based chemistry
,


with half to

Dr

Kary

B. Mullis
, La Jolla, California, U.S.A.,

for his invention of the polymerase chain

reaction (PCR) method,


and half to

Professor
Michael Smith
, University of British

Columbia, Vancouver, Canada,
for his fundamental

contributions to the establishment of

oligonucleotide
-
based, site
-
directed mutagenesis

and its development for protein studies.


Decisive progress in gene technology through two new methods:
the polymerase chain reaction (PCR) method and site
-
directed
mutagenesis

Kary

B. Mullis

Michael Smith

The Nobel Prize in Chemistry 1993

www.nobelprize.org

3.8 PCR and RT
-
PCR


RT
-
PCR

uses reverse transcriptase to convert RNA to
cDNA for use in a PCR reaction.



Real
-
time
, or
quantitative
,
PCR

detects the products of
PCR amplification during their synthesis, and is more
sensitive and quantitative than conventional PCR.


3.8 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.

FIGURE 20: Fluorescence
Resonant Energy Transfer
(FRET)

Fluorescent Tags in Real
-
Time PCR


This fluorescent
-
tagged
oligonucleotide serves as
a reporter probe


Fluorescent tag at 5

-
end


Fluorescence quenching
tag at 3

-
end


With PCR rounds the 5


tag is separated from the
3


tag


Fluorescence increases
with incorporation into
DNA product


TeqMan probe

wikipedia

1.
Primer binding

2.
Probe hybridization

3.
PCR conditions



SYBR Green I Dye

www.currentprotocols.com

1.
Primer binding

2.
PCR conditions



SYBR Green I Dye binds to
the minor groove of ds DNA

Reporter dye are not sequence
specific, spurious products
produced by the reaction may lead
to false positive signals.