• Nucleic Acids separation and detection

techniques
Gene Manipulation Techniques
The major goal of purification is to separate

Nucleic acids separation and detection nucleic acids (DNA and RNA) from cell
techniques contaminating material

DNA cloning - cell lysis

DNA library - deproteinization
- precipatation

1 2

Electrophoresis is a separation method based on the

Separation techniques deferential rate of migration of charged species in an
applied DC electric field.
1- Gel electrophoresis
Electrophoresis has been applied to a variety of
- Agarose Gel Electrophoresis difficult analytical separation problem such as amino
- Polyacrylamide Gel Electrophoresis acid, drugs, vitamins, carbohydrates, peptides,
proteins, nucleic acids, etc.
- Capillary electrophoresis
Strength of GE is its unique ability to separate charged
• Gel staining to visualized the DNA fragments molecules in different fields.
Example
only electrophoresis has sufficient resolving power
to separate polynucleotides that differ by only a
single nucleotide.
3 4

1

Application of electrophoresis is separation of
and analysis of purity and size of
macromolecules
5 6
7 8
- Nucleic Acid Sequencing Gel
- This is PAGE and should be large (40 cm)
- Concentration of the gel depends on number of the
nucleotides to be sequenced (20% for the first 50 -
100 nucleotides)
- Includes urea or formamide as a denaturant reagent.
- Agarose Gel Electrophoresis
- Use agarose for separation which is based on the
size and shape of molecules with different
conformations, such as circular, relaxed, linear, and
supercoiled (covalently close circular) forms
- Use for nucleotide separation (RNA and DNA)
- Pulsed Field Gel Electrophoresis (PFGE)
- Use to separate large DNA fragment of molecular
size smaller than 50 kb (20-30 kb).
- The electric field is not constant in standard method
but is changed repeatedly (pulsed) in direction and
strength during separation.
- Use to isolate intact DNA molecules
2
  type of electrophoresis:
1- Vertical (slab or disc) electrophoresis
2- Horizontal electrophoresis

 Sample preparation:
Bromophenol blue
Glycerol or sucrose

 Staining
1- Ethidium bromide, fluorescent SYBR Green I
• Advantages of fluorescent dyes
- The most widely used is SYBR Green I. it is less
9
toxic and 5 times more sensitive than EtBr 10

 Separation media
1- Agarose gel

 Buffer
TBE, TAE for DNA

11 12

3
 Capillary Electrophoresis
(CE)
Introduction
 CE is a technique in which electrophoresis carried
out in very thin capillary tubes (20-100 µm inner
diameter)
 use of high voltage
 reduce separation time to a few minutes
 extremely high resolution and automated in much
the same way as is HPLC (loading and detection)
 CE can analyze only small samples (5-10 nL), it is
limited to use as an analytical tool.
13
- under the influence of the electric field, components
in the sample migrate along the length of the
capillary column
- detection of the components which depends on the
type of the molecules are UV-VIS fix-wave length
detectors
- the capillary column (tube) is flexible and of 50-100
µm i.d. and 25-100 cm length that may or may not
be filled with chromatographic matrix
- the capillary tube may be coated or filled with a
variety of materials. Example
15
Application
Separation of different molecules (small and large
size), and DNA sequencing
Principle
Migration of the compounds through the medium
(capillary column) under high voltage
In Practice
- CE equipment includes a power supply, two buffer
reservoirs, a buffer-filled capillary tube, and an on-
line detector
14
for separation of small, charged molecules, bare silica
or polyimide-coated capillary are used.
for molecular sieving, the tube is filled with
polyacrylamide or SDS-polyacrylamide
for IEF, the capillary tube is filled with electrolyte and
PH gradient
16
4
 2- Separation by Ultracentrifugation
• If a component is denser than its medium, then the
Capillary Electrophoresis centrifugal force causes it to become concentrated
toward the bottom of a centrifugal tube.
• Large particles sediment more rapidly than smaller
particles of similar shape and density.
• Ultracentrifugation proceeds in a near vacuum to
minimize frictional resistance.
1- Velocity sedimentation
The rate at which a given molecule moves in response
to centrifugal force in a centrifuge is known as its
sedimentation velocity

17 18

• As long as one is dealing with the same type of

molecule, the S (Svedberg) value provides a good
measure of relative size.
• In velocity (or rate-zonal) sedimentation, nucleic acid
molecules are separated according to length.
• The sample is carefully layered on gradient
concentration of sucrose (low at the top and high at
the bottom).
• At high centrifugal forces the molecules move at a
rate determined by their sedimentation coefficient
• The density of the medium even at the bottom (1.2
g/ml) is less than that of the molecules (1.7 g/ml),
these molecules continue to sediment as long as the
tube is centrifuged (never reaches equilibrium) 19 20

5
 2- Equilibrium centrifugation The Buoyant Density of DNA
In this technique, equilibrium (or isopycnic) Density gradient ultracentrifugation is
centrifugation, nucleic acid molecules are separated a useful way to separate and purify
on the basis of their buoyant density. nucleic acids.
• The medium is cesium chloride or sulfate (1.75 g/ml)
mixed with the DNA sample and subjecting to high
rpm for 2-3 days.
• During centrifugation a continuous gradient of cesium
is formed.
• DNA molecules are driven downward or move
buoyantly upward in the tube until they reach a
position that has a buoyant density equivalent to their The net movement of solute particles in an ultracentrifuge is
own and form narrow (sharp) bands. the result of two processes: diffusion (from regions of higher
concentration to regions of lower concentration) and
21 22
sedimentation due to centrifugal force.

Separation of plasmid DNA from the main bacterial

chromosome by CsCl equilibrium centrifugation
Techniques of nucleic acid sedimentation

23 24

6
 Detection teqnique
1- Nucleic acid hybridization
This technique is based on that two single-stranded
nucleic acid molecules of complementary base
sequence can form a double-stranded hybrid.
Example
- a mixture of DNA fragments has a specific DNA
coding for Protein x
- the only way to distinguish between the fragment
encoding for protein x and others is molecular
hybridization using complementary probe.
- incubation of denatured DNA fragments with
protein x mRNA will form ds DNA-RNA hybrid.
25
To perform southern blotting (southern
hybridization)
1- Electrophoresis
2- Blotting
3- hybridization
27
• Now how to separate the hybrid molecules?
by passing the mixture through hydroxyapatite under
ionic conditions in which the hybrids would bind to
calcium phosphate salt while non-hybridized
molecules pass through the column.
the hybrids molecules could be released by
increasing the ionic strength of the elution buffer
• How often molecular biologists perform hybridization?
26
Southren
blotting
and
hybridization
28
7
 Southern blotting (southern hybridization) is the
Determining the location of specific DNA technique used to identify a segment of DNA among
fragments using Southern blot a vast population of different DNA fragments.

The methodology is:

1- Separation of DNA fragments by agarose gel electrophoresis
2- Denaturation of duplex DNA fragment and neutralization of the gel
3- Transfer the DNA to nitrocellulose membrane or nylon membrane (DNA-
absorbing membrane)
4- The transferred DNA on the membrane is fixed to the membrane either by
baking at 80˚C or UV irradiation.
5- Hybridization with a labeled probe (radioactive or non-radioactive RNA,
single-stranded DNA, or oligodeoxynucleotide) which is complementary in
sequence to the specific DNA band.
6- Washing to remove unspecific binding of the probe.
7- Detection of labeled band (region of hybridization) either by X-ray for
radioactive probe or immuno detection for non-radioactive probe.
29 30

Essential Questions
DNA cloning • What are the methods that scientists use to
create recombinant DNA molecules?
DNA library
• Can scientists create genes from recombinant
DNA molecules?
• Can scientists modify the heredity of an
organism using recombinant DNA?

31 32

8
 Outline
• What does it mean: “to clone”?
• What is a DNA library?
• Can the cloned genes in libraries be
expressed?
• What is the polymerase chain reaction (PCR)?
• Is it possible to make directed changes in the
heredity of an organism?
33
Plasmids Are Very Useful in Cloning Genes
• Plamids are naturally occurring extrachromosomal
DNA
• Plasmids are circular dsDNA
• Plasmids can be cleaved by restriction enzymes,
leaving sticky ends
• Artificial plasmids can be constructed by linking new
DNA fragments to the sticky ends of plasmid
• These recombinant molecules can be autonomously
replicated, and hence propagated
35
What Does It Mean “To Clone”?
Clone: a collection of molecules or cells, all
identical to an original molecule or cell
• To "clone a gene" is to make many copies of
it - for example, in a population of bacteria
• Gene can be an exact copy of a natural gene
• Gene can be an altered version of a natural
gene
• Recombinant DNA technology makes it
possible
34
Cloning Vectors
Cloning vectors are plasmids that can be
modified to carry new genes
• Plasmids useful as cloning vectors must have
– a replicator (origin of replication)
– a selectable marker (antibiotic resistance
gene)
– a cloning site (site where insertion of foreign
DNA will not disrupt replication or inactivate
essential markers)
36
9
 Virtually Any DNA Sequence Can Be
Plasmids as Cloning Vectors
Cloned
Nuclease cleavage at a restriction site
One of the first widely linearizes the circular plasmid so that a
used cloning vectors foreign DNA fragment can be inserted.
was the plasmid
pBR322. Recombinant plasmids are hybrid
DNA molecules consisting of plasmid
Note the antibiotic DNA sequences plus inserted DNA
resistance genes elements (pink here).
(ampr and tetr).
Such hybrid molecules are called
chimeric plasmids.

An EcoRI restriction fragment of foreign

DNA can be inserted into a plasmid.
37 38

Short DNA Duplexes With Restriction Sites

Chimeric Plasmids Can Be Used as Linkers

Named for mythological beasts with body parts from The use of linkers to
several creatures create tailor-made ends
• After cleavage of a plasmid with a restriction on cloning fragments.
enzyme, a foreign DNA fragment can be inserted
• Ends of the plasmid/fragment are closed to form a
"recombinant plasmid"
• Plasmid can replicate when placed in a suitable
bacterial host
• See previous (slide) Figure

39 40

10
 Directional Cloning Directional Cloning

Often one desires to insert foreign DNA in a DNA molecules

particular orientation whose ends have
different overhangs
• This can be done by making two cleavages with can be used to form
two different restriction enzymes chimeric constructs in
• Construct foreign DNA with same two restriction which the foreign
enzymes DNA can enter the
plasmid in only one
• Foreign DNA can only be inserted in one orientation.
direction
• See next (slide) Figure

41 42

Biologically Functional Chimeric Plasmids Biologically

Functional
• Plasmids can be used to transform recipient E. coli Chimeric
cells Plasmids
• (“Transformation” means the uptake and replication
of exogenous DNA by a recipient cell.)
• To facilitate transformation, the bacterial cells are A typical bacterial
transformation
rendered somewhat permeable to DNA by Ca2+
experiment. Here
treatment (0.1%) and a brief 42°C heat shock pBR322 is the
• The useful upper limit on cloned inserts in plasmids is cloning vector.
about 10 kbp. Many eukaryotic genes exceed this
size.

43 44

11
 Shuttle Vectors Are Plasmids That Can
Propagate in Two Different Organisms What Is a DNA Library?
Shuttle vectors are plasmids capable of
propagating and transferring (“shuttling”) A DNA library is a set of cloned DNA fragments
genes between two different organisms. that together represent the genes of a particular
organism
• Any particular gene may represent a tiny, tiny
fraction of the DNA in a given cell
• Can't isolate it directly
• Trick is to find the fragment or fragments in the
library that contain the desired gene

A typical shuttle vector. LEU2+ is a gene in the yeast 45 46

pathway for leucine biosynthesis.

What is a DNA Library?

• If your library is from the human genome
• The probabilities are daunting ( 3 x 106 kbp ), you would need ( N ) 1,400,000
• Consider the formula for probability of finding a particular clones ( if the cloned fragments averaged 10 kbp in
fragment in N clones size) to reach 99% probability of finding the fragment
P = 1 – (1 – f )N of interest!
N = number of clones
f = fraction of a particular fragment of the genome in N • For this need a cloning vectors capable of carrying
N = ln (1 – P ) / ln (1 – f ) very large DNA inserts.
Example:
if the library consists of 10 kbp fragments of the E.coli
genome (4640 kbp total), more than 2000 clones must be
screened to have 99% probability (P=0.99) of finding a
particular fragment
f = 10 / 4640 = 0.0022 and P = 0.99, N = 2093 clones
47 48

12

Colony Hybridization
A way to screen plasmid-based genome libraries for a
DNA fragment of interest
• Host bacteria containing a plasmid-based library
of DNA fragments are plated on a petri dish and
allowed to grow overnight to form colonies
• Replica of dish made with a nitrocellulose disc.
• Disc is treated with base or heated to convert
dsDNA to ssDNA and incubated with probes
• Colonies that bind probe (with P-32) hold the
fragment of interest
Probes for Southern
Hybridization Can Be
Prepared in a Variety of
Ways
Cloning genes using oligonuceotide
probes from a known amino acid
sequence.
A radioactively labeled set of DNA
(degenerate) oligonucleotides
representing all possible mRNA coding
sequences is synthesized and is used
to probe the genomic library by colony
hybridization.
Labeling methodologies other than
radioactivity are also available.
What is a DNA Library?
Screening a genomic library by
colony hybridization.
Host bacteria transformed with a
plasmid-based genomic library are
plated on a petri plate and incubated
overnight to allow bacterial colonies
to form.
A replica of the colonies is obtained
by overlaying the plate with a flexible
disc composed of absorbent material
(such as nitrocellulose or nylon).
49 50
Identifying Specific DNA Sequences by
Southern Blotting
• Finding one particular DNA segment among a vast
population of different DNA fragments (e.g., in a
genomic DNA preparation) is to exploit its sequence
specificity to identify it.
• Southern blots (invented by E.M. Southern) do this
• DNA fragments (the “library”) are fractionated by size
with agarose gel electrophoresis
• Gel is blotted to an absorbent support and then
incubated with radioactively labeled oligonucleotide
probes
• An autoradiograph shows the hybridized DNA
51 52
fragments
13
 Identifying Specific DNA Sequences by cDNA Libraries Are DNA Libraries
Southern Blotting Prepared from mRNA
• cDNAs are DNAs copied from mRNA templates.
• cDNA libraries are constructed by synthesizing cDNA
The Southern blotting from purified cellular mRNA.
technique involves the • Because most eukaryotic mRNAs carry 3'-poly(A)
transfer of tails, mRNA can be selectively isolated from
electrophoretically preparations of total cellular RNA by oligo(dT)-
separated DNA
fragments to an cellulose chromatography.
absorbent sheet and • DNA copies of the purified mRNAs are synthesized by
subsequent detection of first annealing short oligo(dT) chains to the poly(A)
the specific DNA tails.
sequences.
• These serve as primers for reverse transcriptase-
53 54
driven synthesis of DNA

cDNA Libraries Are DNA Libraries
Prepared from mRNA
Isolation of eukaryotic mRNA via oligo(dT)-
cellulose chromatography.
cDNA Libraries Are DNA Libraries
Prepared from mRNA
• Reverse transcriptase is an enzyme that synthesizes
a DNA strand, copying RNA as the template
• DNA polymerase is then used to copy the DNA
strand and form a double-stranded duplex DNA
• Linkers are then added to the DNA duplexes
rendered from the mRNA templates
• The cDNA is then cloned into a suitable vector
• Once a cDNA derived from a particular gene has
been identified, the cDNA becomes an effective
probe for screening genomic libraries for isolation of
the gene itself
55 56

14
 DNA Microarrays Are Arrays of Different
Oligonucleotides Immobilized on a Chip
Reverse
• Robotic methods can be used to synthesize
transcriptase-
driven synthesis combinatorial libraries of DNA oligonucleotides
of cDNA from directly on a solid support.
oligo(dT) primers • The completed library is a 2-D array of different
annealed to the oligonucleotides
poly(A) tails of
purified • The final products of such procedures are referred to
eukaryotic as “gene chips” because the sequences synthesized
mRNA. upon the chip represent the sequences of chosen
genes
• The oligonucleotides on such gene chips are used
as probes in hybridization experiments to reveal
57 58
gene expression patterns

The Human Genome Project

• A working draft of the

human genome was
DNA Microarrays Are completed in June,
Arrays of Different 2000 and published in
Oligonucleotides February 2001.
Immobilized on a Chip • The genomes of many
other organisms have
now been sequenced
as well.
• Information about whole
genome sequences has
Gene chips (DNA microarrays) in created a new branch of
the analysis of gene expression. science called
bioinformatics.
59 60

15
 Can the Cloned Genes in Libraries Be
Expressed?
Expression vectors are engineered
so that the RNA or protein products
of cloned genes can be expressed.
Can the Cloned Genes in Libraries Be
Expressed?
To express a eukaryotic protein in E. coli, the eukaryotic
cDNA must be cloned in an expression vector that contains
regulatory signals for transcription and translation.

Expression vectors carrying the

promoter recognized by the RNA
polymerase of bacteriophage SP6 are
useful for the production of multiple
RNA copies of any DNA inserted at
the polylinker.
61 A typical expression-cloning vector. 62

Can the Cloned Genes in Libraries Be Can the Cloned Genes in Libraries Be
Expressed? Expressed?
Some expression vectors carry
Strong promoters have cDNA inserts cloned directly into
been constructed to the coding sequence of a
drive synthesis of protein-coding gene.
foreign proteins to
levels of 30% of total E.
coli protein.

A typical expression
A ptac protein
vector for the
expression vector
synthesis of a hybrid
contains the hybrid
protein.
promoter ptac derived
from fusion of the lac
and trp promoters.
63 64

16
 Can the Cloned Genes in Libraries Be
Reporter Gene Constructs
Expressed?
Reporter gene constructs are
chimeric DNA molecules
composed of gene regulatory
sequences (promoter) next to
an easily expressible gene
product.
This is used to assess
the potential function of the
nucleotide sequence
in regulation.

Green fluorescent
protein (GFP) as a
reporter gene.
65 66

What Is the Polymerase Chain Reaction

(PCR)?

What if you don't have enough DNA for colony

hybridization or Southern blots?

• The small sample of DNA serves as template for

DNA polymerase
Polymerase chain
• Make complementary primers reaction (PCR).
• Add primers in more than 1000-fold excess
• Heat to make ssDNA, then cool
• Run DNA polymerase (usually Taq)
• Repeat heating, cooling, polymerase cycle

67 68

17
 In Vitro Mutagenesis
One method of PCR-based site-
directed mutagenesis.

(1) Template DNA strands are

separate and amplified by PCR.
(2) Following many cycles of PCR,
the DNA product can be used to
transform E. coli cells.
(3) The plasmid DNA can be
isolated and screened for the
presence of the unique
restriction site (by restriction
endonuclease cleavage.
69

18