Gene Manipulation Techniques Techniques: - Nucleic Acids Separation and Detection
Gene Manipulation Techniques Techniques: - Nucleic Acids Separation and Detection
Gene Manipulation Techniques Techniques: - 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
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- Nucleic Acid Sequencing Gel
- This is PAGE and should be large (40 cm)
- Concentration of the gel depends on number of the
Application of electrophoresis is separation of nucleotides to be sequenced (20% for the first 50 -
and analysis of purity and size of 100 nucleotides)
macromolecules - Includes urea or formamide as a denaturant reagent.
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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
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toxic and 5 times more sensitive than EtBr 10
Separation media
1- Agarose gel
Buffer
TBE, TAE for DNA
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Capillary Electrophoresis Application
(CE) Separation of different molecules (small and large
Introduction size), and DNA sequencing
CE is a technique in which electrophoresis carried
out in very thin capillary tubes (20-100 µm inner Principle
diameter) Migration of the compounds through the medium
use of high voltage (capillary column) under high voltage
reduce separation time to a few minutes
extremely high resolution and automated in much In Practice
the same way as is HPLC (loading and detection) - CE equipment includes a power supply, two buffer
CE can analyze only small samples (5-10 nL), it is reservoirs, a buffer-filled capillary tube, and an on-
limited to use as an analytical tool. line detector
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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
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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
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sedimentation due to centrifugal force.
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Detection teqnique
1- Nucleic acid hybridization • Now how to separate the hybrid molecules?
This technique is based on that two single-stranded by passing the mixture through hydroxyapatite under
nucleic acid molecules of complementary base ionic conditions in which the hybrids would bind to
sequence can form a double-stranded hybrid. calcium phosphate salt while non-hybridized
Example molecules pass through the column.
- a mixture of DNA fragments has a specific DNA the hybrids molecules could be released by
coding for Protein x increasing the ionic strength of the elution buffer
- the only way to distinguish between the fragment
encoding for protein x and others is molecular • How often molecular biologists perform hybridization?
hybridization using complementary probe.
- incubation of denatured DNA fragments with
protein x mRNA will form ds DNA-RNA hybrid.
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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.
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?
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Outline What Does It Mean “To Clone”?
• What does it mean: “to clone”? Clone: a collection of molecules or cells, all
• What is a DNA library? identical to an original molecule or cell
• Can the cloned genes in libraries be
expressed? • To "clone a gene" is to make many copies of
it - for example, in a population of bacteria
• What is the polymerase chain reaction (PCR)?
• Gene can be an exact copy of a natural gene
• Is it possible to make directed changes in the
• Gene can be an altered version of a natural
heredity of an organism? gene
• Recombinant DNA technology makes it
possible
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• Plamids are naturally occurring extrachromosomal Cloning vectors are plasmids that can be
DNA modified to carry new genes
• Plasmids are circular dsDNA
• Plasmids can be cleaved by restriction enzymes, • Plasmids useful as cloning vectors must have
leaving sticky ends – a replicator (origin of replication)
• Artificial plasmids can be constructed by linking new – a selectable marker (antibiotic resistance
DNA fragments to the sticky ends of plasmid gene)
• These recombinant molecules can be autonomously – a cloning site (site where insertion of foreign
replicated, and hence propagated
DNA will not disrupt replication or inactivate
essential markers)
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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.
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
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Directional Cloning Directional Cloning
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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
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What is a DNA Library?
Colony Hybridization
Screening a genomic library by
colony hybridization.
A way to screen plasmid-based genome libraries for a
Host bacteria transformed with a
DNA fragment of interest
plasmid-based genomic library are
plated on a petri plate and incubated
• Host bacteria containing a plasmid-based library overnight to allow bacterial colonies
of DNA fragments are plated on a petri dish and to form.
allowed to grow overnight to form colonies
• Replica of dish made with a nitrocellulose disc. A replica of the colonies is obtained
by overlaying the plate with a flexible
• Disc is treated with base or heated to convert disc composed of absorbent material
dsDNA to ssDNA and incubated with probes (such as nitrocellulose or nylon).
• Colonies that bind probe (with P-32) hold the
fragment of interest 49 50
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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-
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driven synthesis of DNA
cDNA Libraries Are DNA Libraries cDNA Libraries Are DNA Libraries
Prepared from mRNA 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
Isolation of eukaryotic mRNA via oligo(dT)- the gene itself
cellulose chromatography. 55 56
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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
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gene expression patterns
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Can the Cloned Genes in Libraries Be Can the Cloned Genes in Libraries Be
Expressed? Expressed?
To express a eukaryotic protein in E. coli, the eukaryotic
Expression vectors are engineered cDNA must be cloned in an expression vector that contains
so that the RNA or protein products regulatory signals for transcription and translation.
of cloned genes can be expressed.
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.
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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.
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In Vitro Mutagenesis
One method of PCR-based site-
directed mutagenesis.
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