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Techniques
Thin-Layer Chromatography Is an
Important Technique for Lipid Analysis
• Lipids can be isolated, separated, and studied using
nonpolar solvents such as acetone and chloroform
• Thin-layer chromatography is used to separate
different kinds of lipids based on their relative
polarities
• A glass plate is coated with silicic acid and lipids are
spotted onto a position near the bottom of the plate
called the origin
Principle of separation of lipids via TLC
• A nonpolar organic solvent moves up the plate by
capillary action, taking different lipids with it to
varying degrees
• Nonpolar lipids have little affinity for the silicic
acid on the plate, and so move readily with the
solvent, near the solvent front
• Polar lipids will interact variably (depending on
how polar they are) with the silicic acid, and their
movement will be slowed proportionately
Figure 7-9
The Lipid Bilayer Is Fluid
• The lipid bilayer behaves as a fluid that
permits the movement of both lipids and
proteins
• Lipids can move as much as several mm per
second within the monolayer
• Lateral diffusion can be demonstrated using
fluorescence recovery after photobleaching
(FRAP)
Measuring lipid mobility with FRAP
• Investigators label lipid molecules in a
membrane with a fluorescence dye
• A laser beam is used to bleach the dye in a small
area, creating a dark spot on the membrane
• The membrane is observed afterward to
determine how long it takes for the dark spot to
disappear, a measure of how quickly new
fluorescent lipids move in
Figure 7-11
The Membrane Consists of a Mosaic of
Proteins: Evidence from FreezeFracture Microscopy
• Support for the fluid mosaic model came from
studies involving freeze-fracturing
• A bilayer or membrane is frozen and then hit
sharply with a diamond knife
• The resulting fracture often follows the plane
between the two layers of membrane lipid
Figure 7-16A
Figure 7-16B
Freeze-fracture analysis of
membranes
• When a fracture plane splits a membrane into its
two layers, particles the size and shape of
globular proteins can be seen
• The E surface is the exoplasmic face and the P
surface is the protoplasmic face
• Artificial bilayers without added protein show no
particles
Gel Electrophoresis
• Electrophoresis is a group of techniques that
use an electric field to separate charged
molecules
• How quickly a molecule moves during
electrophoresis depends on both charge and
size
• Electrophoresis uses various support media
most commonly polyacrylamide or agarose
Agarose Gel electrophoresis
 Positive electrode (anode)
• Negative electrode (cathode)
• One electrode is applied, charged molecules in the
samples migrate through pores of the gel toward their
pole of attraction.
• Mobility is also dependent on size and shape. Smaller
molecules maneuver more easily through pores
• Used to determine mutations in DNA, new genes,
restriction sites in DNA, etc.
Southern Blotting
• Transfer of DNA to a nitrocellulose filter
• Used to identify DNA sequences using a DNA
or RNA probe
Electrophoresis of membrane proteins
• Membrane fragments are solubilized in SDS,
which disrupts protein-protein and proteinlipid associations
• The proteins are thus coated with negatively
charged detergent molecules
• The proteins are loaded onto a polyacrylamide
gel and an electric potential applied
Electrophoresis of membrane proteins
(continued)
• The negatively charged proteins run toward the
positively charged bottom of the gel
• Polypeptides move through the gel with the
smallest moving fastest
• The gel is stopped when the smallest proteins
reach the bottom, and is stained with a dye
such as Coomassie brilliant blue to show the
proteins
Figure 7-22
Additional techniques using
electrophoresis
• Two-dimensional SDS - PAGE (polyacrylamide gel
electrophoresis) separates proteins in two
dimensions, first by charge and then by size
• Following electrophoresis, polypeptides can be
identified by Western blotting
• In this technique proteins are transferred to a
membrane and bound by specific antibodies
PCR cycle
• Each cycle consist of three stages:
– Denaturation
• Reaction tube is heated to ~94-960C causing separation of the
target DNA into single strands
– Hybridization (Annealing)
• Tube is cooled slightly to ~60-650C, which allows the primers to
hydrogen bond to complementary bases at opposite ends of the
target sequences
– Extension (Elongation)
• Temperature is raised slightly to ~70-75°C and DNA polymerase
copies the target DNA by binding to the 3`end of each primer.
• At the end of one cycle- target DNA has doubled – usually run
about 30-40 cycles.
• http://www.dnalc.org/ddnalc/resources/pcr.html
Type of DNA polymerase important
• Must use an enzyme that is suitable for the
various temperature changes.
• Most popular is Taq DNA polymerase.
Isolated from archaea called Thermus
aquaticus, a species that is adjusted to hot
temperature.
• Named the molecule of the year by the
Journal Science in 1989.
Advantage of PCR
• Amplify millions of copies of target DNA from
a very small amount.
• After 20 cycles approximately 1 million copies
are produces -2 20
• New Applications of PCR
• Quantitative Real-time PCR –used primers
with fluorescent dyes to quantify
amplification reactions as they occur
Cloning PCR products
• PCR is often used instead of library screening
• Disadvantage of PCR cloning is that you need
to know something about the DNA sequences
to design primers
• Use PCR to clone gene.
– Gene is amplified using primers
3.4 What Can You Do with a Cloned Gene?
Applications of Recombinant DNA Technology
• DNA Sequencing
– Important to determine the sequence of nucleotides
of the cloned gene
– Reasons for knowing the DNA sequence:
• Deduce the amino acid sequence of a protein encoded by a
cloned gene
• Determine the exact structure of a gene
• Identify the regulatory elements such as promoter
sequences
• Identify differences in genes created by gene splicing
• Identify genetic mutations
DNA sequencing
Common Methods are: PCR sequencing and Computer-automated
DNA sequencing
– Most widely used sequencing method developed
in 1977 by Frederick Sanger
• Chain termination sequencing (Sanger method)
DNA Sequencing Technique
• A DNA primer is hybridized to denatured
template DNA (plasmid-containing cloned
DNA)
• This is added to a reaction tube containing
– Deoxyribonucleotides and DNA polymerase
• A small amount of a modified nucleotide
called a dideoxyribonucleotide (ddNTP) is
mixed in with the target DNA, primer,
polymerase, and deoxyribonucleotides.
Dideoxynucleotide Procedure for DNA
sequencing
• What is a dideoxynucleotide?
– Human-made molecule
– Lacks a hydroxyl group at both the
2’ and 3’ carbons of the sugar
moiety (normal
deoxyribonucleotide has a 3’OH
group)
DNA replication
• Recall normal DNA replication
– Nucleoside triphosphate is linked
by it 5’alpha phosphate group to
the 3’hydroxyl group of the last
nucleotide growing chain.
– If dideoxynucleotide is
incorporated at the end of the
growing chain, DNA synthesis
stops because a phosphodiester
bond cannot be formed with the
next incoming nucleotide.
Steps involved
• Anneal a synthetic oligonucleotide (17-24mer) to a
predetermined segment of a strand of the cloning vector
near the insertion site of the cloned DNA (radioactively
labeled primer)
• This acts as a primer sequence by providing a 3’ hydroxyl
group for initiation of DNA synthesis
continued
• The primed DNA sample is partitioned into four
separate tubes. Each tube contains four
deoxyribonucleoties, DNA polymerase, cloned DNA
to be sequenced, a one modified
dideoxyribonucleotide.
• Recall chain growth stops as soon as a
dideoxynucleotide is incorporated. (at the
3’terminus)
Sequencing continued
• After DNA synthesis, each reaction tube will contain
unique oligonucleotide.
• DNA molecules are separated by polyacrylamide gel
electrophoresis ( good for small sizes up to a single
nucleotide)
• Autoradiograph shows the radiolabeled DNA
fragments that were produced during the
enzymatic DNA synthesis step.
• Each of the four lanes on the autoradiograph
corresponds to a reaction tube that contained one
of the four dideoxynuclotides.
How is it read?
• As accurately as possible, the order
of the bands are read from the
bottom to the top of the
autoradiograph (the radiolabeled
fragment closes to the bottom)
• Remember you are reading the
complementary strand to the
template strand
• Normally can resolve up to 350
bands
Limitation of DNA sequencing
• Used to sequence approximately 200- 400
nucleotides in a single reaction
• Longer than 400 must run multiple reactions
to create overlapping sequencing
• Piece together to determine the entire
sequence
• Cumbersome for large-scale sequencing like
Human Genome Project
Automated DNA Sequencing
• Minimizes manual manipulations
– Dideoxynucleotide method forms the basis of automated
DNA sequencing
– Highly accurate can resolve 20,000 bases per hour
• Sequence analysis carried out with four different
fluorescent dyes (for each dideoxy)
• Samples are still separated with polyacrylamide gel
or polymer-filled capillary tube.
• Fluorescent dye emits a narrow spectrum of light.
The fluorescent signals are read by a computer and
converted to a sequence of nucleotides.
– Computer automated sequencing
• ddNTP’s are each labeled with a different fluorescent
dye
• Samples are separated on a single-lane capillary gel
that is scanned with a laser beam
• Creates different color patterns for each nucleotide
• Converted by computer to the sequence
3.4 What Can You Do with a Cloned Gene?
Applications of Recombinant DNA Technology
• Chromosome Location and Copy Number
– Identify the chromosome location of the cloned gene
– Determine if the gene is present as a single copy in the genome
– Fluorescence in situ hybridization (FISH)
• Chromosomes are isolated from cells and spread out
on glass slide
• cDNA probe for gene of interest is labeled with
fluorescent nucleotides and incubated with slides
Fluorescence in situ hybridization (FISH)
• Identify which chromosomes contains a gene of interest.
– continued
– Probe will hybridize with complementary sequence on
the slide.
– Fluorescently labeled probe is illuminated to indicate
the presence of that gene
– Align chromosomes (karotype) to determine which
one.
– Usually for multiple copies of genes, genetic disorders
(fetal disorders- Downs syndrome)
3.4 What Can You Do with a Cloned Gene?
Applications of Recombinant DNA Technology
• Chromosome Location and Copy Number
– Southern blotting- Developed by Ed Southern in 1975
• Digest chromosomal DNA into small fragments with
restriction enzymes
• Fragments are separated by agarose gel electrophoresis
• Gel is treated with alkaline solution to denature the DNA
• Fragments are transferred onto a nylon or nitrocellulose
filter (called blotting)
• Filter (blot) is incubated with a probe and exposed to film by
autoradiography
• Number of bands on film represents gene copy number
Southern Blotting and PCR
• http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::53
5::535::/sites/dl/free/0072437316/120078/bi
o_g.swf::Southern+Blot
• http://www.sinauer.com/cooper/4e/animatio
ns0411.html
• http://www.maxanim.com/genetics/PCR/PCR.
htm
Setting up the gel sandwich
• Gel is placed under the nylon or nitrocellulose
membrane, filter paper, paper towels, and
weight allowed for wicking the salt solution
through gel
• DNA is transferred from the gel to the filter by
capillary action or by current
• Blot is then incubated with nonradioactive or
radioactive probe.
mRNA analysis
• All cells in the body (except germ cells) carry the same set of
genes, but only a subset of those genes is active in a
particular cell or tissue.
• You can determine which genes are being transcribed into
mRNA (expressed).
• One a gene is cloned out of a library or you make a probe it
can be used in the analysis of mRNA
3.4 What Can You Do with a Cloned Gene?
Applications of Recombinant DNA Technology
• Studying Gene Expression
– Techniques involve analyzing mRNA produced by a tissue
– Northern blot analysis
• Basic method is similar to Southern blotting
• RNA is isolated from a tissue of interest, separated by
gel electrophoresis, blotted onto a membrane, and
hybridized to a probe.
• Amount of RNA from different sites, tissues, exposures
compared.
Northern Blot
Problems with Northerns
• Not particularly sensitive and may not detect
a gene transcribed at a low level.
• Northern blots require a fairly large quantity
of mRNA, which may be difficult to obtain
from a small tissue, such as the embryonic
kidney
RT-PCR
– Sometimes the amount of RNA produced by a tissue is below
the level of detection by Northern blot analysis
– What can we use to amplify genes?
– Can PCR amplify RNA?
– Reverse transcription PCR
• Enzyme reverse transcriptase
• Reverse transcription of mRNA is performed –
converted into double-stranded cDNA
• cDNA is then amplified with a set of primers specific
for the gene of interest
• Products electrophoresed on agarose gel
Real time PCR
• Used to determine the amount of PCR product during an
experiment
• Quantify amplification reactions as they occur in “real time”
or quantitative PCR (qPCR)
• Basic procedure uses a specialized PCR (thermal cycler)
machine
– Laser to scan the PCR tube
– Reaction contains a dye-containing probe or DNA binding
dye that emits fluorescent light when illuminated by the
laser.
– Light emitted correlates to amount of PCR product
amplified
Two approaches to Real-time PCR
• SYBR Green
– Dye that binds double-stranded DNA
– As more dsDNA is copied, more DNA binds to SYBR Green
– Increases amount of fluorescent light emitted
• Taqman
– Probes complementary to specific regions of target DNA between the
primers
– Two dyes: reporter dye at the 5’end of probe and can release
flurorescent light, the other dye is called a quencher, attached to the
3’ end of the probe
3.4 What Can You Do with a Cloned Gene?
Applications of Recombinant DNA Technology
• Studying Gene Expression
– In situ hybridization
• Used to determine the cell type that is expressing the
mRNA
• Tissue of interest is preserved in a fixative solution and
embedded in a wax-like substance
• Tissue can be sliced into very thin sections attached to
microscope slides
• Slides are incubated with a probe to the gene of interest
• Probe hybridizes with mRNA in cells
• Probe is detected
In Situ Hybridization
• Means “in place” hybridization is a more sensitive method for
visualizing gene activity directly in fixed cells or tissues.
• Rather than extracting mRNA from cells, the mRNA is left in
place and the entire cell, tissue, or embryo is fixed using
paraformaldehyde (prevents breakdown of molecules)
Procedure for in situ
• Early procedure – cut tissue into very thin
sections, adhere to microscope slides. Use a
probe to look for mRNA directly in tissue.
• Disadvantages – safety, time consuming
(preparing slides), and difficulty in observing
the gene expression in the overall tissue.
DNA Microarrays
• How do these genes work together?
• Different cells express different sets of genes
at different levels
• DNA array is a means to analyze the
expression patterns of thousands of genes at
a time
• Study all the genes expressed in a tissue
quickly
3.4 What Can You Do with a Cloned Gene?
Applications of Recombinant DNA Technology
• Studying Gene Expression
– Gene microarrays
• DNA microarray analysis
• Single-stranded DNA molecules are attached onto a
slide using a robotic arrayer fitted with tiny pins
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DNA or Gene Microarrays
• A microarray, also known as gene chip, is created using a
small glass microscope slide.
• Single-stranded DNA molecules are attached to the slide
using a computer-controlled high-speed robotic arm called
an arrayer, fitted with a number of tiny pins.
• Each pin contains millions of copies of different DNA
molecules (cDNA’s from different genes).
• DNA is fixed to the slide at specific locations that are
recorded by the computer. (one microarray can have over
10,000 spots of DNA).
• How do we use the microarray?
How do you use a microarray?
• http://www.dnalc.org/ddnalc/resources/dnaarray.html
• Extract mRNA (cDNA) from a tissue of interest
• The mRNA is then tagged with a fluorescent dye and
incubated overnight with the microarray.
• mRNA hybridize to spots on the microarray that contain
complementary DNA sequences.
• Microarray is washed and scanned by a laser that cause the
mRNA hybridized to the microarray to fluoresce.
• Tell you which genes are expressed in your sample and how
much.
Data analysis
• The fluorescent spots reveal which genes are
expressed in the tissue of interest
• Intensity of fluorescence indicates that
relative amount of expression
• Brighter spot – more mRNA expressed in that
tissue
• Microarrays can also be run by labeling cDNAs
from two or more tissues with different
colored fluorescent dyes.
3.4 What Can You Do with a Cloned Gene?
Applications of Recombinant DNA Technology
Uses of Microarray analysis
• Purchase an entire genome on a
microarray- humans, yeast and
bacteria.
• Compare pattern of expressed genes
under different conditions (stress,
inflammation, disease, etc).
• Cancer cells can be compared to
normal cells to look for genes that
may be involved in cancer formation.
Develop new drug therapy strategies.
RNA interference
• 1998, Mello and Fire used double-stranded
pieces of RNA (dsRNA) to inhibit or silence
expression of genes in the nematode
roundworm
• Technique is called RNA interference or RNAi
– ssRNA combines to mRNA
– Degrade mRNA or block translation of RNA
• http://www.wiley.com/college/pratt/0471393
878/student/animations/dna_sequencing/ind
ex.html
• http://www.bio.davidson.edu/courses/genom
ics/chip/chip.html