Download 03 D MURRAY DNA RNA SLIDES 09

Nucleic Acids – Basic Concepts
David Murray PhD
UCD|Mater Clinical Research Centre
UCD School of Medicine and Medical Sciences
Mater Misericordiae University Hospital
Dublin
DNA and RNA are Nucleic
Acids
Outline
• What are DNA and RNA ?
• The Structure and Function of DNA and
RNA
• What is a Gene ?
• What is a Genome ?
DNA and RNA
What's the Big Deal ?
•
•
•
•
•
Hereditary Genetics
Importance of Genetics in Disease
Predisposition
Mutations
Loss of Genetic Control
What is DNA ?
• Contained in the nucleus
• Arranged in 22 chromosomes, plus two sex
chromosomes
• Two copies of each (46)
• 99.9% identical to other humans, 98% to chimp!
• Each cell; 6 feet of DNA
– >billion miles of DNA in the body!
• Therefore, very tightly packed
The Structure of DNA and RNA
• DNA (deoxyribonucleic acid)
• RNA (ribonucleic acid)
– What’s the Difference ?
• Both composed of two different
classes of nitrogen containing
bases:
– the purines and pyrimidines.
The Purines
• The most commonly
occurring purines in
DNA are adenine (A)
and guanine (G)
A
G
The Pyrimidines
• The most commonly
occurring pyrimidines
in DNA are cytosine
(C) and thymine (T)
C
T
RNA
• Contains the same bases as DNA with the
exception of thymine.
• Instead, RNA contains the pyrimidine uracil (U)
• DNA : AGCT
RNA : AGCU
T
U
The building blocks…
• Purines and pyrimidines form chemical linkages
with pentose (5-carbon) sugars.
• The carbon atoms on these sugars are
designated 1', 2', 3', 4' and 5'.
T
A
• It is the 1' carbon of the sugar that becomes bonded to
the nitrogen atom at position N1 of a pyrimidine or N9 of
a purine.
• DNA precursors contain the pentose deoxyribose.
• RNA precursors contain the pentose ribose (which
contains an additional OH group at the 2' position)
T
A
Base
(Purine/Pyrimidine)
+
Pentose
(Deoxyribose/Ribose)
=
Nucleoside
Nucleosides
The resulting molecules are called nucleosides
and can serve as elementary precursors for
DNA and RNA synthesis, in vivo.
Acid
Nucleotides
• Before a nucleoside can become part of a DNA or RNA
molecule it must become complexed with a phosphate
group to form a nucleotide (then termed either a
deoxyribonucleotide or ribonucleotide).
• Nucleotides can posess 1, 2 or 3 phosphate groups,
e.g. the nucleotides adenosine monophosphate (AMP),
adenoside diphosphate (ADP) and adenosine
triphosphate (ATP).
• The phosphate groups are attached to the 5' carbon of
the ribose sugar. Beginning with the phosphate group
attached to the 5' ribose carbon, they are labeled α, β
and γ phosphate.
• It is the tri-phosphate nucleotide which is incorporated
into DNA or RNA
Nucleotide (dNTP)
T
dTTP
Where N = A / G /C / T / U
Polynucleotides
• DNA and RNA are simply
long polymers of
nucleotides called
polynucleotides.
• Only the α phosphate is
included in the polymer. It
becomes chemically
bonded to the 3' carbon
of the sugar moiety of
another nucleotide.
• Phosphate ‘backbone’ is
negatively charged.
Where ‘Base’ = A,C,G or T
Polynucleotides
• The polynucleotide is
connected by a series of
5' to 3' phosphate
linkages.
• Polynucleotide
sequences are
referenced in the 5' to 3'
direction.
• Typically, polynucleotides
will contain a 5'
phosphate and 3'
hydroxyl terminal groups.
Summary of Terms
•
DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) composed
of nitrogen containing bases
– Purines: adenine (A) and guanine (G)
– Pyrimidines: cytosine (C) and thymine (T) (uracil (U) in RNA)
• Link with pentose sugars (DNA: deoxyribose, RNA: ribose) to form
nucleosides
– Nucleosides complex with three phosphate groups (Nucleotides)
– Polymers are incorporated into DNA/RNA (polynucleotides)
Summary of Terms
Base
Nucleoside
RNA/DNA
(Triphosphate)
Code
Adenine
Adenosine
dATP
A
Guanine
Guanosine
dGTP
G
Cytosine
Cytidine
dCTP
C
Thymine
Thymidine
dTTP
T
Uracil
Uridine
dUTP
U
What is the structure of DNA?
How is the structure related to
function?
History
• 1950
– Primary chemical structure of polynucleotides was known
(i.e. the 5'-3' phosphate linkage).
• 1951
– Erwin Chargaff:
• Experiment: To analyse DNA from a variety of species and determine
the relative concentrations of individual pyrimidines and purines (A,
T, C and G bases).
• Result: Although different species had uniquely different ratios of
pyrimidines or purines, the relative concentrations of adenine always
equaled that of thymine, and guanine equaled cytosine.
• Chargaff's Law: A=T, G=C
History
• 1953
– J.D. Watson and F.H.C.
Crick:
• Identified a hydrogen bonding
arrangement between models of
thymine and adenine bases,
and between cytosine and
guanine bases which fulfilled
Chargaff's rule.
• “Double Helix”
_
G=C
A=T
Consequences
• If G always paired with C, and T always paired with A, then
either strand could be regenerated from the complementary
information in the other strand.
• The basis of the complementarity was hydrogen bonding, i.e.
non-covalent interactions which could be easily broken and
re-formed.
• The information which DNA carried was within the unique
base sequence of the DNA.
• From the general interior location of the bases, it would
appear that the double helix would have to dissociate in
order to access the information.
DNA Structure
• Thousands of
nucleotides are strung
together by a
phosphate-sugar
backbone
DNA Structure
• Two strands of DNA
twist around one
another to form a
double helix
• ‘Twisted Ladder’
• Complementary base
pairs form the rungs
DNA Structure
• Two nucleotide
sequences running in
opposite directions pair
with one another.
• Each adenine (A)
pairing with a thymine
(T)
• And each guanine (G)
pairing with a cytosine
(C)
Base Pairs (BP)
5' C-G-A-T-T-G-C-A-A-C-G-A-T-G-C 3'
| | | | | | | | | | | | | | |
3' G-C-T-A-A-C-G-T-T-G-C-T-A-C-G 5'
A Word on DNA function
• Carries the blueprint for life
• Duplication for new cells
• Make proteins for biological functions:
DNA
• Prior to cell division, DNA is replicated
before being it is passed on to daughter
cells
• The DNA within our cells contains the
information for everything which occurs
within each cell,
– every action
– every substance made
– every event
– every response
– everything!
The Genome
The Genome
• The complete set of information in an organism's
DNA is called its genome
• Carries the information for all the proteins the
organism will ever synthesize.
• Typical human cell
– 6 feet of DNA
– Written in the four-letter nucleotide alphabet that
spells out the linear sequence of amino acids in a
protein.
– Carries instructions for ~ 30,000 different proteins
Gene Expression
What are Genes ?
The Cell
Library
Chromosomes Books
Genes
Sentences
• Approx 26,000 human genes
• Made up of DNA
• Coding regions of DNA
Gene Structure
• Like a sentence – beginning (Start) and end
(Stop)
• Ordered structure – not random bunch of
nucleotides linked in some random order
Transcription
• The first step in gene
expression
• DNA (gene) is used
as a template to
synthesise RNA copy
• Transcriptional
Profiling
• A specific gene specifies a polypeptide (protein)
– The DNA is transcribed into message RNA
(mRNA), which is translated into the
polypeptide
DNA
TRANSCRIPTION
RNA
TRANSLATION
Protein
Translation
•
•
•
mRNA used as template to make proteins
Occurs in ribosomes
One codon corresponds to one amino acid
• A specific gene specifies a polypeptide
Gene 1
Gene 3
DNA molecule
Gene 2
DNA strand
TRANSCRIPTION
RNA
Codon
TRANSLATION
Protein
Amino acid
The Genetic Code
• Specific DNA sequences code for specific amino
acids
Transcribed strand
Mutations ?
DNA
Transcription
RNA
Start
codon
Translation
Stop
codon
Faulty Protein
Polypeptide
Summary
An Example
Nucleotide sequence for Human BetaGlobulin gene.
• Haemoglobin subunit
• Caries information for amino acid
sequence of one globulin subunit
molecule.
• Alpha globulin – another gene
• Only one of two complementary strands
of DNA shown
• Written and read from left to right (from 5’
to 3’) like text.
• DNA highlighted in yellow: regions that
specify amino sequence for protein.
Review
• DNA and RNA
– What they are
– Structure and Function
• Genes and Genomes
• Transcription and Translation
• Any Questions ?
Nucleic Acids – Analytical Techniques
David Murray PhD
UCD Clinical Research Centre
UCD School of Medicine and Medical Sciences
Overview…
• Analytical Techniques
• RNA Techniques
– Extraction
– Analysis
• Reverse Transcription
– RNA → DNA
• RTPCR
– Reverse Transcription Polymerase Chain Reaction
• Quantitative Real Time PCR
• RNA interference (siRNA)
• Introduction to Microarrays
RNA Analysis
Extraction
Quantitation
Quality Assessment
Why Analyse RNA ?
•
•
•
•
•
Transcriptional Profiling
Levels of mRNA expression
mRNA: early step in gene expression
Controlled step
Variation between different cases
– Normal Vs Disease
– Responders Vs Non-Responders
ANALYSIS OF GENE EXPRESSION
5’
DNA (gene):
3’
PROMOTER
exon 1
intron
exon 2
transcription
3’
5’
RNA (1o Transcript)
RNA processing
Polyadenylated
RNA analysis
5’
mRNA
3’
AAAAAA
m7GpppN
Note: Non coding Introns are not
included in mRNA molecule
translation
protein
RNA Analysis
RNA
RT-PCR
Quantitative PCR
Microarray Analysis
•Comparison of mRNA expression profiles between two states;
•Disease Vs Normal
•Treated Vs Untreated
•Primary Vs Mets
Working with RNA
RNA is more susceptible to degradation than DNA
The 2´ hydroxyl groups adjacent to the phosphodiester linkages in
RNA are able to act as intramolecular nucleophiles in both base- and
enzyme-catalysed hydrolysis.
DNases require metal ions for activity and so can be inactivated with
chelating agents e.g. EDTA
RNases bypass the need for metal ions by taking advantage of the 2´
hydroxyl group as a reactive species.
Problems with RNases
• RNases
– single-strand specific endoribonucleases
– resistant to metal chelating agents
– can survive prolonged boiling or autoclaving
• But…
– relies on active site histidine residues for activity
– Therefore, it can be inactivated by the histidinespecific alkylating agent diethyl pyrocarbonate
(DEPC).
Avoiding ribonucleases
Exogenous
Introduced during working procedures
Eliminate through good working practices
Endogenous
Released by cells or tissue during extraction
Eliminate through use of inhibitors of RNase
activity
Working with RNA – Dos and Don’ts
Always wear gloves - Skin is an abundant source of
ribonucleases.
Prepare solutions for RNA work using autoclaved glassware,
then autoclave the solutions after they are prepared. Better still
use disposable plastic ware if possible.
If possible use pre-sterilized water.
Use separate solutions for RNA work and only use them for
RNA.
DEPC treatment of water isn’t always necessary. Autoclaving
water and solutions can sometimes be more effective in
removing RNases than chemical treatment.
Working with RNA – Dos and Don’ts
If you do need to treat your solutions with DEPC:
1.make your solution 0.1% DEPC
(500 µl in 500 ml H2O)
2.shake it well
3.keep it overnight at RT
4.autoclave
Take care!
DEPC is highly carcinogenic.
Use a fumehood!
Working with RNA – Dos and Don’ts
Maintain a separate area for RNA work that has its own set
of pipettes.
This is especially important if your work requires the use of
RNase A (e.g. plasmid preps).
Sterile, disposable plasticware can safely be considered
RNase-free and should be used when possible
Use RNase away or RNase zap!!
RNA Extraction
…an example
TRI ReagentTM
•
•
•
•
•
Sigma (Cat# T9424)
RNA, DNA and Protein extraction
Cell/Tissue lysis
Liquid separation
Quick and Effective
Extraction from Cells in Culture
• 80 – 100% Confluent T75
(~1 x 107 cells)
• Remove all media, wash
twice with PBS (saline)
• Add 1ml TRI REAGENT
(cover all cells)
– Scale Down/Up for other
culture vessels
• 10 min at Room Temp (RT)
• Remove to sterile microfuge
tube
Extraction from Tissue
• Remove tissue from
RNAlater into sterile
microfuge tube.
• Add 1 mL TRI REAGENT
• Homogenise at 15,000 rpm
for 1-2 min.
• Wash Tip between samples
in 100% Ethanol, then 0.1 %
DEPC.
Chloroform Separation
• Add 200 µL (0.2 ml)
Chloroform (fumehood!)
• Mix well (vortex), and stand
at RT for 15 min
• Centrifuge at 12,000 x g
(MAX!) for 15 min at 4oC
• 3 layers:
– Upper (aqueous): RNA
– Middle (interphase): Protein
– Lower (organic): DNA
• Remove upper phase to
fresh microfuge tube.
Propanol Precipitation
• Add 500 µL Ice-Cold Isopropanol
and mix
• Stand on Ice for 10 min
• Centrifuge at 12,000 x g for 10 min
at 4oC
• Pellet ?
• Remove Isopropanol
• Add 1 ml 75% Ethanol and vortex
• Centrifuge at 7,500 x g for 5 min
• Remove Ethanol and allow to air dry
(10 min)
• Resuspend in 10 – 50 µL 0.1%
DEPC (60oC 10 min)
RNA Quantitation
RNA Quantitation
• UV Spectroscopy
• 1/100 dilution of RNA
– 5 µL RNA in 495 µL 0.1 %
DEPC
• Absorbance at 260nm and
280nm
– Quartz cuvette
– Blank with 0.1% DEPC
RNA Quantitation Calculation
• A260 = 1.0 (40 µg/mL)
• Concentration (µg/µL) =
A260 x 40 x 100 (diln. factor)
1000 (mL → µL)
• Or Simply: A260 x 4 = Concentration (µg/µL)
• A260/A280 Ratio: RNA Quality/Purity (≈ 1.8)
– Higher: Organic Contaminants
– Lower: Protein Contaminants
RNA Quantitation Calculation
• Eg
– 1/100 dilution of RNA
– Absorbance Values:
• 260nm 0.456
• 280nm 0.250
– Concentration:
• (0.456 x 40 x 100)/1000
• 0.456 x 4 = 1.824 µg/µl
– Purity
• 0.456/0.250 = 1.8 (perfect!)
Newer technologies : BioAnalyzer NanoDrop
Next?
Assessment of RNA Quality
Agarose Gel Electrophoresis
• To assess Quality of
RNA
– Extent of degradation
• Also used to as
standard method for
analysing, identifying
and purifying
fragments of DNA
(later).
“Electrophoresis”
• A technique used to separate and
sometimes purify macromolecules especially proteins and nucleic acids –
based on their difference in size, charge or
conformation.
• When charged molecules are placed in
an electric field, they migrate toward
either the positive (anode) or negative
(cathode) pole according to their charge.
• In contrast to proteins, which can have
either a net positive or net negative charge,
nucleic acids have a consistent negative
charge imparted by their phosphate
backbone, and migrate toward the anode.
V
I
“Electrophoresis”
• Nucleic acids are electrophoresed within a matrix
or "gel".
• The gel is cast in the shape of a thin slab, with wells for
loading the sample.
• Agarose is typically used at concentrations of 0.5 to
2%.
• The higher the agarose concentration the "stiffer" the
gel.
• Agarose gels are extremely easy to prepare: simply
mix agarose powder with buffer solution (TAE/TBE),
melt it by heating, and pour the gel. It is also non-toxic.
• The gel is immersed within an electrophoresis buffer
(same as above) that provides ions to carry a current
and it also maintains the pH at a relatively constant
value.
RNA Electrophoresis
1.
2.
3.
4.
The agarose gel with three slots (S).
Injection of RNA sample into the first slot.
Injection of samples into the second and third slot.
A current is applied. The RNA moves toward the positive anode
due to the negative charges on its phosphate backbone.
-
+
Gel System
Procedure
• Clean Gel System with RNase Inhibitor
Spray
• Mix 50ml 10X TAE (Tris Acetate EDTA)
buffer with 450ml DIW (De-ionised Water)
= 1X TAE
• 0.5g Agarose in 50ml 1X TAE Buffer
– 1% (w/v) solution/gel
– Microwave until dissolved (1-2min @ 650W)
• Pour into casting tray (with combs) and
allow to cool/solidify
Procedure
• Analyse 2µg RNA by electrophoresis
– 2/concentration (µg/µl)
– Eg:
• 1.824 µg/µl
• 2 µg in ~ 1 µl
• Mix with 1 µl DEPC and 0.5 µl RNA
loading buffer
• Heat 65oC 10 min then chill on ice
• Submerge gel in 1X TAE (running buffer)
• Load RNA (2.5 µl) on gel and run at 100V
Procedure
• Remove gel after ~ 40min (blue of buffer almost
at end of gel)
• Visualise under UV light
• Visible ribosomal subunits indicate intact RNA
– 1 Degraded
– 2,3 Good Quality
RT-PCR
Reverse Transcriptase
Polymerase Chain Reaction
The basics…
• Interested in gene expression (mRNA)
• Levels of mRNA (transcripts)
• Comparison between 2 states (normal and
disease)
• mRNA (1-5% of total RNA)
• We use PCR (DNA Technique)
– More on that later
• Must Convert RNA to DNA
– How ?
Reverse Transcription
• mRNA molecule is copied into a double
stranded DNA compliment (cDNA)
• Reverse transcriptase – enzyme that
performs this.
• Used naturally by retroviruses to insert
themselves into an infected organism's
DNA genome
• cDNA contain coding regions only (exons)
Reverse Transcription (RT)
• mRNA Template
• ‘Priming’
– polyA mRNA isolated from total
RNA using oligo dT primer
• Polynucleotide of T’s
– Initiates synthesis
• First strand of cDNA
synthesised using Reverse
Transcriptase (RT) enzyme
– Adds complimentary nucleotide
bases to mRNA to make cDNA
cDNA is then used as a
template in PCR
PCR
• Polymerase Chain Reaction
– Technique for Targeted DNA Amplification
– Starting material ('target sequence’);
• A gene or segment of DNA (cDNA in our case)
– Target sequence can be amplified a billion fold in a matter
of hours
• PCR allows one to take a specimen of genetic
material, even from just one cell, copy its genetic
sequence over and over, and generate a test
sample sufficient to detect the presence or absence
of a specific virus, bacterium or any particular
sequence of genetic material
PCR Applications
• Widely used in molecular biology
• Specific Amplification
• Assuming sequence of target is known;
– Viral Detection
• HIV can be quantitated
– Screening genes for mutations
– Detecting gene expression
– Detection of food pathogens
– Forensic identification
The PCR Reaction
• Template
– Target DNA that Primers will bind
• Primers
– Bind target sequence, making the reaction specific
• Taq
– enzyme which carries out the amplification reaction
– extends the primers from their binding-sites on the target along the
template
• Buffer
– Contains a salt (KCl) and MgCl (cofactor for Taq)
• Nucleotides
– A,T,G and C
– Deoxyribonucleotide triphosphates (dNTPs)
– DNA building blocks
• Water
– High ‘PCR’ grade
PCR: Practicalities
• Always wear gloves
• All reagents must be thawed and mixed
completely before use
• Typical Reaction Mix (50µl);
– 37.5µl sterile water
– 5µl 10X Buffer
– 1µl 10mM dNTP mix
– 0.5µl Taq (5U/µl stock)
– 1µl Primer 1 & 1µl Primer 2 (10 pmol/ul)
– 5µl Template (cDNA)
The PCR Procedure
• Entire genomic double stranded DNA is heated
(denatured)
• Primers (DNA oligonucleotides)
– flank the nucleotide sequence of the gene
– synthesised chemically
– Prime DNA synthesis on single stranded DNA
• In vitro DNA Synthesis catalysed by DNA
polymerase
• Primers remain at 5’ end of new DNA fragments
The PCR Cycle
• Initial Cycle: 1min @ 95oC
• Followed by 40 cycles of following;
– Denature: 1min @ 95oC
– Anneal: 1min @ 50-60oC (depends on
primer)
– Elongate: 1min @ 72oC
• Final extension: 10min @ 72oC
PCR Links
• Calculate Annealing Temperature
– http://www.bioinformatics.vg/bioinformatics_to
ols/oligo2002.shtml
• Primer Design
– http://frodo.wi.mit.edu/cgibin/primer3/primer3_www.cgi
– http://www.basic.nwu.edu/biotools/oligocalc.ht
ml
The Thermocycle
The PCR Procedure
PCR: DNA Amplification
Agarose Gel Electrophoresis
• Analysis of PCR product
• Mix 50ml 10X TAE (Tris Acetate EDTA) buffer with 450ml
DIW (De-ionised Water) = 1X TAE
• 0.5g Agarose in 50ml 1X TAE Buffer
– 1% (w/v) solution/gel
– Microwave until dissolved (1-2min @ 650W)
• Add 1µl 10mg/ml Ethidium Bromide and mix
– Interacts with Nucleic Acids
– Fluorescent Complex – Visible under UV
• Potent Mutagen
– Fumehood, Lab Coat, Safety Glasses, Gloves
– Spills: Absorbed and Decontaminated with soap and water
• Pour into casting tray (with combs) and allow to cool/solidify
Running the Gel
• Submerge gel in 1X TAE (running buffer)
• Mix 3µl PCR reaction with 3µl loading
buffer and load onto gel
• Mix 3µl 100bp DNA ladder with 3µl loading
buffer and load onto gel
• Run at 100V
• Remove gel after ~ 40min (blue of buffer
almost at end of gel)
• Visualise under UV light
• Dispose Gel in Yellow Biohazard Bin
DNA Electrophoresis
1. The agarose gel with three slots (S).
2. Pipette DNA ladder into the first slot.
3. DNA ladder loaded. loading of samples
into the second and third slot.
4. A current is applied. The DNA moves
toward the positive anode due to the
negative charges on its phosphate
backbone.
5. Small DNA strands move fast, large
DNA strands move slowly through the
gel. DNA is not normally visible during
this process, so the marker dye is added
to the DNA to avoid the DNA being run
entirely off the gel. The marker dye has
a low molecular weight, and migrates
faster than the DNA, so as long as the
marker has not run past the end of the
gel, the DNA will still be in the gel.
6. The DNA is spread over the whole gel.
The electrophoresis process is finished.
Real Time PCR
Traditional PCR – Limitations
•
•
•
•
•
•
Qualitative not Quantitative
Gel Required
End point detection
4/5 hrs until result
Non numerical
Non Automated
Real Time PCR
• Monitor Amplification in Real Time
• Measure the kinetics of the reaction in the
early phases of PCR
• Quantitative and Qualitative
• 30/40 min until result
• Numerical output
• Automated
PCR Phases
Reaction components
being consumed.
Reaction is slowing.
Doubling of product at
every cycle.
Area for Real Time Detection
Reaction has stopped.
No more products
are being made.
Real Time PCR Instruments
SYBR Green Chemistry
• A dye that binds the Minor Groove of double
stranded DNA.
• Increasing the intensity of the fluorescent
emissions.
• As more double stranded amplicons are
produced, SYBR Green dye signal will increase.
• Increase in fluorescence directly proportional to
increase in amplicons (amplified product)
produced, which is proportional to the amount of
target template present initially.
SYBR Green Chemistry
Amplification Curve
Real Time PCR
• Pro
– Sensitive
– No gel
• Con
– Expense
– Hardware
Real Time PCR Links
• Good Article
– http://dorakmt.tripod.com/genetics/realtime.html
• Troubleshooting
– http://www.eurogentec.com/module/FileLib/GRTTSGCUST-0304-V4.pdf
Microarrays
Microarray Analysis
Monitor the activity of thousands of genes simultaneously
Compare activity of one gene in many samples.
Compare activity of many genes in one sample.
Take a photo of genes in action!
• Thousands of genes and their products, in
a living organism function in a complicated
and orchestrated way.
• Traditional methods in molecular biology
generally work on a "one gene in one
experiment“.
– Limited throughput.
– “Whole picture" of gene function is hard to
obtain.
• Microarrays
– Monitor the whole genome on a single chip
– Better picture of the interactions among
thousands of genes
Base-pairing or hybridization
(A-T and G-C for DNA)
(A-U and G-C for RNA)
Underlining principle of DNA
microarrays
What is an Array ?
aka Genechips aka BioArrays aka Biochips aka Genomechips
•
•
•
Microarrays involve the immobilization of
defined nucleic acids sequences (probes) on a
solid support…
Subsequent binding of target sequences
complementary to these nucleic acids to
measure gene expression levels.
The DNA sequences at each probe represent
important genes (or parts of genes)
•
1.28 x 1.28 cm glass/silicon wafer
•
•
24 x 24 µm probe site (≈ 500,000 probes)
Lengths of DNA up to 25 nucleotides long
GeneChip
Applications of DNA Microarrays
•
DNA Microarrays are used to study gene activity (expression)
• What proteins are being actively produced by a group of cells?
•
•
•
“Which genes are being expressed?”
Compare expression levels
How?
• When a cell is making a protein, it translates the genes (made of DNA)
which code for the protein into RNA used in its production
• The RNA present in a cell can be extracted
• If a gene has been expressed in a cell
•
•
RNA will bind to “a copy of itself” on the array
RNA with no complementary site will wash off the array
• The RNA can be “tagged” with a fluorescent dye to determine its presence
•
DNA microarrays provide a high throughput technique for quantifying
the presence of specific RNA sequences
Control
Disease
Cells/ Tissues
mRNA
cRNA
Hybridisation
Expression Comparison
The Process
Poly-A
RNA
Cells
AAAA
10% Biotin-labeled Uracil
Antisense cRNA
IVT
L
L L
(In-vitro
Transcription)
cDNA
Fragment (heat, Mg2+)
Labeled
fragments
L
L
L
Hybridize
Wash/stain
Scan
Affymetrix GeneChip
Techonology
Hybridization and Staining
Biotin
Labeled cRNA
GeneChip
Hybridized Array
L
L
+
L
L
L
L
L
+
L
L
L
L
L
SAPE
Streptavidinphycoerythrin
The Result
•A
light source scans the
array, causing the dyes
to fluoresce
•The
glow is picked up by
a sensor and is used to
determine the relative
abundance of the RNA
•This
information must be
processed to determine
the level of activity for
each expressed gene
Data Clustering
Array Applications
•
Basic Understanding
• Arrays can take a snap shot of which subset of genes in a cell is
actively making proteins
•
Medical diagnosis
• Used to determine if a person’s genetic profile would make him or
her more or less susceptible to drug side effects
• Used to distinguish between similar diseases or to define previously
unknown subsets within a disease.
•
Drug design
• Translate the human genome results into new products
• Must figure out what the genes do, how they interact, and how
they relate to diseases.
Microarray Links
•Affymetrix
–http://www.affymetrix.com
RNA interference (siRNA)
Outline
1.
2.
3.
4.
5.
RNA interference (RNAi) – what is it?
Mechanism of RNAi – an overview
Meet the players
Experimental Applications
Therapeutic Applications
what is RNA interference?
•RNAi is a way to silence gene expression
•to perform RNAi, dsRNA homologous
to the targeted gene is made and
then introduced into cells
•mRNA with high sequence homology
to the dsRNA may be silenced
RNA Interference
• Post-transcriptional gene silencing
• First discovered in c. elegans and plants
– Protective role: parasitic and viral resistance
• Mammals
– RNAi occurs – role???
How does RNAi work?
RNAi works postranscriptionally……..
siRNAs have a defined structure
19 nt duplex
2 nt 3’ overhangs
• siRNA binding
• siRNA unwinding
• RISC activation
•(RNAi silencing
complex)
practical aspects of RNAi
• biological research
– defining gene function (gene knockout)
– defining biochemical pathways
• microarray screening of RNAi
knockouts
•
therapeutic treatment
– cancer
– Infection
Although silencing by siRNAs is transient, vectors
can be made to express siRNAs in cells
gene function analysis
cell engineering
in vitro drug target validation
forward genetic screens
future
cell type
of interest
producer
Short hairpin
vector
virus
TISSUE CULTURE
HIGH-THROUGHPUT
tissue type
of interest
ES cell
gene function analysis
in vivo drug target validation
gene interaction
therapeutic testing
tissue or time specific
analysis of gene function
future?
INDUCIBLE
KNOCK-OUT
KNOCK-OUT/ -DOWN
future?
GENE THERAPY
McManus and Conklin
RNAi, 2003
siRNA Delivery
• in vitro
– Chemical transfection (Lipofectamine,
Oligofectamine, TransIT-TKO, Siport Amine,
Siport
• in vivo
– Intramuscular injection
– Hydrodynamic transfection into mammals
siRNA Therapeutics
Therapeutic siRNAs
siRNA target gene
p53 mutant
K-Ras
BCR-ABL
MDR1
C-RAF
Bcl-2
VEGF
PKC-α
Disease
Cancer
Β-Catenin
(Sioud, 2004)
Therapeutic siRNAs
siRNA target gene
Disease
HIV-Tat
HIV-Rev
HIV-Vif, -Hef
HPV-E6 and –E7
HBV-S1, -S2, -S, -X
CCR5, CXCR4
CD4
Viral Infection
(Sioud, 2004)
Therapeutic siRNAs
siRNA target gene
Fas receptor
Caspase-8
TNF-α
Disease
Acute Liver Failure
Sepsis
(Sioud, 2004)
References
Hannon, G.J. (2002). RNA interference. Nature. 418; 244-251. (review)
Agrawal, N. et al. (2003). RNA interference: biology, mechanism and applications. Microbiol. Mol. Biol. Rev. 67; 657-685. (review)
Elbashir et al. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 411; 494498.
Fire, A. et al. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 391; 806811.
Agrawal, N. et al. (2003). RNA interference: biology, mechanism and applications. Microbiol. Mol. Biol. Rev. 67; 657-685.
Tuschl, T. (2002). Expanding small RNA interference. Nature Biotech. 20; 446-448
Donze, O and Picard, D. (2002). RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase. Nucleic
Acids Research. 30; e46
Hannon, G.J. (2002). RNA interference. Nature. 418; 244-251.
McCaffrey, A.P. et al. (2002). RNA interference in adult mice. Nature. 418; 38-39.
Shuey, D.J. et al. (2002). RNAi: gene-silencing in therapeutic intervention. DDT. 7; 1040-1046.
Sioud, M. (2003). Therapeutic siRNAs. TIPS. 25; 22-28.
Wall, N.R. and Shi, Y. (2003). Small RNA: can RNA interference be exploited for therapy? The Lancet. 362; 1401-1403
Extra Links
• NCBI Human Genome:
– http://www.ncbi.nlm.nih.gov/genome/guide/human/
release_notes.html
• Human Genome Project:
– http://www.genome.gov/
• General Protocols
– http://micro.nwfsc.noaa.gov/protocols/protocols.ht
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