Molecular biology
Course structure
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Polymerase Chain Reaction
RNA Isolation
cDNA Synthesis
Amplification
Electrophoresis
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Transfection
Which molecules can be analyzed?
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Genomic DNA
mRNA
Nucleic acid chemistry
DNA and RNA store and transfer
genetic information in living organisms.
DNA:
– major constituent of the nucleus
– stable representation of an organism’s
complete genetic makeup
RNA:
– found in the nucleus and the cytoplasm
– key to information flow within a cell
Why purify genomic DNA?
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Many applications require purified DNA.
Purity and amount of DNA required depends on intended
application.
Tissue typing for organ transplant
Detection of pathogens
Human identity testing
Genetic research
DNA purification challenges
1.
Separating DNA from other cellular components such
as proteins, lipids, RNA, etc.
2.
Avoiding fragmentation of the long DNA molecules by
mechanical shearing or the action of endogenous
nucleases.
Effectively inactivating endogenous nucleases and
preventing them from digesting the genomic DNA is a
key early step in the purification process. DNases can
usually be inactivated by use of heat or chelating
agents.
DNA purification
There are many DNA purification methods. In all
cases, one must:
1.
2.
3.
4.
Effectively disrupt cells or tissues
(usually using detergent)
Denature proteins and nucleoprotein complexes
(a protease/denaturant)
Inactivate endogenous nucleases
(chelating agents)
Purify nucleic acid target selectively
(could involve RNases, proteases, selective matrix and
alcohol precipitations)
Total Cellular RNA
•Messenger RNA (mRNA): 1-5%
Serves as a template for protein synthesis
• Ribosomal RNA (rRNA): >80%
Structural component of ribosomes
• Transfer RNA (tRNA): 10-15%
Translates mRNA information into the appropriate
amino acid
What RNA is needed for?
Messenger RNA synthesis is a dynamic expression of
the genome of an organism. As such, mRNA is central
to information flow within a cell.
• Size – examine differential splicing
• Sequence – predict protein product
• Abundance – measure expression levels
• Dynamics of expression – temporal, developmental,
tissue specificity
RNA isolation
RNA purification
•Total RNA from biological samples
– Organic extraction
– Affinity purification
• mRNA from total RNA
– Oligo(dT) resins
• mRNA from biological samples
– Oligo(dT) resins
Total RNA purification
• Cells or tissue must be rapidly and efficiently disrupted
• Inactivate RNases
• Denature nucleic acid-protein complexes
• RNA selectively partitioned from DNA and protein
RNases
• RNases are naturally occurring enzymes that degrade
RNA
• Common laboratory contaminant (from bacterial and
human sources)
• Also released from cellular compartments during
isolation of RNA from biological samples
• Can be difficult to inactivate
Protecting against RNases
• Wear gloves at all times
• Use RNase-free tubes and pipette tips
• Use RNase-free chemicals
• Pre-treat materials with extended heat (180°C for
several hours), wash with DEPC-treated water
• Supplement reactions with RNase inhibitors
• Include a chaotropic agent (guanidine) in the procedure
that inactivate and precipitate RNases and other proteins
Organic extraction of total RNA
Advantages
• Versatile - compatible with a variety of sample types
• Scalable - can process small and large samples
• Established and proven technology
• Inexpensive
Disadvantages
• Organic solvents
• Not high-throughput
• RNA may contain contaminating genomic DNA
Affinity purification of total RNA
Advantages
• Eliminates need for organic solvents
• Compatible with a variety of sample types (tissue, tissue
culture cells, white blood cells, plant cells, bacteria, yeast,
etc.)
• DNase treatment eliminates contaminating genomic
DNA
• Excellent RNA purity and integrity
RNA analysis
Photometric measurement of RNA concentration
UV 260 nm
Conc=40xOD260
Determination of the RNA concentration
[RNA] μg/ml = A260 x dilution x 40.0
where
A260 = absorbance (in optical densities) at 260 nm
dilution = dilution factor (200)
40.0 = average extinction coefficient of
RNA.
cDNA Synthesis
Template:
- Total RNA
+
- Poly (A) RNA
- Specific RNA
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Priming
Oligo dT (18)
synthesize complete cDNAs, beginning
at the poly A+ tail and ending at the 5
end of the mRNA.
Random hexamer
Hybridizes somewhere along the mRNA
Gene specific primers
Specific mRNA but lower yields
Reverse Transcriptases (RTs)
The reverse transcriptase from the avian myoblastosis
virus (AMV-RT):
Temperature optimum of activity at 45-50°C.
Highly thermostable, can be used at temperatures up to
60°C.
Demonstrates DNA exonuclease and RNase activities.
The reverse transcriptase from the Moloney murine
leukemia virus (MMLV-RT):
The optimal working temperature is about 37°C, with a
maximum of 42°C.
Weaker RNase activity.
Normalization
Most gene expression assays are based on the comparison of two or more
samples and require uniform sampling conditions for comparison to be valid.
Many factors can contribute to variability in the analysis of samples, making
the results difficult to reproduce between experiments: Sample degradation,
extraction efficiency, contamination, Sample concentration, RNA integrity,
reagents, reverse transcription
Housekeeping genes: ß-actin, ß-tubulin, GAPDH, have often been used as
reference genes for normalization. The expression of these genes is
constitutively high and that a given treatment will have no effect on the
expression level.
Amplification (PCR)
The Polymerase Chain Reaction
PCR – first described in mid 1980’s, Mullis (Nobel prize in 1993)
An in vitro method for the enzymatic synthesis of specific DNA sequences
Selective amplification of target DNA from a heterogeneous, complex
DNA/cDNA population
Requires
Two specific oligonucleotide primers
Thermostable DNA polymerase (Taq DNA polymerase from Thermus
aquaticus)
dNTP’s
Template DNA
MgCl2
Sequential cycles of (generally) three steps (temperatures)
MgCl2 (mM)
1.5 2
3 4
5
Magnesium Chloride
(usually 0.5-5.0mM)
Magnesium ions have a variety of effects
Mg2+ acts as cofactor for Taq polymerase
Mg2+ binds DNA - affects primer/template interactions
Mg2+ influences the ability of Taq pol to interact with primer/template
sequences
More magnesium leads to less stringency in binding
Thermal cyclers
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Introduced in 1986.
PCR cyclers available from many suppliers.
heated lids
adjustable ramping times
single/multiple blocks
gradient thermocycler blocks
Reactions in tubes or
96-well micro-titre plates
thin walled tube,
 volume,  cost
 evaporation & heat
transfer concerns0
Temperature control in a PCR thermocycler
94 0C - denaturation
50 – 70 0C - primer annealing
72 0C - primer extension
94 0C - denaturation
Step 1:
Denaturation
dsDNA to ssDNA
Step 2:
Annealing
Primers onto template
Step 3:
Extension
dNTPs extend 2nd strand
Vierstraete 1999
PCR Problems
Mis-priming
Set up reactions on ice
Hot-start PCR –holding one or more of the PCR components until the first
heat denaturation
Manually - delay adding polymerase
Polymerase antibodies
Touch-down PCR – set stringency of initial annealing temperature high,
incrementally lower with continued cycling
PCR additives
0.5% Tween 20
5% polyethylene glycol 400
DMSO
Primer Design
1.
2.
3.
4.
Typically 20 to 30 bases in length
Annealing temperature dependent upon primer
sequence (~ 50% GC content)
Avoid secondary structure, particularly 3’
Avoid primer complementarity (primer dimer)
Primer Design Software
Many free programs available online
OLIGO
PRIMER
PrimerQuest
Primer Design Software
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Many free programs available online
•OLIGO
www.oligo.net
•PrimerQuest
www.eu.idtdna.com/scitools/applications/primerquest
•Primer3
www.frodo.wi.mit.edu/primer3
•GeneWorks
www.geneworks.com.au
•Yeast genome primer design
www.yeastgenome.org/cgi-bin/web-primer
Primer Dimers
• Pair of Primers
5’-ACGGATACGTTACGCTGAT-3’
5’-TCCAGATGTACCTTATCAG-3’
• Complementarity of primer 3’ ends
5’-ACGGATACGTTACGCTGAT-3’
3’-GACTATTCCATGTAGACCT-5’
• Results in PCR product
Primer 1
5’-ACGGATACGTTACGCTGATAAGGTACATCTGGA-3’
3’-TGCCTATGCAATGCGACTATTCCATGTAGACCT-5’
Primer 2
Rules of thumb for PCR conditions
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Add an extra 3-5 minute (longer for Hot-start Taq) to your cycle
profile to ensure everything is denatured prior to starting the
PCR reaction
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Approximate melting temperature
(Tm) = [(2 x (A+T)) +(4 x (G+C))]ºC
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If GC content is < 50% start 5ºC beneath Tm for annealing
temperature
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If GC content ≥ 50% start at Tm for annealing temperature
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Extension @ 72ºC: rule of thumb is ~500 nucleotide per
minute. Use 3 minutes as an upper limit without special
enzymes
Typical PCR Temps/Times
Initial denaturation
90o – 95o C
1 – 3 min
Denature
90o – 95o C
0.5 – 1 min
Primer annealing
45o – 65o C
0.5 – 1 min
Primer extension
70o – 75o C
0.5 – 2 min
Final extension
70o – 75o C
5 – 10 min
Stop reaction
4o C or 10 mM EDTA
Hold
25 – 40
cycles
DNA Gel Electrophoresis
Agarose
Agarose is a polysaccharide consisting of a linear polymer (repeating units) of
D-galactose and 3,6-anhydro L-galactose
The movement of molecules through an agarose gel is dependent on the size
and charge of molecules and the pore sizes present in the agarose gel.
At neutral pH, molecules migrate toward the anode when an electric field is
applied across the gel.
Small, highly negatively charged molecules migrate faster through agarose
gels than large, less negatively charged molecules.
Rates can also be effected by the pore size of the agarose gel. Decreasing
pore sizes increases the separation of small and large molecules during
electrophoresis. Pore size can be decreased by increasing the percentage of
agarose in the gel.
TBE or TAE Buffers
Tris helps to maintain a consistent pH of the solution. EDTA chelates
divalent cations like magnesium. This is important because most
nucleases require divalent cations for activity.
Ethidium Bromide
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A fluorescent dye is added to agarose gels.
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It intercalates between the bases of DNA, allowing DNA
fragments to be located in the gel under UV light.
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The intensity of the band reflects the concentration
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Because of its interaction with DNA, ethidium bromide
is a powerful mutagen. Always handle all gels and gel
equipment with gloves. Agarose gels with Ethidium
Bromide must be disposed as hazardous waste in the
labelled container.
Add loading dye to all samples.
Loading dye contains xylene
cyanol as a tracking dye to
follow the progress of the
electrophoresis as well as
glycerol to help the samples sink
into the well.