BIOMEDICAL RESEARCH TECHNIQUES

(MEDSCI 720)

• PCR
• Real-time qPCR
• In situ hybridisation

Srdjan Vlajkovic
Department of Physiology
Polymerase chain reaction is a technique that amplifies
DNA, enabling scientists to make billions of copies of a
DNA molecule in a very short time.

PCR has been used to:

• detect DNA sequences
• diagnose genetic diseases
• carry out DNA fingerprinting
• detect bacteria or viruses
• research human evolution

• It has even been used to clone the DNA of an Egyptian

mummy!
PCR invented in 1983:
• Kary Mullis at Cetus Corp.
• “Enzymatic application” used to amplify small DNA fragments
• Diagnostic to genotype Sickle Cell Anemia (β-globin gene)
• In 1993 Kary Mullis won Nobel Prize

Revolutionary technique:
• Amplifies > 1 billion copies of DNA from ONE template molecule
• One day to genotype patient (mutant or wild-type allele?)
(much faster than Southern blot which takes days!)
 Polymerase Chain Reaction (PCR)
• PCR is a technique which is used to amplify the number
of copies of a specific region of DNA, in order to produce
enough DNA to be adequately tested.

• The purpose of a PCR is to make a huge number of

copies of a gene. As a result, it now becomes possible to
analyze and characterize DNA fragments found in
minute quantities in places like a drop of blood at a crime
scene or a cell from an extinct dinosaur.

• PCR is valuable to researchers because it allows them

to multiply unique regions of DNA so they can be
detected in large genomes.
 PCR (Cont’d)

• When first developed, multiple cycles of the PCR

process were cumbersome for two reasons:

• First, the DNA polymerases (Klenow fragment) available

at the time were inactivated each time the temperature
was raised to denature the template strand.

• Second, three water baths at three different

temperatures were necessary, which meant that
constant human attention was required.
 PCR (Cont’d)

Two developments were instrumental in the

maturation of the PCR process.

• First was the purification of a heat-stable DNA

polymerase (Taq DNA polymerase).

• The second development was the invention of a

thermal cycler.
Polymerase Chain Reaction
Multiplex PCR
 Reverse Transcriptase Polymerase Chain Reaction
(RT-PCR)

Reverse transcriptase is a common name for an enzyme that functions

as a RNA-dependent DNA polymerase. They are encoded by
retroviruses, where they copy the viral RNA genome into DNA prior to its
integration into host cells. In the laboratory, it is used for analysing gene
expression. i.e. convert mRNA to cDNA by reverse transcription.

Reverse transcriptases have two activities:

• DNA polymerase activity
• RNase H activity

All retroviruses have a reverse transcriptase, but the enzymes that are
available commercially are derived from one of two retroviruses, either
by purification from the virus or expression in E. coli:
• Moloney murine leukemia virus
• Avian myeloblastosis virus
 Deoxyribonucleotides

Nucleotide sequences of the genes are determined by the

precise order of appearance of 4 different
deoxyribonucleotides within a stretch of DNA.

• The four nucleotide bases, the building blocks of every

piece of DNA, are represented by the letters A, C, G, and T,
which stand for their chemical names: adenine, cytosine,
guanine, and thymine.

• The A on one strand always pairs with the T on the

other, whereas C always pairs with G.
In order to use PCR, one must know the sequences which flank
both ends of a given region of interest in DNA. One need not
know the DNA sequence in-between.
 Primers
5’ F 3’

3’ R 5’

• Complementary to opposite strands with 3’ ends

pointing towards each other

• Should have similar melting temperatures

• Be in vast excess
 Some Applications of PCR
• Molecular Research and Biotechnology
1) The Human Genome Project
2) Evolutionary studies
3) Analyse gene expression by measuring RNA levels (RT-PCR)
4) Detect presence of introduced gene (transgene)

• Medical Diagnostics
1) Diagnosis and characterisation of Infectious diseases:
- Detect presence of viral pathogens (HIV, hepatitis B)
- Detect presence of pathogenic bacteria (E.coli, Anthrax)
2) Diagnosis and characterisation of human genetic diseases
3) Diagnosis and characterisation of Neoplasia

• Forensics
1) Identify criminal suspects
2) Paternity cases
References:
• K. Mullis (1990) The unusual origin of the polymerase chain
reaction. Scientific American, April 1990, pp. 81-88
• Erlich HA, Gelfand D, Sninsky JJ (1991) Recent advances in
the polymerase chain reaction Science 251:1643-1651
 Quantitation of mRNA

• Northern blotting
• Ribonuclease protection assay
• In situ hybridization
• cDNA arrays
• PCR
- most sensitive
- technically simple
- can discriminate closely related mRNAs
- but difficult to get truly quantitative results
 Real-Time RT-PCR Chemistry

• DNA-binding dyes (SYBR green)

• Molecular beacons
• Hybridisation probes
• Hydrolysis probes (Taqman assay)
The principle of Taqman qPCR
 Quantitation options
• Relative – normalisation of gene expression
• Ideal internal standard: expressed at a constant level
among different tissues, at all stages of
development, unaffected by the experimental
treatment
• Most commonly used house-keeping genes: GAPDH,
b-actin, ribosomal RNAs (rRNA)

• Absolute – precise determination of gene copy

numbers
• Requires the construction of a standard curve
• Results expressed as copy numbers per cell, total
RNA concentration, or unit mass of tissue
References:
•Bustin SA (2000) Absolute quantification of mRNA
using real-time reverse transcription polymerase chain
reaction assays. Journal of Molecular Endocrinology
25:169-193
•Bustin SA (2002) Quantification of mRNA using real-
time reverse transcription PCR (RT-PCR): trends and
problems. Journal of Molecular Endocrinology 29:23-39
 In situ hybridization
• A method of localizing and detecting specific mRNA
sequences in morphologically preserved tissues sections
or cell preparations by hybridizing a nucleotide probe to
the sequence of interest.

• The principle: specific annealing of a labelled nucleic

acid probe to complementary sequences in fixed tissue,
followed by visualisation of the location of the probe.

• A critical aspect of these procedures is that the target

nucleic acid is retained in situ

• The sensitivity: 10-20 copies of mRNA per cell.

Preparation of material

• Frozen sections
• Paraffin embedded sections
• Cells in suspension
 Choice of probe

Probes are complimentary sequences of nucleotide bases

to the specific mRNA sequence of interest. These
probes can be as small as 20-40 base pairs or be up to
1000 bp.

The strength of the bonds between the probe and the

target decreases in the order RNA-RNA to DNA-RNA.
This stability is influenced by the various hybridization
conditions (salt concentration, hybridization temperature,
concentration of formamide, pH).
 Probe types

• Oligonucleotide probes
• Single stranded DNA probes
• Double stranded DNA probes
• RNA probes (cRNA probes or riboprobes)
 Oligonucleotide probes

Produced synthetically by an automated chemical

synthesis.

Advantages:
• Small (40-50 base pairs; easy penetration into the cells
or tissue of interest).
• Resistant to RNases
• Single stranded (no renaturation).
 Single stranded DNA probes

• Similar advantages to the oligonucleotide probes

• Larger (200-500 bp size range).
• Produced by reverse transcription of RNA or by amplified
primer extension of a PCR fragment in the presence of a
single antisense primer.
• Disadvantages: time to prepare, expensive reagents
used, good repertoire of molecular skills required for
their use.
 Double stranded DNA probes
• The sequence of interest inserted in bacteria, cloned and
the sequence excised with restriction enzymes.
• Because the probe is double stranded, denaturation has
to be carried out prior to hybridization in order for one
strand to hybridize with the mRNA of interest.
• These probes generally less sensitive (DNA strands tend
to rehybridize to each other)
• Not as widely used today
RNA probes (cRNA probes or riboprobes)

The most widely used probes with in situ hybridization.

Advantage: RNA-RNA hybrids thermostable and resistant to
digestion by RNases.
Two methods of preparing:
• RNA polymerase-catalyzed transcription of mRNA
• In vitro transcription of linearized plasmid DNA with RNA
polymerase.
Disadvantage: difficult to prepare, sensitive to RNases, poor
tissue penetration
Benefits of using oligonucleotide probes

1. Stability
2. Availability
3. Faster and less expensive to use
4. Easier to work with
5. More specific
6. Better tissue penetration
7. Better reproducibility
 Labeling your oligonucleotide

Traditionally oligonucleotide probes have been

radiolabeled. 35Sulphur is the most commonly used
radioisotope.
Several non-radioactive labels used successfully:
• Biotin
• Digoxin and digoxigenin (DIG),
• Alkaline phosphatase
• Fluorescent labels (FITC, Texas Red, rhodamine).
Non-radioactive labels have no inherent "decay" kinetics;
can be used immediately or be divided into aliquots,
lyophilized and stored at -20°C for later use.
 Probe Labelling

Biotin
Rhodamine
• 5' end labeling FITC

• 3' tailing S-dATP, DIG-dUTP,

35

Biotin-dUTP, FITC-dUTP
Terminal
transferase (TdT)
• 3' end labeling DIG-ddUTP

• Incorporation of label Biotin-dATP, FITC-dATP

 Detection
• Radiolabeled probes: photographic film or emulsion.
• Fluorescent labels: FISH (fluorescent in situ hybridization); advantage:
two or more different probes can be visualized at one time.
• DIG and Biotin labeled oligonucleotide probes generally require an
intermediate step before detection: anti-DIG antibodies or streptavidin
• The advantage of using a DIG labeled probe: can be detected with
antibodies conjugated to a number of different labels (alkaline
phosphatase, peroxidase)
• Biotin can also be detected with antibodies, but more often with Avidin
from egg white or Streptavidin
• In general, the DIG label is more sensitive than the Biotin label; the DIG
label allows comparable sensitivity to 35S radiolabeled probes.
Localization of ActRIIA activin receptor mRNA in rat
brain using a 35S-dATP 3'-tailed gene probe.
After hybridization the tissue was exposed to
photographic film for 2 days
 Hybridization issues

• Permeabilization
• Pretreatment / prehybridization steps
• Hybridization
• Washes
 Permeabilization
• Common reagents used to permeabilize tissue:
HCl, detergents and Proteinase K.

• HCl. Precise action of the acid not known: extraction of

proteins and hydrolysis of the target sequence may help
decrease the level of background staining.

• Detergents Triton X-100 or SDS frequently used to

permeabilize the membranes by extracting the lipids.

• Proteinase K: an endopeptidase which is used to

remove proteins that surround the target sequence.
Optimal concentration has to be determined.
 Pretreatment / prehybridization steps
Carried out to reduce background staining.

• Peroxidases: 1% H2O2 in methanol for 30 minutes.

• Alkaline phosphatases: levamisole
• Acetylation with acetic anhydride (0.25%) in
triethanolamine; important for decreasing background
and inactivate RNases

Prehybridization: incubate the tissue with a solution that is

composed of all the elements of the hybridization
solution, minus the probe.
 Hybridization
The composition of the hybridization solution is critical in
controlling the efficiency of the hybridization process.

The factors that influence the hybridization of the

oligonucleotide probe to the target mRNA are:
• Temperature
• pH
• monovalent cation concentration
• presence of organic solvents
 Hybridization (Cont’d)
A typical hybridization solution contains:
• Dextran sulphate effectively increases the probe concentration in
solution resulting in higher hybridization rates.
• Formamide and DTT (dithiothreitol): organic solvents which reduce
the thermal stability of the bonds allowing hybridization to be carried
out at a lower temperature.
• SSC (NaCl + Sodium citrate). Monovalent cations decrease the
electrostatic interactions between the two strands.
• EDTA: a chelator that removes free divalent cations from the
hybridization solution, because they strongly stabilize duplex DNA.
• Other components are added to decrease the chance of nonspecific
binding of the probe: ssDNA, tRNA, polyA, Denhardts solution
 Washes

Following hybridization the material is washed to remove

unbound probe or probe which has loosely bound to
imperfectly matched sequences.

Washing should be carried out at or close to the stringency

condition at which the hybridization takes place with a
final low stringency wash.
 Controls

The most important part of any experimental procedure is

the inclusion of controls.

One has to be confident that the hybridization reaction is

specific and that the probe is in fact binding selectively to
the target mRNA sequence and not to other components
of the cell or other closely related mRNA sequences.

If no staining is observed with the probe does this mean

that there really is no expression of that mRNA in the
tissue or does it mean that there may be a problem with
tissue preparation?
 Controls for tissue mRNA quality
If the quality of your tissue is poor and/or your RNA is
degraded it will be very hard to get good results with ISH

• Poly(dT) probe
will detect total mRNA poly A tails. If a very weak signal
is obtained, it is likely that tissue RNA is degraded.

• Probes against house keeping genes

A low signal suggests tissue RNA degradation.

• Positive control
Perform ISH on a control tissue known to have the
sequence of interest (not always possible).
 Specificity controls
• Determine that your probe is only binding to RNA.
The absence of binding after RNase treatment indicates that
binding was indeed to RNA within the tissue.

• Specific versus non-specific binding.

The first control involves hybridization of the tissue with sense
and antisense probes in parallel. The sense control probe
gives a measure of non-specific probe binding due to the
chemical properties of the probe.

• Competition studies with labeled and excess unlabeled

probes can distinguish between specific vs non-specific
binding.

The best way to ensure that your probe is binding to the correct
target sequence is by choosing a correct probe sequence
from the start and having high stringency hybridization and
wash conditions in your experiment.
References:
• Wisden W and Morris BJ (1994) In situ hybridization
protocols for the brain, Academic Press
• Wilkinson DG (1992) In situ hybridization: A practical
approach, Oxford University Press