BIOMEDICAL RESEARCH TECHNIQUES

(MEDSCI 720)

• PCR

• Real-time qPCR

• In situ hybridisation

Srdjan VlajkovicDepartment 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 (-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 ReactionPolymerase 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.

5’

3’

3’ 5’

F

R

Primers

• 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 Project2) Evolutionary studies3) Analyse gene expression by measuring RNA levels (RT-PCR)4) Detect presence of introduced gene (transgene)

• Medical Diagnostics1) 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 diseases3) Diagnosis and characterisation of Neoplasia

• Forensics1) Identify criminal suspects2) 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

• 5' end labeling

• 3' tailing

• 3' end labeling

• Incorporation of label

Biotin

Rhodamine

FITC

DIG-ddUTP

35S-dATP, DIG-dUTP, Biotin-dUTP, FITC-dUTP

Biotin-dATP, FITC-dATP

Terminal transferase (TdT)

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 controlPerform 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