PCR, qRT-PCR, ISH

Question 1

Describe PCR and its purpose:

Answer 1

PCR amplifies DNA, enabling the generation of millions of copies of DNA in a very short time (DNA is very resistant to degradation).
Purpose is to make a huge number of copies of a gene.
Possible to analyse and characterise DNA fragments found in minute quantities.

Question 2

Describe what PCR has been used to achieve:

Answer 2

Detect DNA sequence
Diagnose genetic disease
Carry out DNA fingerprinting
Detect bacteria or viruses
Research human evolution

Question 3

Describe the two factors which made PCR a revolutionary technique:

Answer 3

Amplifies >1 billion copies of DNA from one template molecule
One day to genotype patient (much faster than Southern blot)

Question 4

Describe how PCR can amplify small sections of DNA:

Answer 4

Makes many copies of small DNA fragment
Size range of DNA fragments amplified is 100-10,00bp (size allows for amplification of most genes)
Size and sequence of fragment defined by DNA primers (flank GoI)
>1x10^9 copies of small DNA fragments

Question 5

Describe the two developments instrumental in maturation of PCR process:

Answer 5

1. Heat-stable DNA taq polymerase

2. Thermal cycler development

Question 6

Describe the analysis of PCR products (techniques):

Answer 6

By size fractionate DNA in agarose or polyacrylamide gels
Ethidium bromide stain DNA to determined the size of PCR product (dsDNA) and the amount of product (intensity of bands)
Use gel electrophosis

Question 7

Describe multiplex PCR:

Answer 7

Amplified several DNA sequences at the same time
Uses several primer pairs:
1. All need the same melting temperature
2. All these primers should amplify DNA products that be a different size

Question 8

Describe multiplex PCR:

Answer 8

Amplified several DNA sequences at the same time
Uses several primer pairs:
1. All need the same melting temperature
2. All these primers should amplify DNA products that be a different size

Question 9

Describe reverse transcriptases and their two activities:

Answer 9

Encoded by retroviruses, copy viral RNA genome into DNA (convert mRNA to cDNA)
Has DNA polymerase activity and RNase-H activity (to hydrolyse template)
Taken from moloney murine leukemia virus or avian myeloblastosis virus

Question 10

Which advantages of replication does RT-PCR take advantage of:

Answer 10

A DNA template
Nucleotides
Primers
Polyermase

Question 11

Describe the templates for PCR:

Answer 11

Small amount of template
In theory a single molecules
Do not need to isolate sequence of interest
DNA template doesn’t need to be highly purified
DNA is stable in the absence of nucleases

Question 12

Describe primers:

Answer 12

To use PCR, need to know sequences flanking GoI region
Primers provide specificity
Complementary to opposite strands with 3’ ends pointing towards each other
Should be in vast excess

Question 13

Describe the importance of melting temperatures of primers:

Answer 13

Should have similar melting temperatures
Tm=2(A+T) + 4(G+C)
Tm= temperature at which half possible H bonds are formed (half primer bound to target)

Annealing temperature = Tm-5 degrees

Question 14

List the PCR reagents:

Answer 14

Template (target) DNA
2 ss primers
dNTPs
PCR buffer (KCl, MgCl2, Tris pH 7-8)
Thermostable DNA taq polymerase (KEY)

Question 15

Name the advantages of PCR:

Answer 15

Sensitive: Exponential amplification from trace amounts of DNA
Fast: Can be done in a few hours
Safe: Non-radioactive method without toxic organic solvents

Question 16

Name the disadvantages of PCr:

Answer 16

Contamination can be a serious problem, due to extreme sensitivity
Can sometimes be quite fickle and inconsistent

Question 17

Name the problems with taq polymerase:

Answer 17

Does not have proof reading ability
Error rate 1 in 2x10^4 bases
Seems rare but recovered in cloning a single molecule
Newer polymerase (Pwo, Pfu) have high fidelity

Question 18

List the future uses of PCR:

Answer 18

Molecular research and biotechnology:
Human genome project
Evolutionary studies
Analyse gene expression by measuring RNA levels (RT-PCR)
Detect presence of introduced gene (transgene)

Question 19

List the future uses of PCR:

Answer 19

Molecular research and biotechnology:
Human genome project
Evolutionary studies
Analyse gene expression by measuring RNA levels (RT-PCR)
Detect presence of introduced gene (transgene)

Question 20

Name ways that PCR can be used in medical diagnostics:

Answer 20

1. Diagnosis and characterisation of infectious diseases (presence of viral pathogen, bacteria)
2. Diagnosis and characterisation of human genetic diseases
3. Diagnosis and characterisation of cancer cells

Question 21

Name how PCR an be used in forensics:

Answer 21

Identify criminal suspects

Paternity cases

Question 22

List some variations of basic PCR:

Answer 22

Allele-specific
Assembly
Asymmetric
Digital
Intersequence
Inverse
Ligation-mediated
Nanoparticle assisted
Miniprimer
Nested
Touchdown

Question 23

How can mRNA be quantified?

Answer 23

Northen blotting
Ribonuclease protection assay
In situ hybridisation
cDNA arrays
PCR

Question 24

Describe PCR in quantifying mRNA:

Answer 24

Most sensitive
Technically simple
Can discriminate closely related mRNAs
BUT difficult to get truly quantitative results

Question 25

Describe real-time RT-PCR chemistry:

Answer 25

DNA-binding dyes (SYBR green) Molecular beacons Hybridisation probes Hydrolysis probes (Taqman assay)

Question 26

Describe primer design for PCR:

Answer 26

GC 30-80% No more than 3 consecutive Gs Tm ~60 Forward and reverse primers as close as possible to the probe (not overlapping) 5 nucleotides at the 3' end have only 1/2 G-Cs Amplicon length

Question 27

Describe probe design for PCR:

Answer 27

``` GC 30-80% No more than 3 consecutive Gs Tm ~70 No G on 5' end Select probe from stran with more Cs than Gs Amplicon length ```

Question 28

Describe the amplicon size effect and product size:

Answer 28

200 bp product shows S-shape curve vs. 500 bp has lower efficiency and unpredictable for each amplicon design

Question 29

Describe the Taqman probe:

Answer 29

Hybridises with the target amplicon Is 3' terminally blocked (cannot be extended by the polymerase) Has 2 fluorescent dyes attached (reporter 5' and quencher 3')

Question 30

Describe how the taqman probe operates in PCR:

Answer 30

When the primers reach the prove, the probe is released, and the quencher removes the reporter which results in fluorescence There is an additional reference dye to provide passive internal reference for signal normalisation Fluorescence detection between 520-660

Question 31

Describe real-time PCR detection:

Answer 31

The autonomic detection of PCR product growth throughout the amplification process Collects geometric phase data No post-PCR steps Enables high precision and high throughput

Question 32

Describe amplicon plots:

Answer 32

The large the products, the lower the efficiency | The threshold for detection needs to be constant

Question 33

Describe the Ct (threshold cycle):

Answer 33

The calculated fractional cycle number at which the PCR product crosses a threshold of detection 20 is normal (a good signal) 25-30 is relatively low From >30 only rare genes would be detected

Question 34

What are the quantification options for qPCR:

Answer 34

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 outcome AND use housekeeping genes (GAPDH, B-actin, rRNA)) Absolute - Precise determination of a standard curve, results expressed as copy numbers per cell, total RNA concentration, or unit mass of tissue

Question 35

Describe the general principle of Taqman qPCR:

Answer 35

Sample preparation, put into machine, reverse transcription, PCR, fluorescence detection, quantification on computer

Question 36

Describe in situ hybridisation:

Answer 36

A method of localising and detecting specific mRNA sequences in morphologically preserved tissues sections or cell preparations. The sensitivity - 10-20 copies of mRNA per cell The principle - specific annealing of a labelled nucleic acid probe to complementary sequences in fixed tissue, followe by visualisation of the location of the probe

Question 37

What is the critical advantage of ISH?

Answer 37

Target nucleic acid is retained in situ

Question 38

List the general steps in ISH:

Answer 38

1. Preparation of sample 2. Permeabilisation of tissue/cell 3. Pre-hybridisation (reduce ground signal) 4. Hybridisation with labelled probe (20-40bp oligo) 5. Wash 6. Visualisation of label

Question 39

How can material be prepared for ISH?

Answer 39

Frozen sectons Paraffin embedded sections Cells in suspension

Question 40

Describe general probe characteristics:

Answer 40

Strength of bonds between the probe and the target decreases in the order RNA-RNA to DNA-RNA Stability influenced by various hybridisationn conditions (salt concentration, hybridisation temperature, concentraton of formaide, pH)

Question 41

List the different types of probe types:

Answer 41

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

Question 42

Describe oligonucleotide probes and their advantages:

Answer 42

Produced synthetically by automated chemical synthesis Advantages: Small (20-40 bps, easy penetration into the cells or tissue of interest) Resistant to RNases SS (no renaturation)

Question 43

List the 7 benefits of using oligonucleotide probes:

Answer 43

``` Stability Availability Faster and less expensive to use Easier to work with More specific Better tissue penetrtion Excellent reproducibility ```

Question 44

Describe single stranded DNA probes:

Answer 44

Similar advantages to the oligo probes Larger (200-500bps) Produced by revere transcription of RNA or by amplified primer extension of a PCR fragment in the presence of a single antisense primer Disadvantages: Time to prepare, good repertoire of molecular skills required for their use

Question 45

Describe double stranded DNA probes:

Answer 45

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 hybridisation in order for one strand to hybridise with the mRNA of interest Generally less sensitive probes (DNA strands tend to rehybridise to each other) Not as widely used today

Question 46

Describe RNA probes (cRNA or riboprobes):

Answer 46

Most widely used with ISH RNA-RNA hybrids thermostable and resistant to digestion by RNases Difficult to prepare, sensitive to RNases, poor tissue penetration

Question 47

Describe the 2 methods of preparing RNA probes:

Answer 47

1. RNA polymerase-catalysed transcription of mRNA | 2. In vitro transcription of linearised plasmid DNA with RNA polymerase

Question 48

Describe labelling of oligonucleotides:

Answer 48

``` Traditionally radiolabelled (direct) Non-radioactive labels: Biotin Digoxin and digoxigenin (DIG) Fluorescent labels (FITC, rhodamine, Texas red) ```

Question 49

Describe the advantages of non-radioactive labelling of probes:

Answer 49

No inherent decay kinetics Can be used immediately or be divided into aliquots Lyophilised and stored at -20 for later use

Question 50

Describe indirect non-radioactive labelling:

Answer 50

Nucleotides coupled to a reporter molecule After hybridisation, the reporter molecule is bound by affinity molecule which is conjugated to a marker molecule --> detect the marker molecules

Question 51

Describe detection of probes:

Answer 51

Radiolabelled - photographic film or emulsion Fluorescent labels - FISH (multiple probes can be visualised at the same time) DIG and biotin - require intermediate step before detection (anti-DIG antibodies or streptavidin) - can be detected with antibodies conjugated to a number of different labels DIG label more sensitive than biotin label, DIG label allows comparable sensitivity to 35S radiolabelled probes

Question 52

List the steps in hybridisation in which issues can occur:

Answer 52

Permeabilisation Pretreatment/prehybridisation steps Hybridisation Washes

Question 53

Describe the need for controls in ISH:

Answer 53

Need to be confident that hybridisation 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, this means there is either no expression of that mRNA or there may be a problem with tissue prep

Question 54

Describe controls for tissue mRNA quality in ISH:

Answer 54

``` If the tissue quality is poor or RNA is degraded, it will be hard to get good result Poly(dT) probe will detect total mRNA poly A tails (weak signal=probable degradation) Probes against housekeeping genes (low signal=probable degradation) Positive control (tissue with known sequence of interest) ```

Question 55

Describe the three specificity control parameters for ISH:

Answer 55

1. Determine that your probe is only binding to RNA (absence of binding after RNase treatment indicates that binding was to RNA) 2. Specific vs. non-specific binding (first control involved hybridisation of the tissue with sense and antisense probes in parallel - sense control probe gives a measure of non-specific probe binding due to the chemical properties of the probe) 3. Competition studies with labelled and excess unlabelled probes can distinguish between specific and non-specific binding

Question 56

What is the best way to ensure probe is binding correct target sequence in ISH:

Answer 56

Choose correct probe from the start and have high stringency hybridisation and wash conditions in experiment