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MedSci 720 > PCR, qRT-PCR, ISH > Flashcards

PCR, qRT-PCR, ISH Flashcards

1
Q

Describe PCR and its purpose:

A

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.

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2
Q

Describe what PCR has been used to achieve:

A 
Detect DNA sequence
Diagnose genetic disease
Carry out DNA fingerprinting
Detect bacteria or viruses
Research human evolution
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3
Q

Describe the two factors which made PCR a revolutionary technique:

A

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

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4
Q

Describe how PCR can amplify small sections of DNA:

A

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

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5
Q

Describe the two developments instrumental in maturation of PCR process:

A
  1. Heat-stable DNA taq polymerase

2. Thermal cycler development

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6
Q

Describe the analysis of PCR products (techniques):

A

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

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7
Q

Describe multiplex PCR:

A

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

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8
Q

Describe multiplex PCR:

A

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

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9
Q

Describe reverse transcriptases and their two activities:

A

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

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10
Q

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

A

A DNA template
Nucleotides
Primers
Polyermase

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11
Q

Describe the templates for PCR:

A

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

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12
Q

Describe primers:

A

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

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13
Q

Describe the importance of melting temperatures of primers:

A

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

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14
Q

List the PCR reagents:

A 
Template (target) DNA
2 ss primers
dNTPs
PCR buffer (KCl, MgCl2, Tris pH 7-8)
Thermostable DNA taq polymerase (KEY)
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15
Q

Name the advantages of PCR:

A

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

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16
Q

Name the disadvantages of PCr:

A

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

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17
Q

Name the problems with taq polymerase:

A

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

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18
Q

List the future uses of PCR:

A

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

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19
Q

List the future uses of PCR:

A

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

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20
Q

Name ways that PCR can be used in medical diagnostics:

A
  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
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21
Q

Name how PCR an be used in forensics:

A

Identify criminal suspects

Paternity cases

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22
Q

List some variations of basic PCR:

A 
Allele-specific
Assembly
Asymmetric
Digital
Intersequence
Inverse
Ligation-mediated
Nanoparticle assisted
Miniprimer
Nested
Touchdown
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23
Q

How can mRNA be quantified?

A 
Northen blotting
Ribonuclease protection assay
In situ hybridisation
cDNA arrays
PCR
24
Q

Describe PCR in quantifying mRNA:

A

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

25
Q

Describe real-time RT-PCR chemistry:

A

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

26
Q

Describe primer design for PCR:

A

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

27
Q

Describe probe design for PCR:

A 
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
28
Q

Describe the amplicon size effect and product size:

A

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

29
Q

Describe the Taqman probe:

A

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’)

30
Q

Describe how the taqman probe operates in PCR:

A

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

31
Q

Describe real-time PCR detection:

A

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

32
Q

Describe amplicon plots:

A

The large the products, the lower the efficiency

The threshold for detection needs to be constant

33
Q

Describe the Ct (threshold cycle):

A

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

34
Q

What are the quantification options for qPCR:

A

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

35
Q

Describe the general principle of Taqman qPCR:

A

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

36
Q

Describe in situ hybridisation:

A

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

37
Q

What is the critical advantage of ISH?

A

Target nucleic acid is retained in situ

38
Q

List the general steps in ISH:

A
  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
39
Q

How can material be prepared for ISH?

A

Frozen sectons
Paraffin embedded sections
Cells in suspension

40
Q

Describe general probe characteristics:

A

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)

41
Q

List the different types of probe types:

A

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

42
Q

Describe oligonucleotide probes and their advantages:

A

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)

43
Q

List the 7 benefits of using oligonucleotide probes:

A 
Stability
Availability
Faster and less expensive to use
Easier to work with
More specific
Better tissue penetrtion
Excellent reproducibility
44
Q

Describe single stranded DNA probes:

A

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

45
Q

Describe double stranded DNA probes:

A

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

46
Q

Describe RNA probes (cRNA or riboprobes):

A

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

47
Q

Describe the 2 methods of preparing RNA probes:

A
  1. RNA polymerase-catalysed transcription of mRNA

2. In vitro transcription of linearised plasmid DNA with RNA polymerase

48
Q

Describe labelling of oligonucleotides:

A 
Traditionally radiolabelled (direct)
Non-radioactive labels:
Biotin
Digoxin and digoxigenin (DIG)
Fluorescent labels (FITC, rhodamine, Texas red)
49
Q

Describe the advantages of non-radioactive labelling of probes:

A

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

50
Q

Describe indirect non-radioactive labelling:

A

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

51
Q

Describe detection of probes:

A

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

52
Q

List the steps in hybridisation in which issues can occur:

A

Permeabilisation
Pretreatment/prehybridisation steps
Hybridisation
Washes

53
Q

Describe the need for controls in ISH:

A

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

54
Q

Describe controls for tissue mRNA quality in ISH:

A 
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)
55
Q

Describe the three specificity control parameters for ISH:

A
  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
56
Q

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

A

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

MedSci 720 (9 decks)

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