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6. Methods > Copy Number Detection (Molecular) > Flashcards

Copy Number Detection (Molecular) Flashcards

1
Q

What are the main molecular genetics methods for detecting allele copy number in DNA samples?

A
  1. QF-PCR
  2. RQ-PCR
  3. MLPA
  4. NGS
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2
Q

Breifly describe how QF-PCR is used for copy number detection.

A

Quantification of polymorphic repeat sequences to determine copy number.

Uses fluorescently labelled primers and capillary electriphoresis.

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3
Q

Advantages of using QF-PCR for copy number detection.

A
  1. Detects mosaicism (above 15%), triploidy and aneuploidy
  2. Requires little starting material
  3. Fast turnaround times
  4. Relatively inexpensive
  5. High resolution/High through-put
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4
Q

Disadvantages of using QF-PCR for copy number detection.

A
  1. No positional information obtained
  2. Targeted test
  3. MCC may prevent interpretation of results
  4. May not detect low-level mosaicism (<15%)
  5. Limited ability to detect multiple targets in a single assay due to spectral overlap of dyes
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5
Q

Examples of using QF-PCR for copy number detection.

A
  1. Prenatal aneuploidy screening
  2. Detection of trisomy in pregnancy loss or PND
  3. Post-transplant chimaerism monitoring
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6
Q

Breifly describe how RQ-PCR is used for copy number detection.

A
  • PCR amplification in which the amount of productis measured during each PCR cycle.
  • Uses fluorescent dyes (non-specific e.g. SYBR green) or probes (sequence specific e.g. Taqman).
  • During the exponential phase the amount of amplified product is proportional to the amount of starting material.
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7
Q

Advantages of using RQ-PCR for copy number detection.

A
  1. Quantitative
  2. Very high resolution
  3. Rapid and easy to perform
  4. Fast turnaround time
  5. Requires little starting material
  6. Single cell analysis possible
  7. Detects UPD if methylation specific Res are used
  8. Post-PCR processing is eliminated, reducing labour, cost and possibility of cross-contamination
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8
Q

Disadvantages of using RQ-PCR for copy number detection.

A
  1. No positional information obtained
  2. Targeted test
  3. Specialist equipment required – thermal cycler and optical instrument to measure fluorescence
  4. Unlikely to detect low level mosaicism
  5. Multiple reactions required to examine multiple loci
  6. Sequence-specific probes expensive
  7. Non-specific fluorescent dyes such as a SYBR green intercalate with any dsDNA which may lead to false positive signals
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9
Q

Examples of using RQ-PCR for copy number detection.

A
  1. Quantify gene expression (mRNA)
  2. Minimal residual disease monitoring of gene fusion products e.g. PML-RARA in AML, BCR-ABL1 in CML
  3. Detection of mutations e.g. JAK2 V617F, NPM1/FLT3-ITD
  4. Detection of microdels/dups e.g. 22q11
  5. Detection of subtel del/dup
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10
Q

Breifly describe how MLPA is used for copy number detection.

A
  • PCR-based multiplex reaction allowing amplification of ~40-45 targets in a single reaction.
  • Detects copy number imbalances in gDNA and RNA sequences. Probes anneal to target and are then amplified and quantified
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11
Q

Advantages of using MLPA for copy number detection.

A
  1. Can test for ~40-50 different imbalances in one reaction
  2. High throughput technique
  3. High resolution– detects sequences of only ~60nt-can therefore detect dups/dels of a single exon
  4. Fast turnaround time
  5. Inexpensive
  6. Requires little starting material
  7. Detects methylation
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12
Q

Disadvantages of using MLPA for copy number detection.

A
  1. Targeted test
  2. More labour-intensive than QF-PCR
  3. Can’t be used for single cell screening i.e. PGD
  4. No positional information obtained
  5. Doesn’t detect balanced rearrangements
  6. Cannot detect low-level mosaicism (<20-30%)&unreliable detection at higher levels
  7. Unreliable for detection of maternal cell contamination
  8. Cannot detect triploidy (69,XXX) and unreliable for other triploidy detection
  9. Analysis gives average copy number per cell. Tumour analysis difficult if sample contains <50% cancer cells
  10. SNPs under probes can prevent binding (false positive result)
  11. Sensitive to contaminants
  12. Not easily scaled up
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13
Q

Examples of using MLPA for copy number detection.

A
  1. Aneuploidy screen
  2. Sub-telomeric imbalance screen
  3. Microdeletion screen
  4. MS-MLPA – PWS/AS testing
  5. Commercial gene dosage kits available for BRCA1 and BRCA2 screening, Duchenne/Becker Muscular Dystrophy, Fanconi Anaemia A and many more
  6. Bespoke MLPA
  7. Methylation and cancer screening
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14
Q

Breifly describe how Next Generation Sequencing (NGS) is used for copy number detection.

A

A number of different methods are employed, but all involve step wise addition of nucleotides to fragmented DNA.

Bioinformatic analysis of paired reads then enables the detection of deletions and duplications.

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15
Q

Advantages of using NGS for copy number detection.

A
  1. Very high resolution (single base change detection)
  2. Genome wide or targeted
  3. Provides positional information
  4. Detects UPD and LOH
  5. High throughput
  6. Detects balanced/unbalanced rearrangements (paired-end/mate-pair seq)
  7. More tolerant of poor quality DNA
  8. Detection of both single base variants and CNVs in a single assay
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16
Q

Disadvantages of using NGS for copy number detection.

A
  1. Difficulty with interpreting results-CNVs of unclear significance
  2. Vast amount of data obtained
  3. Requires a lot of starting material
  4. Expensive
  5. Labour intensive
  6. Not suitable for small genes
  7. Need to confirm abnormal results
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17
Q

Examples of using NGS for copy number detection.

A

Copy number detection is a relatively new use of this technology.

Bioinformatic algorithms Canvas and Manta are designed to look for copy number and structural variants in WGS data and can be used to replace array based methods for copy number change.

Cost efficient for disorders that routinely have microarray plus panel sequencing e.g. ID/Devdel.

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18
Q

What does MLPA stand for?

A

Multiplex Ligation-dependant Probe Amplification

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19
Q

What is MLPA?

A

A Multiplex PCR which detects copy number changes in up to 45 gDNA or RNA sequences in a single reaction

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20
Q

What is the principle behind MLPA method?

A
  1. DNA is hybridised to probe sets
  2. The target specific sequences are located directly adjacent to one another, therefore when the probes bind to the DNA they can be joined together using a ligase
  3. This generates a contiguous probe flanked by universal primers.
  4. All ligated probes can then be amplified in a single PCR reaction using the same universal PCR primers.
  5. It is therefore the probes and not the target sequences that are amplified.
  6. Unbound probes will not be amplified, because they only contain one primer sequence.
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21
Q

How are MLPA probes designed?

A

Each probe set consists of 2 halves.

    • 1st half consists of target specific sequence (20-30 nucleotides) (blue) flanked by a universal primer (black).
    • 2nd half consists of target specific sequence (25-43 nucleotides) (blue) flanked by a universal primer (black) BUT in-between is a random fragment of between 19-370 nucleotides, this is known as the stuffer sequence (green).

The stuffer sequence is a different length for each probe pair and allows for the generation of different sized products for electrophoretic resolution.

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22
Q

How does the MLPA result give information about copy number?

A
  • The amount of ligated probe produced is proportional to the copy number of the target region.
  • Following PCR amplification, comparing the relative peak heights to a control sample can indicate changes in copy number.
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23
Q

What are the 6 main stages in the MLPA reaction protocol?

A
  1. Denaturation of the genomic DNA
  2. Hybridisation of the probes to the sample.
  3. Ligation of the probes
  4. PCR amplification of the ligated probes using universal primers
  5. Separation of the amplified products by capillary electrophoresis
  6. Analysis and quantification
24
Q

What control samples should be run in each MLPA reaction and why?

A

For each reaction;

  1. reference samples: should be included to normalise against test samples thus enabling detection of copy number changes in the test samples
  2. Negative (normal) control and positive controls and a “blank” to aid in interpretation and troubleshooting
25
Q

How are MLPA results interpreted?

A
  1. After capillary electrophoresis the quality of the data should be assessed.
  2. The ratios for each probe calculated using data analysis software.
  3. The ratios are generated by comparing a patient and normal control DNA.
26
Q

What are the expected ratios hen analysing MLPA data?

How are abnormal MLPA results confirmed?

A

Ratio;

0.5 (range 0.3-0.7) Heterozygous deletion

1 (range 0.7-1.3) Normal

1.5 (range 1.3-1.7) Heterozygous duplication

Abnormal results should be confirmed by repeating or by another method.

27
Q

What other variations of MLPA exisit for detection copy number?

A
  1. MS-MLPA: CN and methylation status
  2. RT_MLPA: Reverse transcriptase MLPA is used for mRNA expression profiling
28
Q

What are the four main categories of copy number detection with NGS?

A
  1. Read-Pair
  2. Split-Read
  3. Read-Depth
  4. Assembly Based
29
Q

What is the key consideration to be aware of when assessing copy number chnage with NGS data?

A

Many tools have been developed to enable discovery of CNVs from NGS data.

Each of these tools have different strengths and weaknesses in their applicability and suitability for NGS data, and no single tool is capable of identifying the full range of DNA variation.

30
Q

What is the limitations of copy number analysis with NGS data?

A
  • Limited to the length of the reads and NGS data shorter than 1 kb affect the accuracy and precision
  • Reliable only in the unique regions of the genome
  • Accuracy of Read-Pair methods is largely dependent on the insert size
  • small events can be missed with large-insert libraries, insertions larger than the library insert size might be ignored
  • Some methods have poor performance in regions enriched with duplications andrepetitive regions
31
Q

What is real-time PCR (qPCR) and how is it different to standard or QF-PCR?

A
  1. This kinetic reaction enables simultaneous amplification, detection and continuous quantification of DNA templates during each PCR cycle.
  2. In conventional PCR, you see the result of amplification only after the PCR is complete (end-point detection) using agarose gel electrophoresis and staining. The process is time consuming and is not reliably quantitative (size-based discrimination).
  3. With qPCR, amplification of DNA with PCR is monitored in real time (cyclers constantly scan the amplification products).
32
Q

What is the principle underpinning qPCR?

A
  • Accurate data is obtained by quantifying products generated during the exponential phase of the reaction.
  • This is when the reaction is at maximum efficiency and the products are doubling with every cycle.
  • At this stage the amplified product is directly proportional to the amount of template prior to the start of the reaction.
33
Q

What other key considerations are there with qPCR assays?

A
  1. PCR efficiency should be 90 - 100% otherwise quantification is underestimated.
  2. Fluorescence is used as it is a measurable signal directly proportional to amount of amplified product. Thus, specialist equipment is needed: fluorescence-detecting thermal cycler.
  3. A reference gene is used to internally calibrate gene expression of a transcript of interest.
34
Q

What two common methods are used for detection of products in RQ-PCR?

A
  1. Non-specific fluorescent dyes that intercalate with any dsDNA.
  2. Sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter: hydrolysis/ hybridisation/ hairpin:
35
Q

Give an example of a non-specific fluorescent dye used in qPCR reactions?

A
  • SYBR Green I dye
    • Small molecules that when free in solution show very little florescence, but when bound to the minor groove of dsDNA its fluorescence increases by >1000-fold.
    • Detects PCR products as they accumulate during PCR cycles.
    • Indiscriminately bind any dsDNA e.g. primer-dimers etc
36
Q

Give 3 examples of a sequence-specific fluorescent dyes used in qPCR reactions?

A
  1. Taqman® hydrolysis probes
  2. FRET hybridisation probes (Roche LightCycler)
  3. Molecular Beacon hairpin probes
37
Q

What is a Taqman hydrolysis probe?

A
  • Flurophore reporter (e.g. FAM) at the 5’ end and a quencher (e.g. TAMRA) at the 3’ end.
  • The quenching mechanism is based on fluorescence resonance energy transfer (FRET).
  • Advancing Taq polymerase starts to displace nucleotides of the probe by 5’–3’ exonuclease activity à cleavage of the flurophore from the quencher resulting in emission.
38
Q

What is a FRET hybridisation probe?

A
  • Pair of fluorescent probes (one labelled at the 3’ and the other at the 5’ end). When bound these probes are end-to-end allowing a transfer of energy from one fluorophore to the other, a FRET-based increase in fluorescence is emitted.
  • Once PCR extension occurs the donor probe is cleaved and therefore unable to transmit energy to the acceptor probe and emission is lost.
  • This method allows detection during the annealing or extension phase. Can perform melt curve analysis - main advantage over hydrolysis probes.
39
Q

What is a Molecular Beacon hairpin probe?

A
  • Flurophore at the 5’ end and a quencher at the 3’ end. Complementary ends mean they form a hairpin structure. In the presence of a complementary target sequence the probe unfolds and hybridises to the target causing the fluorophore to be displaced from the quencher.
  • Scorpion is another hairpin but combines the primer and probe in same molecule: primers contain an attached fluorescently-labelled tail that hybridises to an amplified target. Method is beneficial for rapid assays with short equilibration times.
40
Q

What are the pro’s of Non-specific intercalating agents used in qPCR?

A
  1. can monitors the amplification of any dsDNA sequence
  2. no probe required, reduces assay setup and running costs
  3. simple and can be used for any reaction
41
Q

What are the con’s of Non-specific intercalating agents used in qPCR?

A
  1. specificity and accuracy - SYBR Green binds to any dsDNA (target or non-target) leading to false positive signals but this may be overcome with melting curve analysis
  2. problems with primer dimers
  3. multiplex assays less achievable
  4. melting curve analysis necessary
42
Q

What are the pro’s of Sequence-specific probes used in qPCR?

A
  1. specific hybridisation between probe and target is required to generate fluorescent signal
  2. probes can be labelled with differential, reporter dyes to allow amplification of two distinct sequences in one reaction tube
  3. detection capable down to a 2-fold change
  4. small amplicon size results in increased amplification efficiency (even with degraded DNA)
  5. post-PCR processing is eliminated, this reduces assay labour and material costs
43
Q

What are the con’s of Sequence-specific probes used in qPCR?

A
  • multiple reactions required to examine multiple loci
  • different probes must be synthesised for each unique target sequence
  • probes are expensive
44
Q

Atwhat stage in the qpCR reaction does quantification take place on why?

A

Can only take place accurately in the exponential or log linear phase of the reaction.

At the beginning of the exponential phase all reagents are still in excess, the DNA polymerase is still highly efficient, and the amplification product (which is present in a low amount) will not compete with the primers’ annealing capabilities, thus contributing to more accurate data.

45
Q

What is the cycle threshold (Ct) value?

A
  • Quantification occurs at the cycle threshold (Ct)
  • This is the number of cycles required for the fluorescent signal to be detected above the background/baseline level,
  • After which an exponential increase is seen.
46
Q

How is the Ct value used and interpreted for DNA quantification?

A
  • Ct values are inversely proportional to amount of template in sample. The lower the Ct value the greater amount of template. Therefore Ct values are used to quantify initial amount of template in reaction.
  • The higher the concentration of starting material the fewer cycles required to reach the Ct threshold.
  • Other factors impact Ct values besides template concentration, e.g. PCR efficiency, artefacts from the reaction mix. Therefore Ct values from PCR reactions run under different conditions or with different reagents cannot be compared directly.
47
Q

What are the ‘baseline’ and ‘threshold’ values in qPCR?

A
  1. Background (or baseline) = signal level during the initial cycles of PCR, in which there is little change in fluorescent signal i.e. the background, or ‘noise’ of the reaction.
  2. Threshold = level of signal that reflects a statistically significant increase over the calculated baseline signal, set to distinguish relevant amplification from the background.
48
Q

Describe the graphical output from a qPCR reaction.

A

ΔRn is reporter signal minus the baseline level, this is plotted against cycle number.

49
Q

What are the two main types of quantification made possible by qPCR?

A
  1. Absolute quantification (standard curve analysis)
  2. Relative quantification
50
Q

Describe qPCR absolute quantification in more detail.

A
  • Relates the PCR signal to input copy number using a calibration curve created by Ct values for a set of dilution standards of known concentration.
  • The test sample Ct is plotted against the log of the standard concentration and the standard curve can be used to determine its concentration.
  • The reliability of this method depends on the condition of identical amplification efficiencies between standards and test sample.
  • Useful for correlating viral copy number with disease state or fusion gene transcripts after treatment in leukaemia.
51
Q

Describe qPCR relative quantification in more detail.

A
  • Based on internal reference/housekeeping genes to determine fold-differences in expression levels of the target gene.
  • Considered easier to perform than absolute quantification as a calibration curve is not necessary and this also eliminates any dilution errors made creating the curve.
  • However the efficiency of the target and the reference PCR must be equal.
  • The most common method is the 2-∆∆Ct method (particularly useful in gene expression analysis comparing normal and tumourigenic cells).
52
Q

Describe how qPCR is used for counting bacterial, viral, or fungal loads.

A
  • RQ-PCR is useful for determining the presence and quantity of pathogen-specific sequences within a sample and is usually quicker than culturing.
  • Within Clinical Microbiology it can measure disease progression and efficacy of antiviral therapies.
53
Q

Describe how qPCR is used for monitoring of neoplastic disorders .

A
  • RQ-PCR (commonly Taqman) is used for monitoring disease-specific prognostic markers of minimal residual disease (MRD) as it is more sensitive than traditional cytogenetic techniques.
  • RNA is first copied to cDNA by RT-PCR and primers are located either side of the breakpoint:
  • CML – BCR-ABL1 transcript
  • ALL – BCR-ABL1, MLL-AF4, TCF3(E2A)-PBX1, and ETV6-RUNX1)
  • AML - PML-RARA, RUNX1-RUNXT1 and CBFB-MYH11
    *
54
Q

Describe how qPCR is used for Single base mutation detection.

A
  • RQ-PCR can be used to detect single base pair changes.
  • This can be utilised to detect the presence of a known mutation, e.g. to perform a quenched FRET based assay where the decrease in fluorescence of the donor fluorophore is measured each cycle.
  • Following the RQ-PCR, melt curve analysis is performed to distinguish between wild-type and mutant alleles.
55
Q

Describe how qPCR is used for SNP Genotyping.

A

This can be achieved by labelling two probes with different fluorophores, a probe specific for each allele and comparing the fluorescence.

56
Q

Describe how qPCR is used for Copy Number Measurement .

A
  • To confirm copy number changes identified by microarray as an alternative to FISH.
  • Although this technique will not provide positional information, it is useful in confirming small imbalances;
  • This is becoming increasingly important as microarray resolution increases beyond the sensitivity of FISH confirmation
57
Q

Describe how qPCR is used for Non-invasive prenatal diagnosis.

A
  • The most popular technique used for the detection and identification of specific cffDNA sequences is currently RQ-PCR using sequence-specific DNA probes e.g. foetal sexing using Y chromosome-specific probes.
  • This is because it combines high sensitivity with a closed detection system, thereby minimising the risk of contamination.
  • However, droplet digital PCR (ddPCR) may supersede RQ-PCR for this application due to greater sensitivity and precision.
  • The use of ddPCR also allows absolute quantification without the need for standards or endogenous controls.

6. Methods (18 decks)

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