Copy Number Detection (Molecular) Flashcards

Question

How are MLPA results interpreted?

Answer 1

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.

Answer 2

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.

Answer 3

1. MS-MLPA: CN and methylation status 2. RT\_MLPA: Reverse transcriptase MLPA is used for mRNA expression profiling

Answer 4

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

Answer 5

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.

Answer 6

* 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

Answer 7

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).

Answer 8

* 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.

Answer 9

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.

Answer 10

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:

Answer 11

* 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

Answer 12

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

Answer 13

* 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.

Answer 14

* 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.

Answer 15

* 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.

Answer 16

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

Answer 17

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

Answer 18

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

Answer 19

* multiple reactions required to examine multiple loci * different probes must be synthesised for each unique target sequence * probes are expensive

Answer 20

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.

Answer 21

* 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.

Answer 22

* 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.

Answer 23

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.

Answer 24

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

Answer 25

1. Absolute quantification (standard curve analysis) 2. Relative quantification

Answer 26

* 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.

Answer 27

* 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).

Answer 28

* 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.

Answer 29

* 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 *

Answer 30

* 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.

Answer 31

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

Answer 32

* 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

Answer 33

* 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.

Copy Number Detection (Molecular) Flashcards

(57 cards)