Molecular

Question 1

How is RNA extraction performed?

Answer 1

1) Ficoll separation and centrifugation is performed to separate the white blood cells.

2) TRIzol is added. This contains:
- GITC (chaotrophic salt) that lyses the cell membrane and denatures DNAses and RNAses.
- Phenol: partitions the sample into aqueous and organic phases
- Chloroform: potentiates the activity of phenol

3) Under acidic conditions, RNA remains in the aqueous phase, DNA and other proteins in the organic phase.

Question 2

How is automated DNA extraction performed?

Answer 2

1) The sample material is placed into the wells of the Sample Cartridge.
2) The samples are then lysed by incubation with a special buffer containing chaotropic salts and proteinase K.
3) Magnetic glass particles are added and the DNA is bound to their surfaces.
4) Unbound substances are removed by several washing steps.
5) Purified DNA is eluted.

Question 3

What are some of the benefits of using RNA rather than DNA when performing PCR?

Answer 3

• mRNA does not contain introns: shorter segment to be transcribed than the corresponding DNA. This is particularly relevant in the case of gene translocations where the translocations occurs over a region of DNA that is often too large to allow direct amplification of that DNA by PCR with a common set of primers.
• A fusion gene can be more readily demonstrated by RT-PCR because, while the intronic-intronic breakpoints are usually highly variable, the exon-exon breakpoints are often highly consistent and thus amenable to amplification using a common pair of PCR primers.
• Quantification of RNA provides an important measure of gene expression which is not only useful for diagnosis but also disease monitoring.

cDNA needs to be made from RNA prior to performing PCR

Question 4

What are the steps of PCR?

Answer 4

1) Reaction temperature raised to 95-98deg for 10-60 seconds to denature the target DNA
2) The temperature is reduced to 50-70deg for 10-60s to enable hybridisation/ annealing of the primers to the target DNA sequence
3) The temperature is increased to 65-75deg and DNA polymerase (generally the heat stable Taq polymerase) forms a complementary strand of DNA from the primer. Each complementary strand generated is termed an amplicon.
4) The process is repeated and an exponential amount of target DNA is formed. By 32 cycle > 1 billion copies are synthesised.

Question 5

What controls should be used in every PCR reaction

Answer 5

• Positive control
• Negative control
• Non-template control

If RNA was the initial nucleic acid isolated for PCR, cDNA is run on electrophoresis prior to PCR to ensure cDNA has been made.

Question 6

What is restriction enzyme digestion?

Answer 6

Restriction enzymes (RE’s) cleave DNA at short, specific sequences.

Point mutations can coincidentally create or destroy an RE recognition sequence.

Question 7

What is allele specific PCR?

Answer 7

In allele specific PCR, the primers overlap the area of interest.

Takes advantage of the fact that a mismatch between the allele and the primer at the 3’ end will prevent PCR amplification.

Question 8

What is multiplex PCR?

Answer 8

• Technique used for the amplification of several discrete genetic loci with multiple PCR primer pairs in a single reaction.
• This can enable the amplification of multiple genes of interest or simply the wild-type and mutant gene to be synthesised on the same PCR run.
• The primers must have similar Tms to enable efficient and sensitive PCR to be performed.
• The generated DNA sequences must also have different sizes to enable the differentiation on gel electrophoresis.

Question 9

What is Gap PCR?

Answer 9

• Large deletions are detected by Gap-PCR.
• Specific primers are designed to flank a known deletion. The principle of gap-PCR is based upon the inability of the primers to generate a PCR product unless a deletion joins the flanking sequences together. - If a deletion is present, PCR amplification will occur and the product is examined by electrophoresis. If the deletion is absent, the primers cannot bind.

Question 10

How does the Taq Man probe work?

Answer 10

• This probe is complementary to a non-primer internal sequence of the PCR product.
• A donor dye is present at the 5’ end and a quencher at the 3’ end. When the probe is intact, the quencher prevents the fluorescent signal from being released.
• As DNA polymerase replicates and moves along a strand of DNA with TaqMan bound, it cleaves off the dye separating it from the quencher and the fluorescence can be measured in real time.

Question 11

What is high resolution melt curve analysis?

Answer 11

• Takes advantage of the principle that different DNA sequences will melt at different temperatures.
• This enables wild type, heterozygous and homozygous mutations to be differentiated.
• In this method, PCR is performed in the presence of a dsDNA intercalating dye.
• After amplification, the temperature is decreased so that dsDNA can be formed. The temperature is then gradually increased while the fluorescence is monitored.

Question 12

What is fragment analysis?

Answer 12

• A labelled primer is used. This generates a fluorescently labelled PCR product.
• The PCR product is then mixed with a size standard that is labelled with a different fluorochrome than the amplified sequence
• The sample is then denatured using a thermocycler then placed in the sequencer for analysis.
• The genetic fragments get separated by size as they migrate through the capillary. Each labelled fragment is detected by the system camera.
• Based on the dye used and the fluorescent signal produced, a peak is generated.
• The peaks are then analysed using software.
• A sizing curve is generated using the sizes of each known DNA fragment in the size standard and their respective migration times.
• The size of the unknown fragment is derived off the sizing curve.

Question 13

What is digital droplet PCR?

Answer 13

• Method of performing digital PCR that is based on water-oil emulsion droplet technology.
• A sample is diluted and fractionated into 20,000 droplets, and PCR amplification of the template molecules occurs in each individual droplet.
• This enables single template molecules to be amplified individually and the products detected with fluorescent probes.
• Can be used to determine absolute template quantity. Can be used for MRD monitoring.
• Doesn’t require generation of a standard curve.

Question 14

How is sanger sequencing performed?

Answer 14

• PCR performed using ddNTPs (which do not have a free 3’ OH end) mixed with sNTPs
• The ddNTPs contain different fluorochromes for different bases
• When the DNA polymerase incorporated a ddNTP , it wont be able to add any further nucleotides. This results in PCR products of varied lengths
• The PCR product is then separated by capillary electrophoresis.
• A laser excites the dye labelled DNA fragments as they pass through a tiny window at the end of the capillary. The excited dye emits a light at a characteristic wavelength that is detected by a light sensor.
• Software can then interpret the detected signal and translate it into a base and generate the sequence of base pairs in the amplified region of DNA.

Question 15

What are the steps of next generation sequencing?

Answer 15

1) DNA sequencing library generation
- DNA fragmented
- Genes of interested are selected (Hybridisation-capture-based target enrichment or PCR amplification)
- Fragmented ends of DNA repaired with an A overhang
- Adapters added

2) Cluster generation
- Target DNA attaches to the flow cell.
- Bridge amplification occurs and clusters are generated
- Sequencing primer anneals

3) Sequencing by synthesis
- Each base has a 3’ chemical block which prevents further bases from being added.
- The first base is added.
- The fluorescence of the base is excited and detected by the optics of the sequencing instrument.
- The base is then unblocked and the fluor cleaved
- The next base is added and the same process is performed until synthesis of the target strand of DNA is complete.

4) Interpretation
- Complex bioinformatics software, matched to a reference genome.

Question 16

How is TCR and Ig gene rearrangement analysis performed at ADHB?

Answer 16

PCR with heteroduplex analysis

o Multiplexed reactions using consensus primers to conserved sequences within the V, D or J gene segments.
o PCR products denatured by heat followed by rapid random renaturation at low temperature. The products are then separated by electrophoresis on polyacrylamide gel according to their length and composition.
o In samples consisting of polyclonal lymphoid populations, the forced duplex formation caused by rapid denaturation and renaturation results mainly in heteroduplexes which gives rise to a background smear of slowly migrating fragments upon electrophoresis.
o Clonal cells give rise to one or two discrete bands.

Question 17

What are the steps of performing conventional cytogenetics?

Answer 17

1) Cells may be stimulated to divide by adding mitogens.
○ Some cells may divide spontaneously.

2) Cells cultured in medium (RPMI) with foetal calf serum.
3) Arrested in metaphase by the addition of colcemid.
4) Fixed in methanol/ glacial acetic acid.
5) Dropped onto clean glass slides and are air dried.
6) Slides aged, put through a weak trypsin solution, stained with Giemsa, cover-slipped and examined under the microscope.
7) Metaphases photographed or captured via an image system and karyotyped.

Question 18

How is FISH performed?

Answer 18

• Fluorescently labelled probes are hybridised to non-dividing cells.
- Need to denature the DNA within the cells to get single stranded DNA to enable to probe to anneal and hybridise to the DNA sequence of interest.

• Signals are visualised using a fluorescent microscope.

Question 19

How is FISH performed in myeloma?

Answer 19

• Malignant plasma cells are very hard to culture in the lab for cytogenetic and FISH analysis.

• When FISH is performed, a plasma cell enrichment strategy must be used to ensure detection of the malignant cell population in plasma cell myeloma.
○ A locus specific FISH probe is hybridised to the cells overnight.
○ The cytoplasm of the plasma cells is labelled by incubation with fluorescently labelled antibody directed against the kappa &/or lambda light chains.

Question 20

How is microarray used in Haematology?

Answer 20

○ Can be used to detect sub-microscopic DNA abnormalities that are not readily detectable by conventional karyotype and/or FISH).
○ Detects loss and/or gain of DNA, and copy neutral loss of heterozygosity (CNLOH).
○ Duplications and deletions of oncogenes such as CDKN2A, ETV6, RUNX1, cMYC are detectable by microarray technology at a greater resolution than conventional techniques.
○ Many (i.e. 526) cancer genes targeted with a resolution 2000-4000 DNA base-pairs (2-4 kb).
○ The high resolution and genome-wide coverage combined with minimal DNA requirements, and relatively short turnaround time allows chromosomal microarray analysis to be advantageous for use in a clinical setting.
○ The test is generally regarded as a complementary investigation

Question 21

What does microarray detect?

Answer 21

• Copy number variants

- Regions of homozygosity

Question 22

What does microarray not detect?

Answer 22

○ Balanced chromosomal rearrangements
○ Small copy number variants (<100kb)
○ Low level mosaicism (<20%)
○ Uniparental heterodisomy
○ Single nucleotide variants

Question 23

What are short tandem repeats?

Answer 23

• Short sequences of DNA, normally of length 2-5 base pairs, that are repeated numerous times in a head-tail manner.
• The polymorphisms in STRs are due to the different number of copies of the repeat element that can occur in a population of individuals.

Question 24

How is chimerism analysis performed?

Answer 24

• Commercial multiplex kits designed for forensic purposes are frequently used; offer a high degree of informativity and standardization.
• Contain fluorescently labelled primers for STRs.
• Multiplex PCR reaction is performed then run on a capillary based genetic analyser.
• PCR is performed in the donor and in the host prior to transplantation to determine if there are informative differences
• PCR is then performed post transplant and the informative differences established pre-transplant can be used to determine how much DNA is from the donor and how much is from the host.
• Whole blood, T-cell separated and bone marrow can be used.

Question 25

What are some of the challenges with NGS

Answer 25

1) Determining the significance of mutations- differentiating leukaemia driver mutations from:
- Polymorphisms
- Passenger mutations
- CHIP
- Germline mutations
- PCR, sequencing, post sequencing artefacts

2) Limited sensitivity for MRD assessment
- Mutations detected at diagnosis may be reidentified at at VAF of 1-2%

3) High financial burden
4) Demand on interdisciplinary approach

Question 26

How would you go about setting up a new molecular test in your laboratory?

Answer 26

• Quality assurance is paramount when setting up a new diagnostic assay. This requires engagement with/ from your organisation (department/ section heads, quality department)
  1) Define the clinical question/ objective

2) Design your test so that it answers the clinical objection
- Literature search- any validated tests in place? recommendations for other groups/ official bodies etc…
- What methodology (will depend on the mutation/ aberration that needs to be detected, sensitivity required)
- If PCR- what method of end point detection is most appropriate. What primers should be used? Are probes necessary?
- What controls are needed?

3) Are systems/ equipment already in place to enable use of your new assay?
- Staff levels/ training/ competency
- Regents and analysers (including availability with other already established testing in your lab)
- Pre- post PCR set-up to minimise contamination

4) Draft a validation protocol
- Outlines objective and analytical method
- Defines pre-acceptance criteria
- Perform validation procedure to determine sensitivity (LLoQ, LLoD), specificity, accuracy, precision, CV, measurement uncertainty
- Ensure pre-acceptance criteria have been met
- Often involves testing 20- 30 normals as well as abnormal samples
- Often requires involvement from a reference laboratory

5) Ensure continued QA of your procedure/ test
- Standard operating procedures/ troubleshooting documents
- Staff training and competency
- Performing minimum number of required tests per year to ensure ongoing competence
- Appropriate QC
- Participation in EQC
- Certification/ accreditation.