Molecular and Genetics Flashcards
(19 cards)
What are the steps of DNA extraction, what are the different methods, what is one overarching consideration
Steps:
- Cell lysis
- Digest the proteins
- Purify the DNA
Method 1: Liquid phase extraction
- Proteinase K is added to sample to remove organic matter and proteins
- Ethanol (removes salts) and isopropropanol (precipitate) is added -> when centrifuged together with the sample this will create a nucleic acid pellet (or DNA bead)
- Can be washed further with ethanol to further purify
Method 2: Solid phase extraction (faster and safer, more commonly used)
- Cell is lysed
- Then either
–> DNA is adsorbed to silica at a specific pH in the presence of specific salts, OR
–> DNA is reversibly bound to magnetic beads coated in anti-DNA-Ab
- No centrifuge = minimisation of shearing forces that may damage the DNA
Consideration = Keep clean and decontaminated (clean area with bleach, change gloves frequently)
How do you evaluate the DNA concentration and quality after DNA extraction
Nanodrop UV spectrophotometer is used to determine the nucleic acid to protein ratio to determine the purity (absorb different nM of light)
Gel electrophoresis is used to determine DNA integrity - single band of high molecular weight substance if intact
What are the indications for cytogenetics
- Diagnosis
- Clinically urgent cases (PML-RARA)
- WHO genetically defined entities
- Demonstrate clonality - Prognosis
- Entity specific risk stratification - Response to therapy
- Including clonal evolution or cytogenetic remission
Steps in Karyotype preparation
- Amount of particles/viscosity determines how much of specimen is used
- Two cultures are set up in different incubators just in case there is an issue/to give different mitogenic stimulus
- Culture set up is lineage specific but generally always have a 24h and then the 2nd culture (another 24h for ALL vs 96h for plasma cells)
- Synchronisation
- Fluorodeoxyuridine is used to arrest cells in S phase to coordinate them in the same part of the cell cycle
- Then thymidine is added to release
- 6-10 hours after release (depends on cell line), cells will approach metaphase
- Then add mitotic inhibitor like colchicine
Simplified steps of karyotype harvest/dropping
- Add colchicine to stop mitosis
- Add hypotonic solution to lyse the red cells
- Add acetic acid to harden wbcs and preserve chromosomes
- Add Carnoys fixative to preserve the WBCs and centrifuge x3 to firm up the cell membrane (dont want there to be holes as then chromosomes escape)
- Dilute cell pellet to optimal concentration
- Wet the slides a few times to help with spread
- Then drop the cells onto the slide
- Fix and dry
Karyotype banding
G banding with trypsin and giemsa
Trypsin
- Eats away at chromosome to produce light bands (euchromatin)
- Too long = overbanding
Giemsa
- Stains dark areas heterochromatin
Silver staining
- Identifies centromere
Phytohaemaglutinin
- Can be added to peripheral blood chromosomes to stretch the chromatin and produce a higher resolution
Resolution in karyotype
Number of bands you see
Typical resolution is ~400 bands
Single chromosome band is ~150 genes
High resolution banding = cells are arrested in prometaphase
How do you determine a clone on karyotype
Clone = found in 2 or more metaphases except if it is a loss of a whole chromosome in which case it must be found in 3 or more metaphases
Loss of Y chromosome is only considered clonal in older men if it is seen in >85% of cells
What are the benefits of a karyotype
- Genome wide
- Best way to identify balanced translocations (where there is no loss of genentic product)
- Can be used to monitor disease (e.g. additional chromosomal abnormalities as seen in CML disease progression)
- Cheap and safe to perform
- Can be used to identify critical disease associated regions on chromosomes (e.g. ch 13 in myeloma)
- Can identify specific WHO entities
What are the disadvantages of karyotype
- Low resolution, can only detect major structural abnormalities
- Often needs to be performed in conjunction with another test (FISH, array, NGS)
- Interpretation is subjective and dependent on operator experience (though note usually 2 people interpreting 1/2 each)
- Only a small number of cells analysed (20) - very low compared to other methods and low level clones can be missed
Discuss quality checks in karyotyping
Add from notes
Discuss FLT-3 ITD wild type and variants
- Mutation in FLT3 gene on chromosome 13
- Results from internal tandem duplications in exon 14 and 15
- This causes activation of the FLT3 protein
- Wild type FLT3 is 329 base pairs
- If there is an ITD there will be >330 base pairs (3 or a multiple of 3)
- Generally patient will have the wild type and the variant (however this would be extremely rare as all cells would be leukaemic and the mutation would be homozygous)
- Different ITD peaks indicate different AML subclones
Discuss the diagnosis of FLT3-ITD
PCR capillary electrophoresis
- Samples are run twice (two separate PCR reactions and then both are run twice through CE) = 4x replicates
- Controls: positive control, wild type control, NTC
- Fluorescently labelled PCR primers amplify exon 14 and 15 of the FLT3 gene
- Following PCR capillary electrophoresis is used to separate ssDNA by size
- Fluorescence detection is used to detect the target ssDNA
- Every patient will have one peak for wildtype and then a further peak if they have a mutant with the height of the peak indicating the amount of mutant ITD
- Variant must be present in 2/4 replicates
- Then calculate ratio and mutational burden (see notes)
Advantages and disadvantages of capillary electrophoresis in FLT-3 ITD testing
Advantages
- Quick
- Accurate detection of almost all ITD mutations regardless of size (whereas NGS can detect smaller but usually not larger ITDs)
- Generally perform 2-4 replicated thereby having a high standard of quality/confidence
Disadvantages
- Level of detection of 5%
- Can be affected by “Bleed through” and “stutter”
- DNA based assay so quantification is not as sensitive as other assays
Bleed through = if one capillary is heavily positive this can affect samples on either side. But this can be detected by an experienced analyst
Stutter = small peaks occur when there should be no peak - this is from bleed through, poor CE protocol or excessive PCR sample loading
NPM1 mutation in AML (general)
- Account for ~1/3 of adult AML
- Most cases is due to a net 4 base pair insertion in exon 12 of NPM1
- 3 most common mutations account for 90% of cases
- A (75%) = Duplication of preceding 4 bases TCTG
- B (15%) = Insertion of CATG
- C (5%) = Insertion of CCTG
- Associated with a normal karyotype
- AML defining
- Morphology: Cup shaped myeloblasts, multi-lineage dysplasia in 25%
- Prognosis = Favourable. If with FLT3-ITD intermediate.
NPM1 mutation in AML (diagnosis)
Screening (determine presence)
- Capillary electrophoresis (cant determine type), OR
- High resolution melt curve analysis, OR
- Myeloid NGS
Subtype determination
- Sanger sequencing, OR
- NGS
MRD
- Reverse transcriptase PCR (either standard RT-qPCR or GeneXpert)
HRM sensitive down to 5%
NGS sensitive down to 1
CE sensitive down to 2%
FLT3-TKD mutations (general)
Mutations occur in the TKD2 domain
90% of mutations are at the D835 site
FLT3-TKD mutations (diagnosis)
- Restriction enzyme (EcoRV) is used to cut the sequence at D835 site
- Digested length of TKD wild type should be 80bp if unmutated
- If D835 is mutated the restriction enzyme fails and the expected shorter fragment is not seen
Significance of CEBPA mutations
Only in frame mutations affecting the basic leucine zipper (bZIP) region of CEBPA (monoallelic or biallelic) have been associated with favourable outcome
