Molecular Techniques Flashcards
(44 cards)
1
Q
What types of specimens are
required for cytogenetic karyotypes ?
A
- live cells
2
Q
What types of specimens can be used
for FISH testing ?
A
- FFPE or air dried aspirate smears
- Inhibited by:
- fixatives containing heavy metals (zinc formalin, B5, Zenker’s)
- standard decalcification
3
Q
What is the process for karyotype analysis ?
A
- a mitotic inhibitor is added to live cells (Colcemid)
- this allows nuclei to be arrested in Metaphase
- then to visualize chromosomes different banding stains are used
- most common Giemsa or G-banding
4
Q
What is the benefit of chromosome analysis?
A
- direct complete visualization of chromosomes
- useful in the detection of:
- translocations
- deletions
- duplications
- aneuploidy
- unsual chromosome formations (ring chromosome)
5
Q
When is something clonal by karyotype ?
A
- need 2 metaphases with the same structural aberration
- OR
- 3 metaphases in the case of single chromosome losses
IMP: remember that only 20 metaphases are analyzed so it is difficult to assess fo low levels of disease with this method.
6
Q
What is another limitation of
cytogenetics/karyotype analysis?
A
- not sensitive in detecting neoplastic cells in disorders which the abnormal cells do not grow in culture or do not enter into metaphase
- ex: Multiple Myeloma and Hodgkin Lymphoma
7
Q
When is karyotype analysis performed ?
A
- it is performed during most bone marrow evaluations for hematopoietic disorders
- karyotype analysis of extra medullary lymphomas is debateable because they often have complex karyotypes
- plus their molecular alterations can be detected by FISH
8
Q
What rearrangements are known to be
difficult to evaluate by cytogenetics ?
A
- inv(16)
- frequently cryptic (below the level of resolution or exchange of regions with similar banding patterns)
- ETV6-RUNX1
- FIP1L1-PDGFRA
- frequently cryptic (below the level of resolution or exchange of regions with similar banding patterns)
IMP: these alterations can be detected by FISH
Note: poor sample preparation can further hinder the identification of these changes
9
Q
What does interphase FISH analysis
look for typically ?
A
- looks for specific chromosomal aberrations
- including those that are too small to detect by karyotype
10
Q
What types of alterations can
interphase FISH detect ?
A
- translocations
- copy number changes (gains or losses)
11
Q
How many cells are usually analyzed by FISH
and what can be said about the sensitivity ?
A
- 200 nuclei
- higher false positive rate (3-4% of nuclei)
- only slightly more sensitive than karyotype for detection of specific aberrations
12
Q
What types of aberrations is FISH
less sensitive in picking up ?
A
- numerical changes, particularly chromosome losses
- this is because of sectioning of cells
- metaphse FISH can be used to confirm the changes
13
Q
What is the basis of chromosomal microarray
and what are the advantages and disadvantages ?
A
- more specialized analysis of chromosomes by using probes that span the length of the whole genome
- Advantage
- rapid detection of copy number changes
- detection of loss of heterozygosity
- Disadvantage
- cant detect translocations because you are not visualizing the chromosomes
14
Q
What are the two types of chromsomal
microarray available ?
A
- microarray-based comparative genomic hybridization
- single nucleotide polymorphism or SNP
15
Q
How is comparative genomic hybridization
performed ?
A
- the patient sample is compared with a control after both are hybridized to the same microarray
16
Q
How is single nucleotide polymorphism evaluated ?
A
- compare the patient sample to a database of controls
- this allows for the detection of single-nucleotide changes including a loss of heterozygosity
17
Q
What is a major down side of chromosomal
microarray ?
A
- because all genomic material is analyzed at once, clonal evolution or multiple clonal popoulations are difficult to analyze
18
Q
What is the typical TAT, pros and cons of
karyotyping ?
A
- TAT: 4-10 days
- Pros
- full genome is visible
- Cons
- low resolution
- usually slower TAT
19
Q
What is the typical TAT, pros and cons of
FISH ?
A
- TAT: 24-72 hours
- Pros
- high resolution
- Fast TAT
- Cons
- only 1 or 2 regions per hybridization
20
Q
What is the TAT, pros and cons of
microarray ?
A
- TAT: 2-5 days
- can use DNA from any source
- Pros
- very high resolution
- full genome
- fast TAT
- Cons
- does not detect balanced translocations
- may not detect tetrasomy
21
Q
What types of specimens are
acceptable for molecular testing
using DNA-based assays?
A
- fresh or frozen peripherl blood, bone marrow or tissue samples
- FFPE
- air-dried aspirate preparations
22
Q
What is the anticoagulant of
choice when submitting for molecular samples ?
A
- IMP: heparin can inhibit PCR reactions
- collect specimens in EDTA or acid citrate tubes
- also cannot used decalcified materials because the nucleic acids are damaged in the acidification process
23
Q
What is the acceptable specimen for
RNA samples ?
A
- must use fresh samples
- < 72 hours from time of collection
- this will prevent significant RNA degradation
24
Q
What is the basis of PCR techniques ?
A
- it involves in vitro DNA duplication
- results in exponential numbers of DNA copies from limited amounts of starting material
25
What are the acceptable specimens
used for PCR?
* tissues
* fresh, frozen, or FFPE
* RNA
* reverse transcription (RNA sequence of interest) is converted to complimentary DNA (cDNA)
* uses an RNA-dependent DNA polymerase
* usually derived from a retrovirus
26
See pg. 151 for review
of PCR technnique
27
What is a major advantage and potential
pitfall of PCR ?
* the large of number of copies allow for good sensitivity
* potential false positives may be present if there is cross contamination from another sample
28
What are the clinical uses
of PCR ?
* hematologic malignancy variant detection
* assessment of B and T cell clonality
* detection of fusion products of specific chromosomal translocations and rearrangements
* NGS
29
How does allele specific
PCR work ?
* primers bind to variant or nonvariant sequences
* if the primers are mutation specific, a PCR prodcut will be generated only in the presence of that particular mutation
30
What is the principle of
RT-PCR ?
* RT-PCR = reverse transcriptase
* random primers convert RNA sequences into cDNA
* once cDNA is formed, amplification is performed with primers specific to that amplification
31
When is RT-PCR used in Hemepath ?
* amplify translciations and fusions with pimers specific to each partner
* ex: BCR-ABL
32
What is the advantage of using
cDNA in RT-PCR ?
* using cDNA avoids the amplification of large intronic regions present at most translocation breakpoints in genomic DNA
IMP: you can use RT-PCR for gene expression profiling as well
33
What does quantitative PCR (qPCR)
tell you ?
* enables quantitation of amplified DNA as it is produced during each PCR cycle
* this method is stometimes called real-time PCR
* terminology is not favored though, prefer to call it qPCR
34
How does qPCR measure the
quantity of DNA ?
* measures the quantity of DNA via the incorporation of fluorescent dyes (SYBR Green)
* these bind to dsDNA
* only allows for detection of completed PCR products
* alternatively could also use a fluorescent hydrolysis probe (Taqman)
* as DNA amplification occurs, Taq polymerase causes hydrolysis of the probes
* the products are quantified based on the cycle number at which the fluorescence intensity is detected above the background
35
qPCR is often used for MRD
quantification, how is that done ?
* cycle threshold (Taq PCR of fluorescent)
* compared patient specimen to standard plotted curve
* has a sensitivity of
* 1X10^-4 to 1x10^-6
* often used for things such as
* BCR-ABL1, CBFB-MYH11, PML-RARA
36
What is the major advantage of
droplet PCR ?
* p. 152 review for technique
* advantages:
* accurate quantitation of nucleic acids in samples with low cellularity/tumor burden without the need of a standard curve
* high tolerannce to difference PCR inhibitors
* ability to detect rare mutations because the PCR effectively concentrates the target of interest in such low volumes
* should use for common alterations or recurrent translocations
37
How is sanger sequencing performed ?
* one of the first molecular methods
* labeled ddNTP lacking a 3' hydroxyl group gets randomly incorporated into the growing strand
* thus the PCR is unable to continue
* generates fragems of a distinct length
* the fragments encompass the region of interest
* then the sequance is deduced based on where the ddNTPs were incorporated
* fragment sizes are run on capillary electrophoresis
38
What are the benefits of sanger sequencing
as well as the disadvantages ?
* inexpensive and rapid
* relatively low sensitivity
* need 30% of cells to have a heterozygous mutation before it is even detected
Note: now largely replaced by NGS
39
Review NGS technique p. 153
40
What sort of molecular alterations
can NGS detect ?
* depends on the primer and assay design:
* point mutations
* small insertions
* deletions (indels)
* structural rearrangements
* copy number variants
* MRD
41
What is the VAF in NGS ?
* VAF = variant allele frequency
* it is the proportion of the variant sequence in relation to the total number of reads generated for a particular gene region
* typically approximates the porportion of variant DNA
42
What is a VAF of a mutation that is germline
or heterozygous somatic ?
43
What is typically the limit of detection in
NGS ?
* 5% for a variant allele out of ~2,000 reads
44
See table on p.154....
