L7&8 Testing and Technology Flashcards
What kind of drug label is LUMAKRAS?
one gene - multiple alleles
- The drug only targets KRAS with mutated G12C allele
What is an example of one gene-multiple drugs?
PDE-1 inhibitors
- PDE-1 is one gene, but there are multiple drugs to target it.
What mutation can LUMAKRAS be used for?
KRAS G12C mutation
- NOTHING ELSE
What is the general procedure for PGx testing?
- Sample collection
- Sent to lab
- Genetic counseling
- Pharmacy works with doctors to document and make decisions regarding results
Who can perform PGx tests?
CLIA certified labs
- clinical laboratory improvement amendments (CLIA) of 1988
find via GTR: genetic testing registry
How can you collect enough information to prescribe a PGx test?
- work closely with therapeutic team
- discuss the patient
- Understand FDA labeling/CPIC guidelines
- know the principles of technologies
How can you make an informed decision for the prescription of PGx test?
- strength of PGx info vs other factors
- cost vs benefit
- selection of technologies
Do all FDA-approved drugs require PGx testing?
No
Which population may CYP2C9*5-11 be more important for?
African descendants
What is the target for samples for PGx testing?
DNA
any nucleated cells/tissue contains germline DNA
What is the gold standard for PGx samples?
Peripheral blood
Have to use white blood cells to get DNA though
What are the characteristics of using white blood cells for PGx testing?
- 2-6 mL as standard amount
- prefer EDTA-anticoagulant tube
- Use sterile technique to prevent contamination
- room temp 1-2 day delivery
Can we get DNA from red blood cells?
NO
What are the disadvantages of using cheek swab/saliva for DNA sample?
- less DNA yield
- possibly contaminated
- poorer quality
rinse your mouth before hand!
What are the characteristics of tissue sample (fresh biopsy) for DNA testing?
- high yield of DNA
- -80 C for long term storage
- Dry ice for transportation
easily goes bad!
What are the characteristics of tissue sample (formalin fixed and paraffin embedded) for DNA testing?
FFPE
- DNA is usually degraded
- detections are still doable
less preferred but more widely available
What was the first FDA approved broad companion diagnostic for all solid tumors?
- FoundationONE CDx
- FFPE testing
Which is more stable (DNA or RNA)?
DNA
- DNA is very stable
What are factors that affect DNA quality?
- neutral pH, avoid oxidants, UV
- repeated freezing and thawing
- bacterial contamination
- 4 C for short term storage (1-2 months)
- -80 C for long term storage (years)
- Aliquot into small volume if possible
What temperature should DNA be stored at for short term (1-2 months)?
4 C
What temperature should DNA be stored at for long term storage? (years)
-80 C
What are the two kinds of sequencing methods?
- Sanger sequencing
- high-throughput sequencing (whole exome/next gen)
What is the fundamental technique for DNA amplification?
PCR
What are the two goals of testing?
- testing known variants
- testing both known and unknown alleles
What are the two critical steps in DNA amplification?
- target DNA amplification
- allele discrimination
What does PCR stand for?
Polymerase chain reaction
most useful technique for DNA amplification
What are the required substrates for PCR?
- DNA template
- dNTPs (dATP, dGTP, dCTP, dTTP)
- Primers
- Buffer: pH, Mg2+
- Enzyme: Taq DNA polymerase
What are primers for PCR?
2 short sequences specific to the region of interest
What is the difference between dNTP and ddNTP?
- dNTPs are nucleotides that are the building blocks of DNA
- ddNTPs are nucleotides used in the Sanger sequencing method
Why did 3 water baths used to be needed?
- First bath needs to be 98 C for denaturation to separate the DNA strands
- Second bath is somewhere between 48-72 C for annealing. This allows primers to base pair to complementary DNA template.
- Third bath is 68-72 C for extension. Polymerase extends primer to form nascent DNA strand.
How may copies are made in PCR?
- From 2 copies to 2^(n+1) copies
- n = number of thermal cycles
How does PCR amplify DNA?
PCR amplifies DNA from both DNA molecules of homologous chromosomes
What technology should be selected for detecting known SNPs or targeted SNPs?
- DNA chip!
- it is high throughput (up to 5M at a time)
- it can ONLY detect known SNPs
What CYPs does the Amplichip CYP450 Array cover?
- CYP2D6
- CYP2C19
What are features of Sanger Sequencing?
- It is conventional sequencing
- Low throughput (higher cost per base pair)
- can detect both known and unknown alleles, SNPs, indel, small CNV
- Targeting sequencing: sequencing one specific DNA fragment
- Labor intensive
What are features of next gen sequencing?
- High throughput
- Low cost per SNP
- Parallel sequencing
- Massive sequencing: sequencing multiple DNA fragments simultaneously
What is the general mechanism of Sanger sequencing?
- It is based on the selective incorporation of chain-terminating ddNTPs by DNA polymerase during in vitro DNA replication
What is the recommended sequencing depth?
10x to 30x
- more expensive with higher depth, but much easier to draw conclusions with higher depth
Define germline:
- sequence of germ cells that may be passed to a child
- exists in the somatic genome
- exists since the individual was born
Define somatic:
- sequence of nongermline cells that is NOT passed to a child
- does NOT exist in the germline genome
- Acquired (cancer, sunshine)
What detection methods are used for somatic mutations?
- Sanger
- next gen
- other methods
just not DNA chips
