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PHRM 847 > L7&8 Testing and Technology > Flashcards

L7&8 Testing and Technology Flashcards

1
Q

What kind of drug label is LUMAKRAS?

A

one gene - multiple alleles

  • The drug only targets KRAS with mutated G12C allele
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2
Q

What is an example of one gene-multiple drugs?

A

PDE-1 inhibitors

  • PDE-1 is one gene, but there are multiple drugs to target it.
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3
Q

What mutation can LUMAKRAS be used for?

A

KRAS G12C mutation

  • NOTHING ELSE
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4
Q

What is the general procedure for PGx testing?

A
  1. Sample collection
  2. Sent to lab
  3. Genetic counseling
  4. Pharmacy works with doctors to document and make decisions regarding results
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5
Q

Who can perform PGx tests?

A

CLIA certified labs
- clinical laboratory improvement amendments (CLIA) of 1988

find via GTR: genetic testing registry

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6
Q

How can you collect enough information to prescribe a PGx test?

A
  • work closely with therapeutic team
  • discuss the patient
  • Understand FDA labeling/CPIC guidelines
  • know the principles of technologies
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7
Q

How can you make an informed decision for the prescription of PGx test?

A
  • strength of PGx info vs other factors
  • cost vs benefit
  • selection of technologies
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8
Q

Do all FDA-approved drugs require PGx testing?

A

No

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9
Q

Which population may CYP2C9*5-11 be more important for?

A

African descendants

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10
Q

What is the target for samples for PGx testing?

A

DNA

any nucleated cells/tissue contains germline DNA

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11
Q

What is the gold standard for PGx samples?

A

Peripheral blood

Have to use white blood cells to get DNA though

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12
Q

What are the characteristics of using white blood cells for PGx testing?

A
  • 2-6 mL as standard amount
  • prefer EDTA-anticoagulant tube
  • Use sterile technique to prevent contamination
  • room temp 1-2 day delivery
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13
Q

Can we get DNA from red blood cells?

A

NO

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14
Q

What are the disadvantages of using cheek swab/saliva for DNA sample?

A
  • less DNA yield
  • possibly contaminated
  • poorer quality

rinse your mouth before hand!

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15
Q

What are the characteristics of tissue sample (fresh biopsy) for DNA testing?

A
  • high yield of DNA
  • -80 C for long term storage
  • Dry ice for transportation

easily goes bad!

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16
Q

What are the characteristics of tissue sample (formalin fixed and paraffin embedded) for DNA testing?

FFPE

A
  • DNA is usually degraded
  • detections are still doable

less preferred but more widely available

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17
Q

What was the first FDA approved broad companion diagnostic for all solid tumors?

A
  • FoundationONE CDx
  • FFPE testing
18
Q

Which is more stable (DNA or RNA)?

A

DNA

  • DNA is very stable
19
Q

What are factors that affect DNA quality?

A
  • 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
20
Q

What temperature should DNA be stored at for short term (1-2 months)?

A

4 C

21
Q

What temperature should DNA be stored at for long term storage? (years)

A

-80 C

22
Q

What are the two kinds of sequencing methods?

A
  • Sanger sequencing
  • high-throughput sequencing (whole exome/next gen)
23
Q

What is the fundamental technique for DNA amplification?

A

PCR

24
Q

What are the two goals of testing?

A
  • testing known variants
  • testing both known and unknown alleles
25
Q

What are the two critical steps in DNA amplification?

A
  • target DNA amplification
  • allele discrimination
26
Q

What does PCR stand for?

A

Polymerase chain reaction

most useful technique for DNA amplification

27
Q

What are the required substrates for PCR?

A
  • DNA template
  • dNTPs (dATP, dGTP, dCTP, dTTP)
  • Primers
  • Buffer: pH, Mg2+
  • Enzyme: Taq DNA polymerase
28
Q

What are primers for PCR?

A

2 short sequences specific to the region of interest

29
Q

What is the difference between dNTP and ddNTP?

A
  • dNTPs are nucleotides that are the building blocks of DNA
  • ddNTPs are nucleotides used in the Sanger sequencing method
30
Q

Why did 3 water baths used to be needed?

A
  1. First bath needs to be 98 C for denaturation to separate the DNA strands
  2. Second bath is somewhere between 48-72 C for annealing. This allows primers to base pair to complementary DNA template.
  3. Third bath is 68-72 C for extension. Polymerase extends primer to form nascent DNA strand.
31
Q

How may copies are made in PCR?

A
  • From 2 copies to 2^(n+1) copies
  • n = number of thermal cycles
32
Q

How does PCR amplify DNA?

A

PCR amplifies DNA from both DNA molecules of homologous chromosomes

33
Q

What technology should be selected for detecting known SNPs or targeted SNPs?

A
  • DNA chip!
  • it is high throughput (up to 5M at a time)
  • it can ONLY detect known SNPs
34
Q

What CYPs does the Amplichip CYP450 Array cover?

A
  • CYP2D6
  • CYP2C19
35
Q

What are features of Sanger Sequencing?

A
  • 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
36
Q

What are features of next gen sequencing?

A
  • High throughput
  • Low cost per SNP
  • Parallel sequencing
  • Massive sequencing: sequencing multiple DNA fragments simultaneously
37
Q

What is the general mechanism of Sanger sequencing?

A
  • It is based on the selective incorporation of chain-terminating ddNTPs by DNA polymerase during in vitro DNA replication
38
Q

What is the recommended sequencing depth?

A

10x to 30x
- more expensive with higher depth, but much easier to draw conclusions with higher depth

39
Q

Define germline:

A
  • sequence of germ cells that may be passed to a child
  • exists in the somatic genome
  • exists since the individual was born
40
Q

Define somatic:

A
  • sequence of nongermline cells that is NOT passed to a child
  • does NOT exist in the germline genome
  • Acquired (cancer, sunshine)
41
Q

What detection methods are used for somatic mutations?

A
  • Sanger
  • next gen
  • other methods

just not DNA chips

PHRM 847 (8 decks)

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