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18 Cards in this Set
- Front
- Back
- 3rd side (hint)
What are the 3 steps in putting together a new molecular diagnostic test?
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-choose target gene
-choose method of amplification -choose detection method |
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What are the criteria for selecting a gene target?
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-DNA/RNA/protein
-short (shorter amplification time) -60% GC content (stable interaction with probe) -highly conserved gene (must be found in organism) -gene-specific locus (must be specific to organism) |
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Which is better for quantification, PCR or RT-PCR? How is RT-PCR different from PCR?
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RT-PCR (for every cycle, 1 molecule will be detected by laser detection)
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-probe has reporter and Quencher molecule that emit signal when separated
-when Taqman pol (5'-5' exonuclease activity too) comes to probe, it will cleave off the reporter, emitting fluorescence for the one strand it just amplified |
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Describe real-time immuno-PCR
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-detects protein
-antibody on plate, then add protein, then add second antibody with biotin -add Si which binds very strongly to biotin -then add DNA with biotin attached to bind to antibody-biotin-Si complex -run PCR |
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What are the standards for determining whether or not a molecular diagnostic test is good or bad?
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-accuracy vs. precision
-specificity vs. sensitivity |
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Differentiate between sensitivity and specificity
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-sensitivity: ability to detect people who actually have the disease
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-specificity: ability to detect people who do not have the disease
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What is the calculation for sensitivity? Specificity?
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sensitivity = true positives/(true positives+false negatives) x 100
Sensitivity is indirectly proportional to # of false negatives |
specificity = true negatives/(true negatives+false positives) x 100
Specificity is indirectly proportional to false positives |
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What is an ROC curve? What does it represent and what are the labels of axes?
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-Receiver-operator characteristic curve
-represents trade-off between sensitivity and specificity, how good the diagnostic test is -further up in left corner the better |
-y axis = sensitivity (1-false negative rate)
-x axis = 1-specificity (false positive rate |
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T or F: specificity is strongly influenced by prevalence of disease population
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TRUE
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What is a linear dynamic ranger?
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linear range that can be used for accurate quantification
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how can we test individual identity?
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-short tandem repeat (STR)
-no individual shares the exact same molecular profile with others |
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what are short tandem repeats?
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-number of repeats at telomeres
-no 2 people have identical number so can be used to identify person of a crime |
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what is the problem with STR testing?
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very sensitive so contamination is likely
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What is a melting curve? How can mutations be detected using a melting curve?
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-melting curve plots % dsDNA against temperature
-melting point is where dsDNA=ssDNA |
-every DNA sequence has characteristic melting curve and Tm due to GC content
-with every mutation, the Tm changes |
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Why is it beneficial to study a single cell rather than a section of tissue?
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-tissue is heterogenous, don't know how much/where in tissue has tumor
-single cell is just one cell so simpler |
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How can a single molecule (eg. DNA) be detected?
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nanoparticle-conjugated fluorophores (bio-barcode assay, BCA)
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How does bio-barcode assay (BCA) work?
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-have magnetic particle that has DNA/RNA/protein of interest
-add gold particle with barcode DNA -capture with magnetic field -run through scanner to find molecule of interest -highest detection known today (zeptomolar) |
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how can molecular based testing be used for personalized medicine?
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-pharmacogenetics (unique prescription for each person)
-theragnostics (determines whether or not a drug should be used for certain therapy) -prognomics (patient prognosis for disease and after treatment |
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