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12 Cards in this Set
- Front
- Back
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what is multiplex ligation-dependent probe amplification used for?
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it's a quantitative method used for detecting large insertions or deletions affecting multiple exons or whole genes and can be run in multiplex (w/ all exons from a gene)
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how many bases can you examine with Sanger sequencing?
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about 500 (as well as small indels)
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explain Sanger sequencing
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1. PCR the sequence of interest
2. a fraction of the NTPs in the PCR are flourescently labeled dNTPs which cause termination when incorporated 3. products are generated that are terminated at every base 4. run a gel electrophoresis to separate the the long from the short pieces 5. convert flurorescent signal to peaks to compare to ref seq |
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what are some limitations of PCR-based mutation detection assays?
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1. large gains and losses won't generally be detected
2. PCR relies on efficient annealing. sequence variants (SNPs) w/ in the primer/probe regions can interfere resulting in allelic dropout |
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what types of mutations are generally deleterious?
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splice-site, frameshift, and nonsense
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what properties should a dz have to be a good candidate for NBS?
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1. clearly defined
2. treatable w/ Rx started in neonatal period 3. reasonable incidence |
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what properties should a screening test have to work well in NBS?
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1. rapid turnaround time
2. high sensitivity and specificity 3. reasonable cost 4. small and easily obtainable sample |
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how does mass spec work?
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separates and quantifies ions based on their mass/charge ratio
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what are some good points about MS/MS spec for NBS?
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1. requires very small sample size
2. accurate and precise 3. fast (2 min per sample) 4. can test for multiple analytes w/o extensive prior separation 5. can screen for multiple very rare d/o in a single sample and one run |
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how do you detect organic acidemias by MS/MS screening?
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by their carnitine intermediates
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what is acyl carnitine a marker for?
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fatty acid oxidation d/o
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What does MS/MS NBS detect?
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abnormal analyte concentrations, not disorders!
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