239 Flashcards
what are the major steps in PCR? How many cycles do you need to go thru? What do you usually do after?
Add a bunch of primers and heat to denature, primers attach before reannealing then you heat again and get amplification via heat stable polymerases. Usually go thru about 30 cycles. Use gel elecrophoresis to separate products (not used for quantification)
what do you do differently to make PCR real time? How many cycles do you need to get through to see it? When is diagnosis most specific?
fluorescence and imaging as you go. 25 cycles. Most diagnostic right after you reach the threshold
branched DNA, hybrid capture, invader technology are examples of what?
signal amplification
what is the advantage and disadvantage of target vs signal amplification
target has greater analytical sensitivity (lower limit of detection) but higher risk of contamination (more false positives). You also need prior knowledge of sequence
what does FISH detect
micro deletions and complications
what do you do in array CGH?
You add both control and test DNA and they compete for probes. If there is more than the control DNA then you see more and if there is less then you see less
advantage of MLPA (multiplex ligation-dependent probe amplification)
detects small deletions at MULTIPLE loci
rt-PCR, MLPA, FISH, chromosome analysis, array CGH: put in order of resolution? which require prior knowledge of sequence? which can illustrate mechanism? which can view whole genome? which cannot detect balanced rearrangements?
rtPCR=MLPA>array>FISH>chromosome
rtPCR, MLPA
FISH, chromosome
chromosome, array
array
what do you target with sanger sequencing?
coding regions only
what has brought down the cost of sequencing
massive parallel or next gen sequencing
when would you use sanger sequencing over NGS?
when the phenotype is only associated with a few genes. Not when there can be multiple mutations in multiple genes
