COBAS Amplicor device - can u help me understand this.. (Jul/23/2007 )
hello All
in this device, PCR is performed by amplifying target
nucleic acids in the presence of a thermostable DNA polymerase,
AmpErase, dNTPs, and biotinylated primers. After amplification
is complete, the resulting amplicons are hybridized to
paramagnetic microparticles coated with a test-specific deoxyribonucleotide
probe. At the conclusion of hybridization, the
reaction mixture is separated magnetically, the unincorporated
primers and dNTPs are aspirated, and the microparticles remaining
are washed. Detection is accomplished by incubating
the microparticles with avidin-HRP conjugate and reacting with
TMB substrate. The resulting colorimetric product is read at
660 nm by the on-board photometer.
in order to make sure that no contamination present from previous samples,
our amplicon contains dUTPs rather than dTTPs.by this,
the amplicons would be recognized by AmpErase which is going to cut them at 50/52C (done at the begining)
my question is how our amplicon (DNA) contains dUTPs rather than dTTPs,knowing that it is still DNA????
no one knows
let me reform my qs ;
in this device amplification is done in the thermal cycler portion.
in which dUTPs are used instead of dTTPs.
the reason why uracil is used instead of thymidine is because
the enzyme AmpErase recognizes uracil and cuts the DNA where it is found.
knowing this...
before loading your sample in the device,u "prime" it;
i mean the temperature is set to 50C in the device,which is the optimum temperature
for this enzyme to work....
this priming step removes any remaining DNA from previous samples,
making sure that no contamination present.
my question is how can this device incorporate uracil (which is a distinctive nitrogenous base to RNA) in DNA ????
if you don't know the answer can you lend me a hand finding any helpful source ?
thanks