Gerwyn, Reply #79 on: Today at 08:04:09 AM:
if anyone is wondering why an assay that can find a low copy number of the VP-62 clone cant find wt virus the answer is relatively simple
The chemophysical properties of the DNA are different
high levels of oxidative stress caused by hgrv infections and commonly found in studies investigating patients with ME cause oxidative modification of nucleotide bases. The most common is the addition of a hydroxyl functional group or deamination. This (for reasons involving sterochemistry and something called twisting) means that the stacking energy and hydrogen bonding which hold complimentary nucleotide bases together are dramatically reduced. This can be compensated for by using increased levels of magnesium and lowering annealing temperatures.Another strategy is to use an RNA template to begin with. mlv viruses cant replicate in resting cells so xmrv will exist in such cells as preintegrative complexes and integrated highly methylated proviruses. ROS oxidation induces a pattern of base substitution or inversion in the provirus affecting primer annealing.The presence of certain oxidised bases or even abasic sequences can stop taq polymerase "in its tracks" leading to an absence of product.Unless PCR reagent concentrations and cycling conditions are adjusted accordingly the virus will escape detection. silverman was able to adjust his assays to find gag sequences which were already present and was fortunate enough to isolate XMRV from RNA hence largely avoiding these issues
The effect of activating PMBcs would be to allow the integration of "fresh" proviruses as the preintegrative complexes are nowhere near as prone to oxidative damage.This would also allow the creation of viral RNA
I will post the detailed version but I wanted to post this to demonstrate what a BS decision the Science editors are making
"Science" has abandoned Science. I hope that its subscribers take note
Dr Mikovits: "it is premature to retract our paper"
Doctor Speedy and ME in search of medical honesty
"If anyone is wondering why an assay that can find a low copy number of the VP-62 clone cannot find wild-type virus, the answer is relatively simple.
ReplyDeleteThe chemophysical properties of the DNA are different.
High levels of oxidative stress caused by HGRV infections and commonly found in studies investigating patients with ME cause oxidative modification of nucleotide bases. The most common is the addition of a hydroxyl functional group or deamination. This (for reasons involving sterochemistry and something called twisting) means that the stacking energy and hydrogen bonding which hold complimentary nucleotide bases together are dramatically reduced. This can be compensated for by using increased levels of magnesium and lowering annealing temperatures.Another strategy is to use an RNA template to begin with. MLV viruses can't replicate in resting cells so XMRV will exist in such cells as preintegrative complexes and integrated highly methylated proviruses. ROS oxidation induces a pattern of base substitution or inversion in the provirus affecting primer annealing. The presence of certain oxidised bases or even abasic sequences can stop taq polymerase "in its tracks" leading to an absence of product. Unless PCR reagent concentrations and cycling conditions are adjusted accordingly the virus will escape detection. sSlverman was able to adjust his assays to find gag sequences which were already present and was fortunate enough to isolate XMRV from RNA hence largely avoiding these issues.
The effect of activating PMBCs would be to allow the integration of "fresh" proviruses as the preintegrative complexes are nowhere near as prone to oxidative damage. This would also allow the creation of viral RNA."
"... the answer is relatively simple."
ReplyDeleteCrumbs, I'm glad it wasn't complicated...
Not meaning to be flippant - I do understand this is positive news for us, just wish I understood it.
Thanks for the explanation, however ignorant it made me feel and thanks Dr Speedy for posting it.