Tuesday, July 12, 2005

Confusion over PCR

Confusion over PCR

It's no wonder that the dissident movement continues so long as this kind of confusion, and misinformation, continues.

The question is - if PCR copies fragments, doesn't that mean the virus is already fragmented?

Sadly this is a case of a little knowledge being a dangerous thing... PCR is MADE to copy fragments. Simply put, two small "primers" are used that are short chunks of DNA designed to match a desired sequence. One primer binds at the start of the sequence of interest, the other binds at the end of the sequence. You then start to copy DNA, and hopefully the reaction will proceed long enough so that the fragment overlaps. You then heat everything up so it all falls apart, then cool it to re-anneal the primers. Some of the "start" primers will now bind to the short sequence you amplified up from the "end" primer, and complete the double-stranded DNA. The same thing happens to the short fragment amplified up from the start primers.

After several rounds of amplification you have your original template, which may be several hundreds or thousands of bases long, several single-stranded or partially double-stranded fragments of DNA, and a large number of perfect double-stranded fragments which contain the start sequence, the end sequence, and everything in between.

As such, even if you were to start with an entire chromosome from a human, you could only PCR up a fragment. That's how it works.

The later commentators to the thread on DAG only serve up more twaddle, but talking about replicating RNA in cell culture. Frankly, that's ludicrous. RNA would be rapidly degraded in cell culture if it wasn't being made by the cells or an infection of the culture. RNA cannot replicate on its own. Also the cultures used to grow HIV are NOT frequently stimulated, in direct contradiction to what the dissidents will tell you. Some of the early culture systems were stimulated in order to activate the T cells (HIV can infect but cannot grow in non-activated T cells - the explanation is that it hijacks the same transcription factors that activated T cells rocket science). As time went on, the techniques improved. I've grown HIV in long and short term cultures (anything from 10 to over 190 days) in unstimulated T cells called Jurkats. So they're lying on two counts - RNA won't be replicated in a culture, and the virus cultures don't need stimulating anyway.

In fact they're lying on three counts, by suggesting that the stimulation will result in the appearance of virus-like particles and activity. A simple thing called a control culture, performed repeatedly and published alongside much of the HIV culture literature, put the lie to that.

The other thing about PCR is that they complain that these fragments only copy fragments of the virus, not the entire genome. How do we know that these fragments ARE the virus? They're forgetting that whether you copy 100 bases or 10,000 it's irrelevant - the accuracy of the reaction depends in both instances on these primers, which are only around 20 bases long.

It all just goes to show that they really don't understand what it is they're criticising, but then if they did understand it, they wouldn't criticise it.


Post a Comment

<< Home