(dr @ 21.12.2008 11:42)
В плане дискуссии к "Ъ IP-штамп: frNwv0U1khjS": "...не имея собственных разработок, защищенных патентами и прочим, я бы не посмел ..." - вот и не смейте
А как появятся собственные разработки - тогда сразу и напишете
И патентами защищайте почаще, чтобы мы ваши идеи сразу со схемами с ФИПСа качали.
Про цену книги у нас: Коллеги, конечно, надо брать по 165 в издательстве. Разве что кому до Каширской на 135 руб ближе ехать, чем до Аваимоторной. А так - смысла нет преплачивать.
Денис
Real-Time PCR
—Russ Higuchi
When late in 1986 I joined Cetus, the birthplace of PCR, "closed-tube" PCR using a thermostable DNA polymerase had just been achieved (1)(2). Before this, the thermolabile polymerase had to be added at each cycle. Tom White, our vice-president in charge of Research and Development, told me that what was needed now was some way to visualize the amplification product, also without opening the tube. Such closed-tube detection was termed "homogenous".
A few years later I was working with Gavin Dollinger on his idea of "tagging" things that needed to be traced (e.g., explosives, money, and pharmaceuticals) with specific, amplifiable DNA sequences (3). We noticed that very high molecular weight DNA was occasionally generated in PCRs from such tagged substances. If we could reproducibly promote such large DNA, Gavin reasoned, it might be detected directly in the tube by light scatter. We set out to use biotinylated primers and streptavidin in the PCR to try to catalyze the formation of long branched chains of amplicons. We succeeded only in forming precipitates. To determine whether DNA was in the precipitates, we added ethidium bromide to the completed PCRs and held the tubes up to ultraviolet (UV)1 light. The precipitates were fluorescent, but the fluorescence was not specific to the presence of double-stranded DNA (dsDNA). Bob Griffith, who was helping us with these experiments, was directed to try some different conditions. He showed me the gel with the expected DNA band from the control without streptavidin and mentioned that, without thinking, he had added the ethidium bromide at the beginning of the PCR.
I had two reactions. One was surprise that the PCR had worked at all in the presence of ethidium bromide, a known DNA polymerase inhibitor. Indeed, PCR would have been inhibited if the concentration of ethidium bromide had been somewhat higher. The second was to realize that because PCR creates much more dsDNA than is put into it, there should be more ethidium bromide fluorescence created as well. Such fluorescence would be detectable without opening the tube.
The PCRs were repeated without streptavidin. When held up to a UV light, the tube containing the amplification target glowed brightly compared with negative controls (Fig. 1A ). Although this "endpoint" reading of PCR was of itself very useful, we thought that taking fluorescence readings on a cycle-by-cycle basis might provide a quantitative assay with a wide dynamic range (4). The fewer PCR cycles it took to detect an increase in fluorescence, the more copies of the target DNA sequence there were to begin with. To demonstrate this "real-time" reading of the PCR (which we later called "kinetic"), we attached one end of a bidirectional fiber optic cable to the open top of a PCR tube in a thermocycler and the other to a fluorescence spectrophotometer. With excitation at 500 nm, a fluorescence trace at 600 nm was recorded (Fig. 1B ). The ups and downs in fluorescence that corresponded to the downs and ups in temperature from thermocycling were seen, as well as the net gain in fluorescence at low temperature attributable to the accumulation of dsDNA PCR product.
Figure 1. Real-time visualization of PCR.
(A), three completed PCRs in microfuge tubes illuminated by UV light. All tubes contained ethidium bromide. The first tube on the left had target sequence cognate to the primers; the two others did not. (B), fluorescence traces from ethidium-bromide-containing PCRs taken with a fiber optic cable and a spectrophotometer. The upper trace is from a PCR begun with 20 ng of human male DNA and primers specific to a Y-chromosome sequence. The lower trace is from a control PCR with the primers but without the target DNA added. Fig. 1B was reprinted with permission from Biotechnology 1992;10:413–7.
To assess the quantitative performance of PCR in this mode, it soon became clear that we needed a way of reading multiple amplifications in parallel. One way, ultimately adopted in the first commercial real-time PCR instrument, was to use multiple fiber optic cables (one per tube) and a rapid multiplexing system for sequentially exciting and reading the fluorescence from each tube. I thought that a simpler way would be to use digital imaging; Bob Watson and I therefore implemented such a system using a charge-coupled device (CCD) camera from a home-built, digital gel-documentation system (5)(6). The camera was set up in a darkroom looking down over the exposed tops of the closed PCR tubes set into the thermocycler block. UV lights were used to illuminate the tubes in the block, and at every annealing/extension phase of the PCR an image was "grabbed". At every well position in the image, the pixel values were summed. The cycle-by-cycle plot of these sums generated "growth curves" for each PCR.
realtimepcr_content.pdf
Другое дело, если это ликбез для покупателей... 
- книга действительна нужна для тех кто не читвет "по-ихнему". 
Так ведь принято во всем мире что-то, да своё продвигать любыми путами, но в защищенном 
