tag:blogger.com,1999:blog-36768584.post4466607251604701536..comments2024-03-03T18:49:34.382-05:00Comments on Omics! Omics!: A qPCR (aka RT-PCR aka rRT-PCR) ExplainerKeith Robisonhttp://www.blogger.com/profile/04765318239070312590noreply@blogger.comBlogger7125tag:blogger.com,1999:blog-36768584.post-48254862678287917772020-03-08T14:25:18.050-04:002020-03-08T14:25:18.050-04:00Is this the same technology that used for HIV test...Is this the same technology that used for HIV testing? yurihttps://www.blogger.com/profile/02221754119038537718noreply@blogger.comtag:blogger.com,1999:blog-36768584.post-63875073548007436962020-02-07T01:52:02.328-05:002020-02-07T01:52:02.328-05:00"Most thermocyclers use Peltier elements also..."Most thermocyclers use Peltier elements also known as thermocouples"<br /><br />No, Peltiers are not also known as thermocouples. These are two distinct pieces of hardware with distinct purposes. Thermocouples are usually used to control the operation of Peltier elements. The operation of both relies on a thermoelectric effect, but the construction is vastly different Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-36768584.post-43028758529221553722020-02-06T21:03:49.480-05:002020-02-06T21:03:49.480-05:00Much egg on my face -- thank you for noting that. ...Much egg on my face -- thank you for noting that. It seemed odd but then I just rechecked my arithmetic, failing to check that I had the units rightside up! Oy!Keith Robisonnoreply@blogger.comtag:blogger.com,1999:blog-36768584.post-85468107343680957132020-02-06T19:29:25.413-05:002020-02-06T19:29:25.413-05:00The ramp rates you cite are in degrees C per secon...The ramp rates you cite are in degrees C per second, not seconds per degree C.Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-36768584.post-2149465619075139072020-02-04T09:16:17.979-05:002020-02-04T09:16:17.979-05:00Thank you for the constructive comment.
I agree...Thank you for the constructive comment. <br /><br />I agree that diagnostic PCR amplicons should be kept short and generally are. I have a colleague who is a fiend on this point. <br /><br />When I wrote that, I hadn't yet found the actual CDC primers and probes, which are indeed much shorter. Tried to get to clever doing the calculations.<br /><br />It would be interesting to see how the test would perform with much shorter cycle times, though in the end it is the ramp times that dominate the overall time. Given the short amplicons, Taq polymerase should be processive* enough to finish such short amplicons most if not every time<br /><br /><br />Definition for non-experts:<br />processivity is the property of a polymerase of how long it polymerizes before spontaenously stopping and falling off the template. Highly processive polymerases will rarely stop; less processive polymerases will stop more frequentlyKeith Robisonnoreply@blogger.comtag:blogger.com,1999:blog-36768584.post-11421202121500253792020-02-04T08:59:08.046-05:002020-02-04T08:59:08.046-05:00Good overall summary but the rate/size description...Good overall summary but the rate/size description is off, for diagnostic qPCR amplicons are always very short, tending to be 100 bp or less. You want maximum speed and efficiency, no need to use the DNA product for anything afterwards so who cares how long it is. Theoretically you could use the rate of synthesis of a polymerase and extension time equal to number of seconds required...but in practice that's not how it works, there's no 100% efficient reaction, processivity means multiple association/dissociation events, etc. so all PCR uses way more extension time than it "should". Shorter amplicons always better for Dx. The CDC assay "N1" for example is 72 bp end to end with the hydrolysis probe in the middle.Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-36768584.post-88346761904918173492020-02-02T20:27:45.778-05:002020-02-02T20:27:45.778-05:00Very nice description.Very nice description.Anonymoushttps://www.blogger.com/profile/03657588285977411163noreply@blogger.com