In the last few weeks we have been covering DNA analysis and PCR.
Not only am i getting a good hands on feel for the PCR process I am now, thanks to my class, getting a good hold on the whys and wherefores of my actions.
Here are a few conference posts from the last 2 weeks.
In each case I am expressing things learned only in the last few weeks.
PCR is the man made simulation and amplification of the natural process of mitosis.
DNA is doubled and doubled again until there are enough copies. The man made version of this mitosis is called Polymerase Chain Reaction.
Polymerase is an enzyme that causes DNA or RNA to form.
Chain refers to the cyclical and repeating nature of the operation
Reaction in this case illuminates the forced and controllable nature of the process.
As with DNA’s natural in cell cycle only small sections of the full DNA strand are replicated at any one time.
In normal Mitosis this copying is done through RNA replication.
I am going to crib from my own entry from a previous post here.
“The DNA in the center (the nucleus) pulls apart into two independent strands. Imagine a ladder in which the two long sides (strands in the case of DNA) pull apart. As the independent strands are separated by an enzyme called helicase, one strand, called the leading strand, is copied forwards and continuously and the other strand, called the lagging strand, is copied backward and in pieces. Proteins called DNA polymerases build new strands of DNA from the original (parental) strand serves as a template for the new strand. This will allows the new DNA structure to replicate exactly the original because each rung of the ladder (the bases or nucleotides called A,T,C or G aka. Adenine, Thymine, Cytosine and Guanine) attracts one very specific partner nucleotide and only that one.”
In the case of PCR, the action of helicase (that of breaking the strands apart) is preformed through simple heat. The heat is high enough to melt the DNA strands apart but not high enough to actually destroy the individual strands.
At this point as with Mitosis sections of the DNA strands are replicate. Unlike normal Mitosis or even normal RNA replication, very specific strands are desired. To eliminate the undesirable DNA, or even contamination, very specific primers are added to the mix.
Primers are prearranged nucleotides (ATGC’s) that are meant to match up only with the DNA sections the lab wants to see. These primers come in pairs. There is one primer for the beginning of the desired section and one for the ending.
As with normal DNA replication this process is very fast (about 30 sec.). In PCR repeated cycles are forced through repeated heating and cooling back down of the sample DNA.
My PCR runs usually take about 2 hours and can have up to 30 of these cycles.
The DNA replicates exponentially. I have attached a picture of what 6 cycles look like to give you an idea of how effective this process is. In a nutshell a single DNA can go from 1 double helix of DNA to over a billion in under 4 hours.
The second part of the question “What do the following mimic: Buffer, Taq polymerase. Magnesium. Add any other components you wish.” Speaks to the things scientist do to add speed and specificity to the process.
Buffer is a more liquid version of cytoplasm. It is easier for the relevant particles to move about and to be healthy. Buffer is sort of like climate control.
Taq polymerase is by far the most interesting thing about PCR and the most original most mentally innovative element of PCR. Like the DNA polymerase builders in normal mitosis Taq polymerase is a construction machine on the molecular level. The remarkable thing is that Taq polymerase comes from (or in synthesized using the model of) the bacteria Thermus Aquaticus. T. Aquaticus is an extremophile. This bacteria lives near and on thermal vents (under water volcanoes) in extreme heat. It is the ability of Taq polymerase to thrive in and build nucleotides in such crazy (relative to normal DNA polymerase) temperatures that gives PCR its major start stop cycle boost. Every time the heat is turned up the Taq polymerase starts building and overtime the solution is allowed to cool it stops again, eliminating the need to wait for normal DNA building cycles.
Magnesium is required to maintain healthy levels of Taq polymerase. If the concentrations are too high the DNA will stabilize and not come apart and if concentrations are too low the Taq will not be active enough.
If you remember how PCR works this is not terribly complicated. RtPCR is PCR with an extra step. As you recall in PCR a sample is heated up and cooled down many times (cycled). The same thing happens here. In PCR 2 primers are needed, these mark the beginning and end of each desired segment. In RtPCR the same two primers are used but this time a probe is added. The probe looks and acts a bit like a primer. It bonds to the DNA strand in the same way, by lining up the ATGC bases. The difference is that a probe is meant to be destroyed during each cycle.
Each probe has a pair of florescent reactive particles (this is the same kind of florescent light you see in offices or in TV police procedurals). The first part of the pair is florescent light emitting and the second part is florescent light absorbing. Think of it like a star and a black hole right next to each other. The black hole absorbs all the light from the star so that it can’t be seen.
Some PCR machines have florescent light emitters and detectors in addition to their thermo cyclers (that is the part that heats up and cools down in each cycle)
During DNA division and replication the primers at the start and finish stay right where they are but the probes are in the way of the normal polymerase building action, so the polymerase just breaks them up and shoves them out of the way. The breaking up of the probe allows half of it to become florescent light emitting without interference. The black hole in this case, is far away enough from that star that the star can shine brightly.
This light is of course very very small. It takes many cycles before there is enough light to actually be seen.
The point at which there is enough light to first be detected is called the threshold cycle.
Threshold cycles are very predictable. If you start with 1 DNA copy it will take about 40 cycles to get enough copies to be detectable. If you start with 32 copies it will take 35 cycles. If you start with 1024 copies it will take 20 cycles.
There is a chart in each of the videos below to illustrate this threshold point.
Using this method you can get a very good estimate of the number of copies you have initially or “how much DNA has been extracted’.
A standard curve is used for a control in a RtPCR run. The stand curve is made up of a known concentration of a known DNA sample. If your standard curve comes out looking like you expect it to then you know your process is working the way you want it to.
It is possible for machines to malfunction or for improper handling of samples to destroy their contents. Only by always including a standard curve can you reassure yourself as to the viability of your RtPCR results.
I have two videos here that will probably be either helpful or entertaining for anyone interested in seeing RtPCR explained through animation.
This guy speaks very slowly. His English is good but accented. The slowness may be a little boring but his explinations and animations are great for the basics of how RtPCR works
This guy is a very animated presenter and a little technical but if you understand PCR it is a fun presentation.
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