what are the limitations of PCR?

Answer 1

The website that Ashley V sent is very good and lists most of the limitations of PCR. Another one that is not mentioned, is (in the normal PCR) the lack of a quantitative measure of the mRNA expression. This happens because the amplification of the portion of cDNA goes exponentially and might reach a plateau, after which the differences between templates are cancelled out. A way of solving this is by using semi-quantitative PCR, by limiting the number of cycles (time-course or "cycle-course"), or, even better, a real-Time PCR. For this, the procedure follows the general pattern of polymerase chain reaction, but the DNA is quantified after each round of amplification; this is the "real-time" aspect of it. Two common methods of quantification are the use of fluorescent dyes that intercalate with double-strand DNA, and modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA.

Answer 2

This Site Might Help You.

RE:
what are the limitations of PCR?

Answer 3

Polymerase chain reactions?

I believe about 1 in 10,000 bases have an error, due to the inability to check and remove bases that may be added incorrectly. Also, sometimes the DNA gets tangled or folded (I forget exactly), I think they have fixed this though.

If the number of base pairs exceeds around 2500, the polymerase sometimes falls off, there are some that can be used for over 40,000 bases though.

I'm not positive on all of these numbers, it's been a while.

Answer 4

Well, PCR is a way to use biological processes to generate lots of copies of a piece of DNA. It's a chain reaction, because every cycle of copying generates something up to an equal amount of templates to make copies of in the next cycle. 1 -> 2 -> 4 -> 8 -> 16 -> 32, etc. etc. The more you have, the more copies you can make in a single cycle. Sometimes you already have loads of DNA of the kind you need, and it would not be useful to do PCR. PCR requires a starting piece of DNA called a 'primer' for the copying enzyme to work from, and we generally use one primer to copy the counterpart to the 'top' strand and one primer to copy the counterpart to the 'bottom' strand. The resulting double-stranded copies start at the beginning of the 'sense' primer (the one that begins the counterpart to the 'bottom' strand, going from left to right) and end at the beginning of the 'antisense' primer (the one that begins the counterpart to the 'top' strand, going from right to left). This lets us control what piece of DNA we make, because the primers we use determine where the DNA copies start and end. If that is confusing to you, try making a diagram to help you out. Start with a double-stranded piece of DNA, top and bottom strands together, then separate them and see how the primers would attach to each strand. Each primer would have a nucleotide sequence which must match a corresponding section on the template strand. So, if you know that the 'top' strand has a stretch that goes CCTTACAAAG, you could make an anti-sense primer which would go CTTTGTAAGG. This would stick 'upside-down' onto the top strand, and read from right to left, the way the 'bottom' strand is read. The first C of the primer would bind to the last G of the template, then the TTT would bind to the AAA, and so forth. The DNA polymerase enzyme would start building the copy strand from the G at the end of the primer, and continue going from right to left until it either ran out of template, or ran out of nucleotides or ran out of time. The sense primer would match a sequence on the 'bottom' strand the same way, and produce a copy of the part of the 'top' strand that goes from the beginning of the sense primer until the template ends (if it's a copy of the 'bottom' strand that starts with an anti-sense primer), or it runs out of nucleotides to build with, or it runs out of time. You do this process in a 3-part cycle: First, you 'melt' the DNA by heating it up so hot that the bonds holding the strands together break and the strands separate. Then you allow the primers to 'anneal' to the template strands by cooling the temperature down enough that those short pieces can stick to their counterpart sequences. Then you heat the reaction soup up again to the temperature that the DNA polymerase likes to work at, and it builds your copies for you. The annealing temperature depends on the particular mix of template and primer, but is usually somewhere between about 55 -65 degrees Centigrade. The melting temp is either 94 or 95 degrees C, and depending on the polymerase, the copying temp is somewhere around 72 degrees C. When you're done copying, you go back to the melt step and separate the strands again, anneal the primers to the strands, and make more copies. After a few cycles, some of your earlier copies are now being used as templates. Every copy of a strand uses up a batch of nucleotides and a primer, so at some point, you're going to run out of raw materials. Of course, this assumes that the enzyme doesn't run out of steam first. You can make a butt-load of DNA copies from a very tiny starting amount (all you theoretically need is one DNA molecule to start with), and you can decide what piece of the DNA you want the copies to include. Of course, it kind of helps if you know some parts of the original DNA sequence so that you can make the primers, but there are ways around that problem. There is another little thing, though - nothing is perfect, of course, and our polymerase doesn't always grab the right nucleotide to build the copy with. Suppose we get an error built in early on in the copying process? Then most of our copies will also have that same error. We can't eliminate that problem, but we can reduce it by using a special kind of polymerase which has something called a 'proofreading' function that basically checks the copy for mismatches as it goes along and repairs the mistake. This takes a lot longer, though, so it can be a trade-off of sorts between speed and accuracy. There's a whole lot more that's very cool about PCR, but I've already written half a page, so that's enough. One thing, though - we don't use human polymerase to do this, because it burns out at the melting temperature; it just can't handle that. Instead, we use polymerases from bacteria that live at (and copy their DNA at) very high temperatures - in places like hot springs and deep-sea volcanic vents. Those polymerases can handle cycle after cycle

Answer 5

check this website, it gives the limitations and disadvantages, assuming you are talking about Polymerase Chain Reaction!
science, it's great isnt it?
http://www.pcrstation.com/pcr-limitations/

Answer 6

You must already know part of the sequence you are trying to amplify (i.e. you need to be able to make primers which will bind upstream and downstream of your target).

Answer 7

Probably the biggest limitation is the risk of contamination.

Answer 8

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https://www.youtube.com/watch?v=rbvvvLWE8jI