PCR (Polymerase Chain Reaction) - theory & practical tips скачать в хорошем качестве

PCR (Polymerase Chain Reaction) - theory & practical tips

PCR (Polymerase Chain Reaction) is a way to amplify (make lots of copies of) specific parts of DNA from larger parts of DNA. You specify the region you want copied using PRIMERS which are short fragments of DNA that bind to where you want the copier (DNA Polymerase) to start & stop. It’s kinda like the original, bigger, DNA template is like a transcontinental railroad & you only want to copy the stretch from Utah to Colorado - the primers act as “train stations” that the DNA Pol “train” travels between, laying down “tracks” (DNA nucleotides) ahead of it as it goes based on the sequence of the other strand. To expand that analogy... I like to think of nucleotides as train tracks and DNA Pol as a train. This train can only travel on double-stranded track, so it has to lay the track down ahead of it as it goes (and it knows what track to lay down by making it “match” the other side of the track (e.g. if the next track across from it (on the other strand) is a T, lay down an A). In PCR, we use short pieces of DNA (oligonucleotides, or “oligos”) which we design to “bookend” our region of interest (AMPLICON) and starting stations for the train - since DNA Pol needs double-strandedness to start, it’ll only start where you make it double stranded (but shorter than the other strand so there’s still stuff to copy). Basically, you want something like this:⠀ ⠀ ——====—- to make this ——========⠀ You need one primer per strand - one starting at your "start" and one starting at your "stop" (see figures). And be aware DNA is "directional" - its ends (which we call 5' and 3') are different. DNA Pol can only copy DNA in one direction, 5’ to 3’, you always have to keep in mind which way the “train tracks run." PCR is run in cycles of 1️⃣ MELT (heat up dsDNA to unzip strands) 2️⃣ ANNEAL (cool down slightly to allow primers to bind and 3️⃣ EXTEND (starting where primers leave off, add nucleotides complementary to template strand until you reach end of template strand). After the 1st cycle (where Pol goes till it runs out of steam or out of time), this end is determined by other strand’s primer because DNA can only copy it cannot “compose” so it’ll run off the track corresponding to the position that strand started being copied from in the 1st round. (easier to explain in pics)⠀ ⠀ You do this over & over 🔁 (30 or so times) to get lots of copies (each time you get 2X as many copies because each new strand becomes another template strand). ⠀ much more detail and technical tips in this post: http://bit.ly/pcrtrain