PCR (Polymerase Chain Reaction) overview, design, and troubleshooting tips скачать в хорошем качестве

PCR (Polymerase Chain Reaction) overview, design, and troubleshooting tips

PCR (Polymerase Chain Reaction) - a comprehensive overview with tips to help it work well for you! 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.     blog form: http://bit.ly/pcrtrain   The instructions for making proteins (long chains of amino acid letters that fold up into cool shapes and act as molecular workers) are written in a different biochemical language - DNA. Stretches of DNA that hold “protein recipes” are called “genes” and, since the genetic code is universal, you can stick that gene to “any” cell and that cell’s ribosomes (protein-making machinery) will “understand” it and make the corresponding protein. Similarly, if you know what genes an organism (Including a person) has, you can know what proteins they make, whether they have mutated versions of some genes, how they relate to other organisms.     So, DNA’s really important and kinda like how you might love your IKEA table but don’t have a particular fondness for the instruction manual, but you need it, in order to better understand proteins I often have to deal with their DNA instructions, and to do this I need to make lots of copies of specific stretches of DNA, and thankfully PCR provides a way! The discovery of PCR is attributed to Karry Mullis (working for a biotech firm called the Cetus Corporation) who got the first glimmers of it in 1983 and published it as a legit technique in 1985 (and in 1993 won the Nobel Prize for it).    As with basically all “breakthrough” scientific discoveries, he couldn’t have done it with out the work of countless other scientists working before him. (and we couldn’t do the work we do today without the work of him and others!).     For example, the stage was set in part by Arthur Kornberg, who won the Nobel Prize for discovering an enzyme (reaction speeder-upper) that could copy DNA, using a single strand of DNA letters (deoxynucleotides (dNTs)) as a template for linking up (polymerizing) “opposite” letters into a complementary chain (which can then be used as a template for recreating that original template sequence thanks to the 1:1 “oppositeness” of DNA letters (more below). He called this enzyme DNA Polymerase (DNA Pol) and he could get it to make single complementary copies of DNA, but Mullis’ contribution was using that DNA Pol to make lots and lots of copies.    And we still do. But we typically use super-hardy versions of DNA Pol that can withstand the high temps we cycle them through in the process I’m gonna tell you about. And that choice of polymerase is just one decision you have to (I mean get to) make when you go to the DNA copy machine! So hopefully today’s post will help you iron out your routine. I’m going to start with an overview of how it works (so hopefully not too jargony/wonky) but then I’ll get into some details for those who want them. (apologies in advance for formatting - full-time grad student doing full-time lab stuff…) So let’s go!    DNA (DeoxyriboNucleic Acid) is the biochemical language our genetic info’s written in & its alphabet consists of 4 deoxynucleotide (dNT) “letters,” A, T, C, & G which have a “generic” part made up of a deoxyribose sugar with phosphate(s) hooked up on the “left arm” (5’ position) has a hydroxyl (-OH) group as a “left leg” (3’ position) & then the different letters have different “nitrogenous bases” (bases) that stick off as a “right arm” - these bases are the single- or double-ringed parts.    dNTs use their generic parts to link together through phosphodiester bonds to form long single stranded DNA (ssDNA) & 2 complementary single strands “zip together” using their unique base parts (A across from T, C across from G) to form double-stranded DNA (dsDNA). This double-strandedness protects it from damage (the bases are facing in) and allows for easy copying since if you unzip it one strand can be used as a template for making another.    This “unzip and copy” is what happens in replication - before a cell divides, it needs to copy all its DNA (its entire genome) so that it can pass on a full set to each daughter cell, and it does this with the help of DNA Pol, which brings together the freely-roaming nucleotides, holding the right ones together & helping them link up, while rejecting the wrong ones (ones that don’t complement each other (e.g. don’t let an A bind a C!)    Finished in comments