Solid state oligonucleotide synthesis (phosphoramidite method) скачать в хорошем качестве

Solid state oligonucleotide synthesis (phosphoramidite method)

Phosphoramidite is pretty cool - am I right or am I right? It helps us DNA or RNA write! Solid-state oligonucleotide synthesis, including the phosphoramidite method, can be used by companies to make large amounts of short DNA or RNA sequences (oligonucleotides or “oligos”). So I thought I’d do you a solid and tell you about what might happen when you order your PCR primers! blog form (text old, video): http://bit.ly/solidstateoligo “Nucleic acids” include DNA & RNA and they’re made up of nucleotide letters.Nucleotides are made up of of a sugar (ribose in RNA & deoxyribose in DNA) connected to a unique nitrogenous base (A, C, G, or U (or T in DNA)) and phosphate(s). If you don’t have the phosphate(s), you call it a nucleoSide. There’s a big difference between how nucleotides are added in cells and how they’re added through chemical synthesis… Your cells can only copy existing DNA (into DNA or RNA) because they rely on an existing template. When using solid-state synthesis methods to custom-make short pieces of DNA or RNA (oligonucleotides or “oligos”), there is no template, you just introduce letters one at a time, so you can write “anything.” But you have to tightly control it because, since there’s no template there’s no way to “proofread” - so you have to make sure excess letters don’t linger – and you have to make sure that the letters get linked correctly – in your cells, proteins ensure this specificity because when they hold the letters in addition position only the right way is available. It involves multiple steps and some longish chemical names but just like most chemical synthesis reactions, at its heart, the logic’s “simple” → hide what you don’t want changed (PROTECT) & make what you do want changed more easily changeable (SELECTIVELY DEPROTECT) → add something to change it (COUPLE) → remove unreacted things (CAP) → repeat cycle until you’re satisfied → remove any “hiders” you don’t want in your final product (FULLY DEPROTECT) → clean it up (DESALT, HPLC/PAGE purify, etc.) Similar methods can be used for DNA & RNA, but if you want RNA, it’s a lot more $ you’ll have to pay! DNA & RNA differ by a single oxygen (that D’s for “deoxy”), yet custom DNA’s cheap, but RNA’s expensive! It’s not just because RNA’s cooler (though it is) or because oxygen’s super pricey, it’s what that oxygen can do… it likes to kick off its neighbors, breaking the chain of letters being written or stealing it for itself… So when companies synthesize RNA using SOLID-STATE OLIGONUCLEOTIDE SYNTHESIS, they have to protect the RNA from itself – and protect their profit margins… We use numbers with a “prime” sign (‘) to indicate where things stick off of the sugar - I like to imagine nucleosides as little people with the left arm being the 5’, right arm 1’, right leg 2’ and left leg 3’. The difference between RNA & DNA (other than that U vs T thing) is that RNA has another OH as its “right leg” whereas DNA just has a hydrogen (H) which often isn’t even drawn in, just implied. What difference could an oxygen make? A LOT! In solid-state oligo synthesis, you chemically hide (block/protect) or “unhide” (deprotect) these arms & legs to direct how they link up. But in RNA you have to “boot” 2 legs, not just one, and you have to keep one of those boots on while you remove the other.  Because that “right leg” can kick off the growing chain or steal it from the left! In the form of nucleosides your cells use, a phosphate (PO4-) “hand” is attached at the 5’ arm & there’s a hydroxyl (-OH) foot at the 3’ leg. In cells, like the bacteria we use to amplify plasmids, or in PCR, a POLYMERASE protein helps link nucleotides together by forming phosphodiester bonds between the phosphate of 1 nucleotide (attached at the 5’ location on the sugar) & the 3’ -OH of the next. Chemical synthesis instead adds them as modified “nucleoside phosphoramidites” – instead of having the phosphate group come from the “left arm” (5’) it comes from the “left leg” (3’). And they’re added “right to left” 3’→5’ but the end products are the same. note: an important consequence of this is that the beginning of your DNA or RNA oligo will have a 5’OH instead of 5’ phosphate(s) unless you pay them more to add it on That’s not the only difference - since you don’t have a polymerase relying on a template, you have to direct the show & make sure things go where they’re supposed to. For both DNA & RNA you need “gloves” - a DMT (4,4’-dimethoxytrityl) group hiding the “left arm” and things like benzoyl or isobutyryl groups to protect the “right arm” (bases) because the same atomic arrangements that allow bases to bind to one another also allow them to bind to things you don’t want. And both DNA & RNA need a “sandal” on their left leg - a 2-cyanoethyl-diisopropylamino group partially protecting the phosphate group of the 3’ leg so it doesn’t “overreact” but leaves it available for the oxygen from the next letter to attack it finished in comments