Understand the principle of dideoxy sequencing.
Dideoxy sequencing, also known as the Sanger method after the guy who invented it, is a pretty reliable technique for sequencing DNA.
In traditional dideoxy sequencing, you need the following materials:
- Template strands for the DNA that you want to sequence
- A primer
- DNA Polymerase I
- All four nucleotides- at least one of these should be attached to a radioactive phosphate
- Dideoxynucleotides. These are nucleotides with two -H groups as opposed to -OH groups (hence di- and deoxy-). The other H group is on the 3' carbon. Yup, that means that there's no 3'-OH, which means that new nucleotides cannot be added after the addition of a dideoxynucleotide. This is pretty important, as we're about to see.
Four reactions need to be carried out, each with a different dideoxynucleotide (i.e. one reaction needs to be carried out with dideoxyadenosine, one with dideoxythymidine etc.). Essentially, in each reaction, DNA Polymerase I is used to synthesise a new strand, but it will terminate whenever a dideoxynucleotide is added. (The concentration of dideoxynucleotides is kept reasonably low so that the chain won't always stop at the first incidence of whatever base you're looking at.)
The fragments created from the previous step are then run through an electrophoresis process. Fragments from each reaction are run through different lanes so that you can keep track of which fragments end with dideoxyadenosine, which fragments end with dideoxycytidine etc. The gel used is usually 4-6% polyacrylamide and 6M urea (the urea is used in order to denature the DNA so that its secondary structure won't interfere with electrophoresis). Like in normal electrophoresis, smaller fragments migrate through the gel faster. Thus by reading "up" the gel you can see what the sequence of bases are.
Understand how PCR can be used in the dideoxy sequencing reaction and how the sequencing is automated.
PCR can be used in the first part of the sequencing reaction (i.e. the bit with the synthesising of fragments ending in dideoxynucleotides) in a technique known as "cycle sequencing," or "linear amplification sequencing." In this process, only one strand is primed so that a linear (rather than exponential) amplification of products is achieved- hence "linear amplification sequencing."
In automated sequencing, the process is carried out in a single tube (rather than four tubes for the four different dideoxynucleotides). This is achieved by fluorescently labelling the ddNTPs (sorry, got sick of typing out "dideoxynucleotides") with different colours. After running the fragments through electrophoresis (only one lane required this time), a machine can detect the different wavelengths of emitted light to give a reading of the sequence.
Be familiar with new and rapid “next generation sequencing”.
Nowadays, there are faster and cheaper methods of sequencing DNA, though they are not necessarily as accurate as the dideoxy/Sanger method outlined above. Most forms of "next generation sequencing" involve creating billions of tiny fragments of DNA (30-70 base pairs long) which are immobilised to beads or chips. This immobilisation is done by adding an oligo-dA tail to the fragments, which bind to oligo-dT molecules in the beads or chips.
Fluorescently labelled dNTPs (not ddNTPs) are used for "next generation sequencing." They will bind to oligo-dT anchors if the fragment next to it has a complementary base. This will then cause that strand to fluoresce. The fluorescent tag is then removed, and a different fluorescently labelled dNTP is added. The fluorescence can be picked up by a computer to sequence the DNA.
Be familiar with how DNA can be chemically synthesized and the uses of synthetic DNA.
One thing you might have wondered throughout all of this is how primers and so forth are synthesised. One method of synthesising oligonucleotides is the phosphoramidite procedure, which synthesises oligonucleotides from 3' to 5' (opposite of the normal direction).
The phosphoramidite procedure starts by anchoring a nucleoside to a glass support at the 3' end. This nucleoside also has a blocking group (usually dimethyoxytrityl, a.k.a. DMTr) attached to the 5' end to prevent spontaneous reaction. Throughout this procedure, most of the bases added also have blocking groups added to them until they are washed off at the end.
The first step of the main part of the procedure is washing off the DMTr group by using trichloroacetic acid. Next the second base is added, except it's not added in the form of a nucleotide: it's added in the form of a nucleoside phosphoramidite derivative. This nucleoside phosphoramidite derivative is essentially just a nucleoside with a DMTr blocking group attached to the 5' end and a phosphoramidite group attached to the 3' end. Phosphoramidite groups centre around a trivalent phosphorus (i.e. a phosphorus atom that only forms 3 bonds rather than 5 like in phosphate). They also have other crap attached to them but I'm not going to go into that because it's not important. What's important is that this phosphoramidite group can be activated by a weak acid, such as tetrazole, allowing it to rapidly react with the 5' end of the first nucleotide. Aqueous iodine is then added to form a stable phosphate group. These steps are then repeated until the desired chain length is reached.
At the end of the process, the oligonucleotide is cleaved from the support by using ammonium hydroxide, which also removes blocking groups from the bases.
We're now done for this topic! Whew! (I might make a short mention on His tagging and GST-Fusion tagging in a later post, since that was covered in an earlier lecture but not in my posts here.)
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