Oligonucleotides are those smallish bits of DNA or RNA that we rely so heavily on for many of our molecular biology experiments. In their naked form, they are single, inert strands of DNA or RNA bases. But if you dress them up, you can increase their functionality.
Here are some of the common oligo wardrobe additions:
Fancy it up with phosphorylation
One of the most common oligo additions is phosphorylation. Anytime you are going to use the oligo with DNA ligase, you want to make sure your oligos are wearing 5’ phosphates. For example, if you are going to anneal two oligos and clone them into an unphosphorylated vector to change the multiple cloning site, your oligos will need to be phosphorylated.
You can also phosphorylate the 3’ end of your oligo. This will prevent degradation of the oligo by 3’-exonucleases and will block extension by polymerases.
Note: Some dyes are added post-oligo synthesis which can result in lower yields. In addition, some fluorescent modifications require a higher level of purification which can increase the cost.
Black is always in style
You can tone things down by adding a dark quencher onto your oligo. They can be added either to the 5’ or 3’ end. Use these with fluorescence-quenched probes and improve the sensitivity of your real-time PCR assays.
Stretching out your oligo
Sometimes you need to give your oligo a little room to stretch its wings. Spacers can be incorporated into the oligo either internally or at the end of the oligo. Ending spacers are ideal when you are attaching a bulky group such as a fluorophore.
Spacers can also be photo-cleavable. Shine a little UV light on your experiment and a portion of your oligo will be released.
A little change can go a long way
Maybe you love your oligo the way it is and only want to make minor changes. Then consider base modifications. The naturally fluorescent base, 2-aminopurine, can be substituted for dA and allow you to study the structure of DNA.
If your oligo is under attack by nucleases, you can help it out by incorporating a phosphorothioate bond modification. This substitutes a sulfur atom in place of a non-bridging oxygen in the phosphate backbone. Like a coat of armor, it will protect the linkage from nuclease degradation.
There are many ways you can dress up your oligo. And most of the time you can order your oligo with the modification already attached – it will arrive already dressed up for the ball.
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