The DNA on the slide is sense, so that it can match anti-sense DNA from the samples produced by the reverse transcriptase. How is this sense DNA produced for the DNA library?
At present there are two ways of doing this, in both cases in order to make sufficient material to spot onto the slides you have to amplify the material using the polymerase chain reaction (PCR). • If one of the oligonucleotides used for PCR is labelled with a tag then one strand will end up with this `tag’ on its end. If your slide is then coated with a ‘hook’, you can attach the strand of your choice onto the slides (this is how, for example, National Institute of Aging slides are made). • The alternative approach is to put amplified double stranded cDNA onto the slides you can then use hybridisation conditions in your reaction that will a) cause the double stranded cDNA to break on the spot and b) allow the DNA in your sample to preferentially bind to the spot DNA – the amount of labelled DNA in your sample is in vast excess to that on the spot, probably several thousand times more.
Related Questions
- In HIV, if reverse transcriptase cobbles together strands of DNA without much accuracy, why doesn’t the same thing happen in all cells? What is so special about HIV?
- Why does it inhibit reverse transcriptase and not the cells own DNA polymerases?
- How does reverse transcriptase make double stranded DNA from RNA?
