Learn about S1 mapping.
The principle behind this technique is to label a single stranded DNA probe that can hybridize only to the transcript of interest, rather than labeling the transcript itself. The probe must span the sequence where the transcript starts or ends. After hybridizing the probe to the transcript, we apply S1 nuclease which degrades only single stranded DNA and RNA. Thus the transcript protects part of the probe from degradation. The size of this part can be measured by gel electrophoresis, and the extent of protection tells us where the transcript starts or ends.
To find the transcription start site of a transcript using S1 mapping we would do the following:
- We label our DNA probe at the 5' end with 32P-phosphate. The 5' end of a DNA strand usually already contains a non radioactive phosphate, so we can remove this phosphate with an enzyme called alkaline phosphatase before we add the labeled phosphate. Then we use the enzyme polynucleotide kinase to transfer the 32P-phosphate group from [γ-32P]-ATP to the 5' hydroxyl group at the beginning of the DNA strand.
- We then label the fragment on both ends, so we can potentially have two labeled single stranded probes. This will confuse the analysis so we remove one of the labels on the end. This is accomplished by re-cutting the DNA with another restriction enzyme, then using gel electrophoresis to separate the short fragment from the long fragment we want. Now the double stranded DNA is labeled on only one end and we can denature it to yield on only one end, and we can denature it to yield a labeled single-stranded probe.
- We hybridize next the probe DNA and the single stranded RNA.
- We then apply the enzyme: S1 nuclease. This enzyme specifically denatures single-stranded DNA or RNA but leaves double stranded polynucleotides, including RNA-DNA hybrids intact. Therefore the part of the DNA probe including the terminal label that is hybridized to the transcript will be protected.
- Lastly we determine the length of the protected part of the probe by high resolution gel electrophoresis alongside marker DNA fragments of known length. Since we know exactly the location of the right hand end of the probe, knowing the length of the protected probe automatically tells us the location of the transcription start site.
We can also use S1 mapping to locate the 3' end of a transcript. All we have to do is prepare a 3' end labeled probe and hybridize it to the transcript. All other aspects are the same as for the 5' end mapping. 3' end labeling is different from 5' labeling since polynucleotide kinase will not phosphorylate 3'-hydroxyl groups on nucleic acids. One way to label 3' ends is to perform end-filling. (When we cut a DNA with a restriction enzyme that leaves a recessed 3' end, that recessed end can be extended in vitro until it is flush with the 5'end. If we include labeled nucleotides in this end filling reaction, the 3' ends of the DNA will become labeled.)
We can use S1 mapping not only to map the ends of a transcript, but to tell us how much of the transcripts we have. This is because the intensity of the band on the autoradiograph is proportional to the concentration of the transcript that protected the probe. The more transcript, the more protection of the labeled probe, so the more intense the band on the autoradiograph. Thus once we know which band corresponds to the transcript whose concentration we want to measure, we can use its intensity to give us the answer. This assumes that the probe is in excess. If there is more transcript than probe, then its concentration can increase without a corresponding increase in the intensity of the characteristic band.
A variation to S1 mapping is RNase mapping. This procedure is analogous to S1 mapping except that the probe is RNA and can therefore be degraded with RNase instead of S1 nuclease.
See the DNA Molecule in 3-Dimensions