Question

To study the molecular mechanism of DNA replication, you incubated soluble E. coli extracts with a mixture of dATP, dTTP, dGTP, and dCTP, all of which are labeled with the α-phosphate group. After a while, the incubation mixture was treated with trichloroacetic acid, which precipitate the DNA but not the nucleotide or very short oligonucleotides. The precipitate was collected and the extent of the radioactively label nucleotide incorporation into the DNA was determined.

a. Would this assay identify DNA? Explain.

B. Would this assay identify RNA? Explain.

c. Suppose that you want to prepare a nucleic acid in which the backbone phosphorus atoms are uniformly labeled with 32P. At which position in the nucleotied precursor should the 32P be located? Explain.

Answer 1

Answer: To study the molecular mechanism of DNA replication, you incubated soluble E. coli extracts with a mixture of dATP, dTTP, dGTP, and dCTP, all of which are labeled with the α-phosphate group. After a while, the incubation mixture was treated with trichloroacetic acid, which precipitate the DNA but not the nucleotide or very short oligonucleotides. The precipitate was collected and the extent of the radioactively label nucleotide incorporation into the DNA was determined.

Answer a: Yes this assay would identify DNA. This can be explained as follows:

If DNA contains dTTP whose α-phosphate group is labelled with 32P, then it can be detected in DNA very easily by radioactivity techniques. Since, labelled dTTP can pair with 'A' nucleotide in DNA; whereas the other three nucleotides i.e. dATP, dGTP, and dCTP, all of which are labeled with the α-phosphate group is present in both DNA and RNA. Only dTTP is present in DNA whereas dUTP is only present in RNA. So if the sample contain DNA it can be easily identified if dTTP whose α-phosphate group is labelled with 32P is present in DNA and would shows radioactivity exhibited by α-phosphate group of labelled dTTP since it would only be present in DNA and not RNA. Thus DNA present in the sample can easily be identified by this experiment. And it would only be shown by DNA since only DNA contains dTTP and not RNA since in RNA dTTP is replaced by dUTP.

Answer b. No, this assay would not identify RNA.

This can be explained as follows:

Since only nucleotide to differentiate and identify DNA and RNA in sample; it can be done by identification of specific radioactive labelled nucleotide present in the sample (presence of dTTP whose α-phosphate group is labelled with 32P i.e. specific for DNA identification and presence of dUTP whose α-phosphate group is labelled with 32P i.e. specific for RNA identification). So if the sample contain labelled dTTP and radioactivity is found it shows the presence of DNA and if the sample contain labelled dUTP and radioactivity is found it shows the presence of RNA. But in the above case we do not have labelled dUTP i.e. even if RNA is present in the sample we cannot identify RNA since no radioactivity is found that demarcates between DNA and RNA. But as we have seen radioactivity in the above sample then we can say that DNA is present (because all DNA nucleotide is labelled) and we cannot identify whether RNA is present or not in the sample.

Thus in the above case the presence of RNA can only be identified when we also have labelled dUTP in the sample. But since we do not have labelled dUTP in sample we cannot identify RNA in the sample.

c. Suppose that we want to prepare a nucleic acid in which the backbone phosphorus atoms are uniformly labeled with 32P. At alpha or terminal phosphates position in the nucleotide precursor should the 32P be located.

This can be explained as follows:

Mostly radioactivity can be detected in DNA or RNA when the alpha or terminal phosphates position of DNA or RNA are labeled. It is difficult to detect radioactivity when phosphate groups other than alpha like beta or gamma phosphates are labeled.