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 -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.) If any one of the four nucleotides is omitted from the incubation mixture, would you detect radioactivity in the precipitate? Explainb.) Would 32P be incorporated into the DNA if only dTTP is labeled? Explainc.) Would radioactivity be found in the precipitate if 32P labeled the β and γ rather than the α phosphate of the deoxyribonucleotides? Explain.
A) Since, mixture of dATP, dTTP, dGTP, and dCTP all were labeled; radioactivity can be detected even if one of the four nucleotides is omitted from the incubation mixture. This is because all other three nucleotides contain labeled phosphate and hence, radioactivity can be detected.
B) Yes. If 32P is labeled only dTTP, it can be detected in DNA. Since, dTTP can pair with 'A' nucleotide in DNA.
C) Mostly radioactivity can be detected in DNA when the alpha or terminal phosphates are labeled. It is difficult to detect radioactivity when phosphate groups other alpha like beta or gamma phosphates are labeled. Even if the radioactivity is detected it can be detected in rare cases like 5`end labeling of DNA on gamma phosphate.
