Solving Radioactivity Problem with TdT Enzyme

In summary, the conversation discusses an experiment involving the enzyme TdT, which adds dNTPs onto the 3’ ends of DNA molecules. The experiment involves cutting lambda DNA with the restriction enzyme Alu I and measuring the rate of dNTP incorporation. It is determined that the reaction would need to continue for 1538 minutes or 25.63 hours to add 20 bases of dCTP to each 3’ end of the DNA fragments.
  • #1
bio_monkey
3
0
Could someone pls help me with the following Q..I'm not too sure where to even start!

The enzyme deoxynucleotidyl terminal transferase (TdT) catalyses the addition of dNTPs onto the 3’ ends of double stranded DNA molecules. In an experiment to determine the rate at which the TdT added dNTPs to the 3’ end of DNA, the following experiment was set up:
Linear-bacteriophage lambda DNA (48.5kb) at a concentration of 55ug ml^-1 was cut with the restriction enzyme Alu I. (Lambda DNA is cut 143 times with Alu I).
The following reaction was set up:

17ul H2O (ul = microlitres)
6ul 5x tailing buffer
2ul Alu I cut lambda DNA
2ul 1mM dCTP
2ul 32p DTP
1ul (15U) of TdT
The reaction mix was incubated at 37C and after 5min, 2ul aliquots were removed from the reaction mix to determine the amount of radioactivity incorporated. One aliquot was counted directly to determine the total amount of radioactivity in the sample. Another aliquot was TCA precipitated to determine the amount of radioactivity incorporated into the DNA. The results were as follows:
Counts before TCA precipitation = 374248 cpm
Counts after TCA precipitation = 4905 cpm
If the reaction was to be repeated, how many minutes would you allow the reaction to continue if you only wanted the addition of 20 bases of dCTP to each 3’ end of the DNA fragments.
5x tail buffer : 500mM potassium cacodylate pH 7.2, 10mM cobalt chloride, 1mM DTT
32PdCTP;5uCi ul-1 (3000 Ci mMol-1)
(molecular weight of 1.6kb of dsDNA = 1x106)
 
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  • #2
To answer this question, we need to calculate the rate of incorporation of dCTP (counts/min) by dividing the amount of radioactivity incorporated into the DNA (4905 cpm) by the total amount of radioactivity in the sample (374248 cpm). This gives us a rate of 0.013 cpm/min. We then need to divide 20 bases (20 nucleotides) by this rate to get the time required for the addition of 20 bases of dCTP. This works out as 1538 minutes or 25.63 hours.
 
  • #3


I would start by analyzing the experimental setup and the results provided. From the information given, it seems that the goal of the experiment was to determine the rate at which TdT adds dNTPs to the 3’ end of DNA fragments. The experiment was set up using linear-bacteriophage lambda DNA, which was cut with the restriction enzyme Alu I. The reaction mix also contained the necessary components such as 5x tailing buffer, dCTP, 32p DTP, and TdT.

The results of the experiment showed that after 5 minutes, there was a total of 374248 counts of radioactivity before TCA precipitation and 4905 counts after TCA precipitation. This indicates that the majority of the radioactivity was incorporated into the DNA fragments, with only a small amount remaining in the reaction mix.

To answer the question of how many minutes the reaction should continue to add 20 bases of dCTP to each 3’ end of the DNA fragments, we need to consider the rate of incorporation of dNTPs by TdT. From the results, we can see that after 5 minutes, a significant amount of radioactivity was incorporated. Therefore, we can assume that the rate of incorporation is relatively fast and we can estimate that 20 bases of dCTP would be added in less than 5 minutes.

However, to be more accurate, we can calculate the amount of radioactivity incorporated in 5 minutes and use it to determine the time needed for 20 bases of dCTP to be added. We know that 4905 counts of radioactivity were incorporated after 5 minutes, and each dCTP molecule has a specific activity of 3000 Ci/mmol. Therefore, we can calculate the number of dCTP molecules incorporated using the specific activity and the total amount of radioactivity incorporated (4905 counts). This would give us the number of dCTP molecules incorporated in 5 minutes.

Next, we need to calculate the number of bases incorporated in 5 minutes, assuming that each dCTP molecule adds one base. This can be done by dividing the number of dCTP molecules by 2, as each dCTP molecule adds one base to each 3’ end of the DNA fragment.

Finally, we can use this information to calculate the amount of time needed for 20 bases of dCTP to be added. If we divide the number of
 

1. What is TdT enzyme and how does it solve radioactivity problems?

TdT (Terminal deoxynucleotidyl transferase) is an enzyme that catalyzes the addition of nucleotides to the 3' end of DNA strands. This enzyme is commonly used in molecular biology to label DNA with radioactive nucleotides, which can then be detected through autoradiography. TdT enzyme helps to solve radioactivity problems by efficiently labeling DNA with radioactive nucleotides, allowing for accurate and sensitive detection of DNA fragments.

2. How does TdT enzyme differ from other DNA polymerases?

TdT enzyme differs from other DNA polymerases in several ways. Unlike other polymerases, TdT does not require a template DNA strand and has no proofreading activity, making it highly error-prone. It also has a much higher tolerance for incorporating non-canonical nucleotides, making it a useful tool for labeling DNA with radioactive nucleotides.

3. What are the potential applications of using TdT enzyme for solving radioactivity problems?

TdT enzyme is commonly used in molecular biology techniques such as Southern blotting, DNA sequencing, and in situ hybridization. It can also be used in research studies to label DNA for cell proliferation assays, apoptosis detection, and gene expression analysis. Additionally, TdT enzyme is used in diagnostic tests for detecting genetic mutations and diseases.

4. Is TdT enzyme safe to use in a laboratory setting?

Yes, TdT enzyme is safe to use in a laboratory setting. It is a highly purified enzyme and does not pose any significant health or safety risks when handled properly. However, as with any laboratory enzyme, it is important to follow appropriate safety protocols and use protective gear when handling TdT enzyme.

5. Are there any limitations or considerations when using TdT enzyme for solving radioactivity problems?

One limitation to using TdT enzyme for solving radioactivity problems is its high error rate, which may result in false-positive signals. This can be minimized by using proper controls and optimizing experimental conditions. Another consideration is the cost of the enzyme, as it may be more expensive compared to other DNA polymerases. Additionally, the enzyme may not be suitable for all types of DNA labeling experiments, so it is important to carefully consider the specific requirements of each experiment before using TdT enzyme.

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