60. A linear double stranded DNA is self-circularized by ligation with T4 DNA ligase. At which concentration
(μg/ml) of DNA would the self-circularization yield be maximum?
1. 30
2. 10
3. 5
4. 0.5
DNA Concentration for Self-Circularization Using T4 DNA Ligase
In molecular cloning, self-circularization of a linear double-stranded DNA is often needed to create plasmids or closed circular constructs. This process relies on T4 DNA ligase, an enzyme that catalyzes the formation of phosphodiester bonds between DNA ends. The efficiency of this ligation reaction heavily depends on DNA concentration.
Key Concept: Intramolecular vs. Intermolecular Ligation
T4 DNA ligase can catalyze two types of ligation:
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Intramolecular ligation: Self-circularization (joining ends of the same DNA molecule)
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Intermolecular ligation: Joining two different DNA fragments
At high DNA concentrations, intermolecular ligation is more likely (leading to concatamers or dimers).
At low DNA concentrations, the ends of the same DNA molecule are more likely to meet and ligate, favoring self-circularization.
Ideal DNA Concentration for Maximum Circularization
Empirical studies and molecular biology protocols suggest:
Self-circularization is maximized at low DNA concentrations, typically around 1–5 μg/ml.
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At 0.5 μg/ml, the DNA ends are sufficiently dilute to favor intramolecular interactions over intermolecular.
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Above 10 μg/ml, intermolecular ligation dominates, reducing circularization efficiency.
Let’s Review the Options:
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30 μg/ml – Too high; favors intermolecular ligation.
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10 μg/ml – Still high; not optimal for circularization.
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5 μg/ml – Acceptable, but not the maximum yield.
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0.5 μg/ml – Correct. Ideal for maximizing self-circularization.
Final Answer:
4. 0.5 μg/ml
This is the optimal DNA concentration for promoting self-circularization of linear double-stranded DNA using T4 DNA ligase, making it the best choice during cloning or construct formation where intramolecular ligation is desir
