Question 1

What is DNA ligation and how does it work in cells?

Accepted Answer

DNA ligation is an enzymatic reaction that joins two DNA molecules by forming a covalent phosphodiester bond. DNA ligases identify and seal breaks in the DNA backbone by catalyzing bond formation between the 3'-hydroxyl and 5'-phosphate groups. In cells, ligation occurs during DNA replication and repairs single and double strand breaks as part of normal cellular processes.

Question 2

Why are sticky ends preferred over blunt ends in DNA ligation?

Accepted Answer

Sticky ends contain complementary overhanging base pairs that stabilize the ligation reaction, making the process more efficient and easier for the enzyme to join DNA fragments. Blunt ends lack complementary base pairing, resulting in less efficient ligation that is more difficult for the enzyme to complete. Sticky and blunt ends cannot be ligated together under normal circumstances.

Question 3

How does the Klenow fragment convert sticky ends to blunt ends?

Accepted Answer

The Klenow fragment, derived from DNA polymerase 1 digested with subtilisin, possesses both 3' to 5' exonuclease activity and polymerase activity. The exonuclease activity removes 3' overhangs, while the polymerase activity extends the 3' end of the complementary strand to blunt 5' overhangs, effectively converting sticky ends to blunt ends.

Question 4

What is the purpose of alkaline phosphatase treatment in ligation reactions?

Accepted Answer

Alkaline phosphatase removes 5' phosphate groups from both ends of vector DNA after restriction enzyme digestion. This prevents self-ligation, an undesirable outcome where the vector reseals itself without incorporating the insert DNA, thereby increasing the probability that the insert will be ligated into the vector.

Question 5

Why is a 3 to 1 insert to vector ratio used in ligation reactions?

Accepted Answer

A 3 to 1 molar ratio of insert DNA to vector DNA increases the probability that the insert will be ligated into the vector rather than the vector ligating to itself. This ratio is calculated based on DNA concentrations measured by spectrophotometer and the molecular weights of the purified vector and insert fragments to determine appropriate volumes for the reaction.

Question 6

How do temperature and duration differ between sticky and blunt end ligations?

Accepted Answer

Sticky end ligations with six base pair overhangs can be performed near room temperature for approximately one hour because complementary ends stabilize fragment joining. Short overhangs or blunt end ligations require lower temperatures between 14-20°C and should be incubated overnight to allow sufficient time for the less efficient joining process.

Question 7

What are common applications of DNA ligation in molecular biology research?

Accepted Answer

DNA ligation is used to insert PCR-amplified fragments into linearized plasmids for creating gene libraries, attach oligonucleotide linkers containing PCR primer binding sites to purified DNA fragments for sequencing, and perform proximity ligation assays on formaldehyde-fixed cells to identify chromatin interactions at various scales.

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