The restriction enzyme EcoRI recognizes the sequence:

5'-G/AATTC-3'

3'-CTTAA/G-5'

It cuts between the G and A as noted by the slashes. Following the digestion of chromosomal DNA and vector DNA with EcoRI, let's suppose that one fragment of chromosomal DNA gets inserted into a vector. No DNA ligase has been added. If both of the sticky ends of the chromosomal DNA fragment are binding to the sticky ends of the vector, what is holding these two pieces of DNA together?

Question

The restriction enzyme EcoRI recognizes the sequence:

5'-G/AATTC-3'

3'-CTTAA/G-5'

It cuts between the G and A as noted by the slashes. Following the digestion of chromosomal DNA and vector DNA with EcoRI, let's suppose that one fragment of chromosomal DNA gets inserted into a vector. No DNA ligase has been added. If both of the sticky ends of the chromosomal DNA fragment are binding to the sticky ends of the vector, what is holding these two pieces of DNA together?

BlackTom AI · Accepted Answer

A 4-base 5' overhang "AATT" produced by EcoRI yields four A–T base pairs at each cohesive end; each A–T pair contributes 2 hydrogen bonds, so two annealed sticky ends give 16 hydrogen bonds in total.

1) Relevant knowledge points and concepts
- EcoRI cuts between G and A, leaving a 5' overhang of 4 bases: 5'-A A T T-3'.
- Complementary base pairing: A pairs with T via 2 hydrogen bonds; G pairs with C via 3 hydrogen bonds.
- Without DNA ligase, annealing of complementary sticky ends is held together noncovalently (hydrogen bonds and base stacking), not by phosphodiester bonds.

2) Step-by-step reasoning
- Write the overhang sequences produced by EcoRI on a fragment and on the vector:
  Top strand overhang: 5'-A A T T-3'
  Bottom complementary overhang: 3'-T T A A-5'
- When the insert sticky end anneals to the vector sticky end, each position forms an A–T pair.

3) Intermediate calculation steps
- Number of base pairs formed at one cohesive end = 4 (A–T, A–T, T–A, T–A — all A–T pairs).
- Hydrogen bonds per A–T pair = 2.
- Hydrogen bonds per end = 4 pairs × 2 H-bonds/pair = 8 H-bonds.
- Two annealed ends present when an insert is ligated or annealed into a vector = 2 × 8 = 16 H-bonds.

4) Why the correct answer is correct
- Because EcoRI leaves a 4-base AATT overhang, annealing produces four A–T base pairs per end; two hydrogen bonds per A–T yield 8 per end and 16 total for both ends. In the absence of ligase no covalent bonds form, so these 16 hydrogen bonds (plus base stacking and ionic interactions) are what hold the pieces together.

5) Why the other choices are incorrect
- 8 hydrogen bonds would correspond to only one end annealing (not both ends).
- 11, 14, 22, 28 are inconsistent with integer multiples of 2 H-bonds per A–T (and none match 2 × 4 × 2 = 16). No G–C pairs are present here to change the count.

6) Conclusion
The two annealed EcoRI sticky ends are held together by 16 hydrogen bonds, confirming the provided answer: 16 hydrogen bonds.

类似问题

From the DNA sequence 5' ATGGCGT 3', what is expected to be its complementary DNA sequence?

What is the complementary DNA strand of 3’….ATGGCTTGA….5’?

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What is the sequence of the complementary DNA strand of the sequence shown below. 5’….CCAGATTTCAGCG….3'?

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