Question

Which of the following components of the strategy ensures that sequence reads are obtained from the ends of cloned fragments?

1) The use of fluorescently labeled primers
2) The use of primers that anneal to vector sequences near vector/cloned fragment boundaries
3) The use of primers that anneal to cloned fragment sequences near vector/cloned fragment boundaries
4) Use of a restriction enzyme such as SmaI that generates blunt ends

Answer 1

The use of primers that anneal to vector sequences near vector/cloned fragment boundaries provides sequence reads from the ends of cloned fragments, essential for verifying correct DNA insertion.

Step-by-step explanation:

The component of the strategy that ensures sequence reads are obtained from the ends of cloned fragments is the use of primers that anneal to vector sequences near vector/cloned fragment boundaries.

In molecular biology, cloning DNA fragments into vectors is a fundamental technique used for studying genes, producing recombinant proteins, and many other applications. A critical step in the cloning process involves sequencing the DNA to confirm the presence and correct orientation of the cloned fragment within the vector. To achieve this, primers are designed to anneal, or bind, to the known sequence of the vector close to where the vector and inserted DNA fragment meet.

The choice of primers is crucial as it ensures that the sequencing reaction starts exactly at the ends of the cloned DNA fragment. This is because DNA polymerase, the enzyme responsible for copying DNA during sequencing, will extend the primers along the adjacent DNA therefore capturing the sequence of the insert. By using primers that anneal to the vector sequences, researchers can obtain sequence reads that accurately reflect the DNA composition at the vector/cloned fragment boundaries, which is essential for verifying the correct insertion of the target DNA within the vector.