Question

Digesting away protruding 3' overhangs: This is another method for producing ___ ends on DNA; ends generated from restriction enzymes that cleave to produce 3' overhangs. The 3' -> 5' exonuclease activity of Klenow will digest away the protruding 3' overhang. Removal of nucleotides from the 3' ends will continue, but, in the presence of nucleotides, the polymerase activity will balance the exonuclease activity, yielding blunt ends.

Answer 1

Blunt ends on DNA are produced by using the Klenow fragment's exonuclease activity to trim 3' overhangs created by restriction enzymes like HaeIII. These blunt ends can then be modified and ligated to create recombinant DNA molecules for cloning into plasmids.

Step-by-step explanation:

Digesting away protruding 3' overhangs to produce blunt ends is a method involving the Klenow fragment of DNA polymerase I which possesses 3'→5' exonuclease activity. This activity can be utilized to trim the 3' overhangs given by specific restriction enzymes to transform them into blunt ends. For example, both target DNA and vector DNA can be acted upon by the restriction endonuclease HaeIII to generate blunt ends. Subsequent modification with enzyme terminal transferase allows for the addition of poly 'G' tails and poly 'C' tails to aid in recombinant DNA (rDNA) formation. This is a crucial step in molecular cloning where DNA ligase can then be used to permanently bind these blunt-ended molecules together, allowing for the creation of modified DNA sequences that can be inserted into plasmids and used for a variety of research purposes.