19.1.4—Enzymes and vectors in gene transfer

Different enzymes perform different jobs in preparing and joining a desired gene: restriction endonucleases cut DNA at specific sequences, reverse transcriptase makes cDNA from mRNA, DNA polymerase completes the cDNA into double-stranded DNA, and DNA ligase joins DNA backbones.

Restriction endonuclease — cuts the DNA or DNA construct at a recognised base sequence. An uneven cut can leave sticky ends, whose exposed bases can hydrogen-bond with complementary ends made by the same enzyme.
Reverse transcriptase — uses the mRNA for the desired gene as a template to make single-stranded cDNA; because processed mRNA lacks introns, the cDNA also lacks those introns.
DNA polymerase — adds complementary nucleotides to the single-stranded cDNA to make a double-stranded gene sequence.
DNA ligase — forms phosphodiester bonds in the sugar–phosphate backbone so the isolated gene can be joined into the DNA construct.

A typical enzyme order is:

1. Obtain or isolate the desired sequence.
2. Use compatible restriction cuts when a DNA construct is being prepared.
3. Use reverse transcriptase and then DNA polymerase if the source is mRNA.
4. Use DNA ligase to seal the joined DNA.
   The exact branch depends on the gene source.

Restriction endonucleases cut the surrounding DNA or DNA construct, not “the gene” as an enzyme role; the same restriction enzyme is used to make compatible ends. These enzymes are not interchangeable: cutting, copying and joining are separate jobs. The matched SME page does not require vector, promoter, marker, PCR or gel mechanisms here; those are separate cards.