Recombinant DNA

Recombinant DNA technology rests on a small toolkit. Restriction endonucleases cut double-stranded DNA at specific palindromic recognition sequences, often leaving single-stranded overhangs called sticky ends. DNA ligase seals the phosphodiester backbone between fragments whose ends are compatible. Because the same enzyme cuts every DNA molecule at the same sequence, a gene from one organism and a vector from another can be cut with the same enzyme and joined together.

The joined molecule is placed in a vector — commonly a bacterial plasmid, but also viral vectors or artificial chromosomes for larger inserts. Plasmids carry an origin of replication so they copy independently, a selectable marker such as an antibiotic resistance gene, and a multiple cloning site. After transformation into a host such as E. coli, only cells that took up the plasmid survive on selective media, and each one multiplies the insert as it divides. If expression is the goal, an appropriate promoter drives transcription of the inserted gene, and the host translates the resulting mRNA into protein.

Some steps require additional tools. Because bacteria cannot splice introns, eukaryotic genes are usually introduced as cDNA, synthesized from mature mRNA by reverse transcriptase. PCR amplifies a target sequence before cloning and can add restriction sites to the ends of a product. Gel electrophoresis separates fragments by size to confirm that a construct is correct.

The practical payoff is large: human insulin, growth hormone, clotting factors, and the hepatitis B vaccine are all recombinant products, and the same methods underpin gene therapy and molecular diagnosis of genetic disease. The MCAT covers recombinant DNA and biotechnology in the biological and biochemical foundations section, expecting familiarity with restriction enzymes, vectors, cDNA libraries, and PCR. USMLE Step 1 applies the diagnostic side of these techniques to inherited disorders such as the thalassemias.