Biotechnology

To clone a gene: (1) cut both the target DNA and the plasmid with the same restriction enzyme; (2) mix and let sticky ends anneal; (3) join with DNA ligase to form recombinant plasmid; (4) transform the plasmid into host bacteria; (5) grow on selective medium so only cells that took up the plasmid survive; (6) each colony is a clone that amplifies the insert as it divides. This produces many identical copies of the gene — distinct from reproductive cloning (e.g., Dolly the sheep by somatic-cell nuclear transfer).

In-vitro amplification that doubles a target DNA segment each cycle (exponential: ~2ⁿ copies after n cycles). Requires template DNA, two primers, dNTPs, and heat-stable Taq polymerase (from Thermus aquaticus, so it survives the denaturation step). Each cycle has three temperature steps:

• Denaturation (~95°C) — strands separate.
• Annealing (~50–65°C) — primers bind their complementary sequences.
• Extension (~72°C) — Taq synthesizes new strands 5′→3′.

Invented by Kary Mullis (Nobel 1993). RT-PCR first converts RNA to cDNA (used to detect RNA viruses); qPCR quantifies the product in real time.

Separates nucleic acids (or proteins) by size in an agarose gel under an electric field. DNA’s phosphate backbone is negatively charged, so fragments migrate toward the positive electrode (anode). The gel acts as a molecular sieve: smaller fragments travel farther, larger ones lag near the well. A size ladder run alongside lets you estimate fragment length. It is the read-out step for restriction digests, PCR, and fingerprinting.

Sanger “chain-termination” sequencing reads the order of bases. A single-stranded template, a primer, DNA polymerase, and normal dNTPs are mixed with a small amount of fluorescently labeled dideoxynucleotides (ddNTPs). A ddNTP lacks the 3′-OH, so whenever one is incorporated the growing strand terminates. This yields fragments of every possible length, each ending in a known base; separating them by size (capillary electrophoresis) and reading the terminal-base fluorescence reconstructs the sequence. Next-generation sequencing now reads millions of fragments in parallel.

A programmable editing tool adapted from a bacterial antiviral defense system. A guide RNA (gRNA) base-pairs with a chosen genomic target and directs the Cas9 nuclease to make a precise double-strand break there (a short PAM sequence must sit next to the target). The cell repairs the break by error-prone NHEJ (knocks a gene out) or, if a donor template is supplied, by homology-directed repair (inserts or corrects a sequence). CRISPR is faster, cheaper, and more precise than older editing tools and is in clinical trials for sickle-cell disease. Its programmability comes from the RNA guide — a favorite MCAT point.

The first genetically engineered human therapeutic.

• Human insulin gene is isolated and cut with a restriction enzyme.
• A bacterial plasmid is cut with the same enzyme to give matching sticky ends.
• DNA ligase joins the insulin gene into the plasmid, forming recombinant DNA.
• The plasmid is inserted into E. coli, which is grown in fermenters.
• Bacteria express insulin, which is purified for diabetic patients.

Recombinant insulin replaced the older porcine and bovine insulin, which often caused allergic reactions.

The hepatitis B vaccine is produced by inserting the HBsAg (hepatitis B surface antigen) gene into yeast. The yeast secretes the antigen, which is purified and used as a safe, non-infectious vaccine. Other recombinant products: human growth hormone (somatotropin), erythropoietin, factor VIII (hemophilia), interferons.

Insertion of a functional gene into a patient's cells to treat a genetic disorder. Somatic gene therapy targets body cells (not heritable). Germline gene therapy targets gametes/embryos (heritable; banned in humans). Most often delivered by viral vectors — retroviruses integrate into the genome, adeno-associated viruses give durable non-integrating expression. Pioneered for ADA-SCID ("bubble boy" disease) in 1990.

Identical antibodies that all bind a single epitope, made by hybridoma technology: fuse an antibody-producing B cell with an immortal myeloma cell to get a hybrid that both secretes one specific antibody and divides indefinitely. Uses include pregnancy tests and blood typing (diagnostics) and targeted therapy — e.g., trastuzumab (Herceptin) against HER2+ breast cancer and rituximab against B-cell lymphoma. The -mab suffix flags a monoclonal antibody drug.

Developed by Sir Alec Jeffreys (1984). Uses variable number tandem repeats (VNTRs) or short tandem repeats (STRs) that differ between individuals. Each person (except identical twins) has a unique pattern. Applications: paternity testing, forensic identification, mass-disaster victim ID.

Stem cells are unspecialized cells capable of self-renewal and differentiation. Sources: embryonic stem cells, adult/somatic stem cells, induced pluripotent stem cells (iPSCs). Used or studied for leukemia (bone marrow transplant), Parkinson's disease, spinal cord injury, type-1 diabetes.

ELISA uses an enzyme-linked antibody (color change = positive) to detect a specific antigen or antibody (HIV screening, COVID-19 antibody testing). PCR detects pathogen DNA/RNA at very low copy numbers (TB, HCV, COVID-19). Together they are the backbone of modern infectious-disease diagnosis.