18.3 Recombinant DNA & Genetic Technologies

Recombinant DNA & Genetic Technologies

PA-CAT Bulletin of Information, rev. 20240815, Table 8, lists Genetic Technologies as a core genetics competency. These tools let researchers cut, copy, read, and edit DNA, and they appear in both molecular-biology and medical-genetics items.

Restriction Enzymes

Restriction endonucleases recognize specific palindromic DNA sequences (commonly 4–8 bp) and cut at defined positions, producing sticky ends (5' or 3' single-stranded overhangs) or blunt ends. Example: EcoRI recognizes GAATTC and cuts between G and A, leaving 5' AATT overhangs. Sticky ends allow fragments from different sources to anneal by base pairing and be joined permanently by DNA ligase. The collection of fragments from a single genome cut with one enzyme is its restriction map.

Cloning Vectors

A vector carries foreign DNA into a host cell for propagation or expression. Common vector types:

Essential vector features include an origin of replication, a multiple cloning site, a selectable marker (e.g., ampicillin resistance), and often a blue-white screen based on lacZα complementation so that colonies carrying recombinant plasmids can be distinguished from those with empty vector.

PCR (Polymerase Chain Reaction)

PCR amplifies a defined target sequence exponentially. A reaction contains template DNA, two primers flanking the target, a thermostable DNA polymerase (originally Taq, from Thermus aquaticus), dNTPs, and buffer with Mg²⁺. Each cycle has three steps:

1. Denaturation (~95 °C) — the double helix melts into single strands.
2. Annealing (~50–65 °C) — primers bind to their complementary sites.
3. Extension (~72 °C) — Taq synthesizes new strands 5'→3'.

After n cycles, the target copy number is approximately 2ⁿ. Applications include clinical diagnostics, forensic identity, cloning, and sequencing-library preparation. RT-PCR (reverse transcription PCR) starts from RNA and uses reverse transcriptase to make cDNA first, measuring expression. qPCR (real-time quantitative PCR) tracks fluorescence each cycle to quantify starting template.

Gel Electrophoresis

DNA is negatively charged because of its phosphate backbone and migrates toward the anode (+) through an agarose (large fragments) or polyacrylamide (small, high-resolution) matrix. Smaller fragments migrate faster; a DNA ladder provides size standards. Pulsed-field gel electrophoresis (PFGE) separates very large molecules by periodically reorienting the electric field. DNA is most often visualized with ethidium bromide (intercalates and fluoresces under UV) or safer alternatives such as SYBR Safe.

DNA Sequencing

• Sanger sequencing — chain-termination method using dideoxynucleotides (ddNTPs) that lack the 3'-OH needed for chain extension. A small fraction of ddNTPs in the reaction produces fragments ending at every base; fragments are separated by capillary electrophoresis and read as a sequence chromatogram. Read length ~800–1000 bp.
• Next-generation sequencing (NGS) — massively parallel sequencing of millions of templates. Common platforms:
  • Illumina — sequencing-by-synthesis with reversible terminators; dominant for short-read whole-genome, exome, and RNA-seq work.
  • Ion Torrent — detects pH changes (H⁺ release) on incorporation.
  • Oxford Nanopore — measures electrical changes as DNA passes through a protein pore; very long reads.
  • PacBio SMRT — single-molecule real-time sequencing; long reads for structural variation and repetitive regions.

CRISPR-Cas9

CRISPR-Cas9 is a genome-editing system adapted from a bacterial adaptive immune defense. A synthetic guide RNA (gRNA) base-pairs with a ~20-nt target sequence; the Cas9 nuclease then creates a blunt double-strand break adjacent to a PAM (protospacer adjacent motif, NGG for Streptococcus pyogenes Cas9). The break is repaired by non-homologous end joining (NHEJ), which is error-prone and produces insertions/deletions that knock out a gene, or by homology-directed repair (HDR) using a donor template, which enables precise edits. Variants include catalytically inactive dCas9 for CRISPR interference/activation (CRISPRi/CRISPRa) and base editors.

Libraries and Blotting

A genomic library is a collection of cloned DNA fragments covering an organism's entire genome. A cDNA library is made from mRNA using reverse transcriptase, so it represents only expressed genes (no introns, no promoters). The three classic blotting techniques identify specific macromolecules from a mixture separated by gel electrophoresis:

Mnemonic: SNOW DROP — Southern = DNA, Northern = RNA, Western = Protein. Each blot's first letter cues the molecule it detects.