Molecular Biology Principles, Genetics, and Basic Techniques
Nucleic acid chemistry
Every assay rests on base-pairing rules: adenine pairs with thymine by two hydrogen bonds, guanine with cytosine by three. Higher GC content therefore raises melting temperature (Tm) and buoyant density, and lowers the salt concentration needed to destabilize a duplex. Polymerases synthesize only 5'→3', extending a 3'-OH, and proofreading activity is a 3'→5' exonuclease. Reverse transcriptase copies RNA into cDNA and carries RNase H activity. Restriction endonucleases cut palindromic recognition sites and are blocked by methylation.
Gene structure and variation
A typical gene has a promoter, 5' untranslated region, exons and introns bounded by GT-AG splice sites, and a 3' untranslated region. Variant classes are silent, missense, nonsense, frameshift, splice-site, and expansion (repeat) variants. Candidates must also handle inheritance patterns, penetrance, variable expressivity, imprinting, and mosaicism, and read HGVS nomenclature (c. for coding DNA, p. for protein).
Techniques
Southern blot detects DNA, Northern blot RNA, Western blot protein. Hybridization stringency rises with temperature and falls with salt or formamide; mismatches lower Tm. Electrophoresis separates by size (agarose for fragments, polyacrylamide for small products and sequencing). Sanger sequencing uses dideoxy terminators and capillary electrophoresis.
Traps
Questions often confuse the blot types, assume silent variants have no effect (they may alter splicing or codon usage), or ask for the direction of polymerase synthesis. Watch for Tm items where salt or formamide is changed.
Rule of thumb
For short oligos, Tm ≈ 2(A+T) + 4(G+C) °C; for long duplex DNA each 1% GC adds roughly 0.4 °C. Example: a 20-mer with 8 A/T and 12 G/C gives 2(8) + 4(12) = 64 °C, so annealing at about 59 °C is appropriate.