Base Pairing, the Double Helix, and Melting Temperature

Complementary base pairing

Two antiparallel strands are held together by hydrogen bonds between facing bases, and the pairing is fixed: A with T, G with C ¹.

Because each base has one partner, one strand’s sequence completely determines the other’s. That is why replication is faithful and why a known strand can find its complement in a sample - the basis of every probe, primer, and capture method.

Every pair is one two-ring purine plus one one-ring pyrimidine, so every rung of the ladder is the same width and the backbones stay evenly spaced regardless of sequence ².

Writing a complement is a daily skill. The complement of 5’-ATGGCA-3’ is 3’-TACCGT-5’, which written in the standard 5’→3’ direction is 5’-TGCCAT-3’ (the reverse complement). Primers, probes, and sequence reads are always handled as reverse complements.

The double helix

The paired strands twist into a right-handed helix. Under physiological conditions DNA takes the B form: bases stacked nearly perpendicular to the axis, about 10 base pairs per turn ¹. The two backbones aren’t directly opposite each other, so the surface has a wide major groove and a narrow minor groove. Base edges are exposed in the grooves, letting proteins read the sequence without unzipping it; the major groove carries more information.

Base stacking

Hydrogen bonds pair the strands, but much of the helix’s stability comes from stacking: the flat aromatic bases pack face-to-face along the axis, held by van der Waals contacts and by keeping their hydrophobic faces out of water ². Stacking depends on which bases are neighbors, so sequence order - not just G+C content - affects stability. This is why accurate Tm calculators use nearest-neighbor tables.

Denaturation and renaturation

Heat, high pH, or chemicals like formamide break the hydrogen bonds and stacking, and the strands separate: denaturation (melting). The covalent backbone is untouched, so each strand keeps its sequence ³.

Remove the denaturing condition - cool slowly - and complementary strands find each other again: renaturation or annealing. PCR does both every cycle: 95 °C to melt, a lower temperature to anneal primers.

Single strands absorb more UV at 260 nm than paired strands (the hyperchromic effect), so melting can be followed on a spectrophotometer.

Melting temperature

Tm is the temperature at which half the duplexes in a sample are melted ³. It summarizes how stable a duplex is.

Rule of thumb: anything that strengthens the grip between strands raises Tm.

Mg²⁺ matters in PCR: the magnesium in a master mix raises primer Tm as well as feeding the polymerase, so changing Mg²⁺ changes specificity.

Estimating Tm for a primer

For short oligonucleotides (roughly 14-20 bases) the Wallace rule gives a quick estimate:

Tm ≈ 2 °C × (A + T) + 4 °C × (G + C)

A 20-mer with 10 G/C and 10 A/T: 2(10) + 4(10) = 60 °C. It ignores salt and neighbor effects, so real assay design uses nearest-neighbor software, but it’s good for sanity checks: PCR primers are usually designed with Tm in the high 50s to mid 60s °C, matched within a few degrees of each other, and about 40-60% GC. The annealing temperature is typically set a few degrees below the primers’ Tm.

Hybridization and stringency

Hybridization is annealing between strands from different sources: a probe and a target, or DNA and RNA. Any two sufficiently complementary strands can hybridize.

Stringency sets how perfect the match must be ³:

Hybridization assays usually bind at moderate stringency and then wash at high stringency to strip off mismatched probe.

What to take from this

A pairs with T by two hydrogen bonds and G with C by three, purine to pyrimidine; one strand dictates the other, so think in reverse complements. Stacking adds much of the helix’s stability. Melting separates strands without breaking the backbone, and Tm is the halfway point; G-C content, length, and salt raise it, formamide and mismatches lower it. The Wallace rule (2 × AT + 4 × GC) gives a rough primer Tm, and stringency (temperature and salt) decides how much mismatch a probe tolerates.