DNA and RNA Structure

Overview

Every molecular test - extraction, PCR, electrophoresis, sequencing - works by exploiting the chemistry of two polymers, DNA and RNA. Both are long, unbranched chains of nucleotides, and the order of the nucleotides carries the genetic information ¹.

The nucleotide

A nucleotide has three parts: a five-carbon sugar, one to three phosphates, and a nitrogenous base ². Sugar plus base alone is a nucleoside; add a phosphate and it’s a nucleotide.

The sugar. Its carbons are numbered 1’ to 5’ (“one prime”), the primes separating them from the base’s atoms. The base hangs off 1’, the phosphate off 5’, and the next nucleotide links at 3’.

Deoxy- means exactly that missing oxygen at 2’ ².

The phosphate. Free nucleotides carry one, two, or three phosphates. The triphosphates (dATP, dGTP, dCTP, dTTP for DNA) are what polymerases consume: breaking off two phosphates pays for adding the nucleotide to a chain ¹. These are the “dNTPs” in every PCR master mix.

The bases.

Thymine is uracil with a methyl group added ¹. The standard hooks: “Pure As Gold” (purines are A and G) and “CUT the Py” (pyrimidines are C, U, T).

The backbone

Phosphodiester bonds join the 5’ phosphate of one nucleotide to the 3’ hydroxyl of the next, making a sugar-phosphate backbone with the bases sticking out to the side ².

Because every link runs 3’ to 5’, a strand has a direction: a free 5’ phosphate at one end, a free 3’-OH at the other. Sequences are always written 5’ to 3’, and polymerases only add to the 3’ end, so all synthesis runs 5’→3’ ¹. That one rule explains primer design, the lagging strand in replication, and why a primer with a mismatched 3’ end fails to extend.

Double-stranded DNA pairs two strands running in opposite directions (antiparallel):

5'-ATGC-3'
3'-TACG-5'

RNA is usually single-stranded but folds back on itself into hairpins and other local double-stranded regions.

Three properties the lab depends on

A negative backbone. Each phosphate is negatively charged at buffer pH, so a nucleic acid has a uniform negative charge proportional to its length. In a gel every fragment runs toward the positive electrode (anode), and the gel matrix sorts them by size ³. The same charge is what lets nucleic acids bind silica in chaotropic salt during extraction.

Fragile RNA. Under alkaline conditions RNA’s 2’-OH attacks the adjacent phosphodiester bond and cuts the chain; DNA, without the 2’-OH, resists this ³. RNA is also attacked by ubiquitous, hardy RNases, so RNA work needs RNase-free reagents, gloves, and cold handling. Alkaline treatment is used deliberately to remove RNA from DNA preparations.

Absorbance at 260 nm. The base rings absorb UV light maximally near 260 nm, so A260 measures how much nucleic acid is present; proteins peak near 280 nm, so the A260/A280 ratio flags protein contamination ³. Spectrophotometry and its purity ratios come back in the nucleic acid prep course.

DNA vs. RNA at a glance

What to take from this

A nucleotide is a sugar, phosphate, and base. DNA differs from RNA at the sugar’s 2’ carbon (H vs OH) and in using thymine instead of uracil. The backbone runs 5’→3’, strands pair antiparallel, and synthesis always adds to the 3’ end. The negative backbone drives electrophoresis, the 2’-OH makes RNA fragile, and base rings absorbing at 260 nm let you measure nucleic acid.