Reverse complement: strands, 5′→3′ and why it matters

Double-stranded DNA is antiparallel: the two strands run in opposite directions. So the sequence of the other strand is not just the complement — it is the complement read backwards. That is the reverse complement.

Complement, then reverse

Every base pairs with one partner: A with T, and G with C (N, an unknown base, stays N). Swapping each base for its partner gives the complement. But because the opposite strand runs 3′→5′ against your 5′→3′ strand, you flip the order to write it the conventional way, 5′→3′. Complement and reverse.

Take ATCGGA. Complement each base and you get TAGCCT; reverse that and you get TCCGAT. So the reverse complement ofATCGGA is TCCGAT — a result you can check by hand.

Why it comes up constantly

Reverse primers: a reverse PCR primer is designed as the reverse complement of the template’s top strand at the 3′ end of your amplicon.
Minus-strand features: a gene annotated on the minus strand reads in the reverse-complement direction relative to the reference’s top strand.
Checking oligos: to confirm a probe or guide binds its target, compare it against the reverse complement of the target site.

Where it trips people up

The two easy mistakes are complementing without reversing (which gives the strand read the wrong way), and reversing without complementing (which gives the same strand backwards). You need both. A palindrome hides this: some sequences are their own reverse complement — GAATTC (the EcoRI site) returns GAATTC — which is exactly why many restriction enzymes cut palindromic sites on both strands at once.