Why the reverse complement matters
DNA has two strands that run in opposite directions. Sequences are written 5′ to 3′, so the other strand, read in its own 5′ to 3′ direction, is the reverse complement. You need it to design a reverse primer, to read a gene that sits on the minus strand, or to compare a sequencing read that came off the opposite strand.
Work it out by hand
- Swap every base for its partner: A ↔ T and G ↔ C.
- Read the result backwards.
For 5′-ATGCCTAG-3′ the complement is TACGGATC and the reverse complement is 5′-CTAGGCAT-3′. Reversing without complementing gives GATCCGTA, which is a different sequence and a common mistake.
Ambiguity codes and RNA
IUPAC codes have partners too: R (A or G) pairs with Y (C or T), K with M, and B, D, H and V pair with V, H, D and B. S, W and N stay as they are. In RNA, U takes the place of T.
Do it in the browser
- Open Reverse Complement.
- Paste one sequence, or many in FASTA format.
- Pick reverse complement, reverse or complement. Titles, case and ambiguity codes are kept.
To change T to U instead, use DNA to RNA.
Designing a reverse primer
A reverse primer binds the end of the region you want to copy, on the other strand. Take the last 18 to 25 bases of the top strand and reverse complement them. If the region ends with 5′-ATGGCTAGCAAGGAGGTCTTA-3′, the reverse primer is 5′-TAAGACCTCCTTGCTAGCCAT-3′.
Sequences that are their own reverse complement
Many restriction sites read the same on both strands. GAATTC, the EcoRI site, complements to CTTAAG, which read backwards is GAATTC again. Such palindromes are why a site appears once whichever strand you search.
Common problems
- Numbers and spaces in the result. Sequences copied from GenBank or a paper may carry position numbers and spaces. Clean them out first, or use a tool that ignores them.
- Ordering the complement, not the reverse complement. A primer ordered as the plain complement reads 3′ to 5′ and will not work. Always write and order primers 5′ to 3′.
- Lowercase letters. Lowercase bases often mark repeats or primer tails; a good tool keeps the case on each base when it reverses.