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Short answer
A heteroduplex is a double-stranded DNA molecule whose two strands are not perfectly complementary, so it carries one or more mismatches. The mismatch distorts the helix, which is why heteroduplexes migrate more slowly than matched homoduplexes on polyacrylamide gels and why certain enzymes cut them at the mismatch. Labs use both properties to detect sequence differences.
Definition and scope
Bio-Rad’s technical note on heteroduplex analysis describes heteroduplex molecules as having “a mismatch in the double strand, causing a distortion in its usual conformation.” A homoduplex, by contrast, pairs two fully complementary strands. The term applies to any mismatch, from a single-nucleotide substitution to a short insertion or deletion, and the same physical idea underlies two families of bench methods: gel-mobility analysis and enzyme mismatch cleavage.
This page is a conceptual explainer for researchers and core-facility staff. It does not give a runnable protocol; consult the instructions of the instrument or kit you use.
How heteroduplexes arise in practice
The Bio-Rad note lists the routes by which a sample ends up containing heteroduplexes. Each one needs two different versions of the same sequence present in one tube:
- PCR of a heterozygous individual. Amplicons from both alleles can cross-anneal, producing mismatched strands alongside the two homoduplexes. This was first reported as a PCR artifact (Nagamine et al., Hum Genet, 1989).
- Mixing mutant and wild-type DNA in the same PCR. The analyst deliberately combines the two templates.
- Denaturing and renaturing a mixture. Mutant and wild-type DNA are heated apart and allowed to re-anneal, so strands from different sources pair up.
In all three, a sample containing only one sequence cannot form a heteroduplex, which has a practical consequence discussed under common misunderstandings.
How labs detect heteroduplexes
Gel mobility (heteroduplex analysis)
According to the Bio-Rad note, heteroduplexes are detected on polyacrylamide gels because they migrate more slowly than the corresponding homoduplexes, and “as little as one mismatch can show a different mobility.” Matrices such as Bio-Rad’s detection-enhancing matrix (DEM) are polyacrylamide analogs that improve detection. In the note’s cystic fibrosis example, a gel was run in 0.6x TBE at 100 V for 16 hours at room temperature with 200-300 ng of DNA per lane and ethidium bromide staining. Heterozygous mutant samples showed two upper heteroduplex bands and a lower homoduplex band. For background on the gel format, see what gel electrophoresis is and polyacrylamide gels in SDS-PAGE.
Enzyme mismatch cleavage
Mashal, Koontz and Sklar (Nat Genet 1995;9:177-83) showed that bacteriophage T4 endonuclease VII and T7 endonuclease I cleave heteroduplex DNA at mismatches. In their tests, 3 of 3 short deletions and 13 of 14 point mutations were cleaved, in fragments up to 940 bp; all four classes of single-nucleotide mismatch were cut, and the cleavage product sizes correlated with mutation location. Vouillot, Thelie and Pollet (G3 2015;5:407-15) describe enzyme mismatch cleavage (EMC) as using enzymes, bacteriophage resolvases or single-strand nucleases, that cut at mismatches and at extrahelical loops from one or several nucleotides.
This is the basis of the T7 endonuclease I (T7E1) and Surveyor assays used to check for nuclease-induced edits; see sgRNA design and base editing for the editing context.
Comparison of the two readouts
Every row below is drawn from the three sources cited at the end of this page.
| Feature | Gel mobility (Bio-Rad note) | Enzyme mismatch cleavage (Mashal 1995; Vouillot 2015) |
|---|---|---|
| What reports the mismatch | Slower migration of the heteroduplex band on a polyacrylamide gel | Cleavage at the mismatch by a resolvase or single-strand nuclease |
| Sensitivity stated | As little as one mismatch can change mobility | 13 of 14 point mutations and 3 of 3 short deletions cleaved in the original study |
| Location information | Not described in the note | Cleavage product sizes correlated with mutation location |
| Needs two sequence versions in the tube | Yes: a heteroduplex must form | Yes: the enzymes act on heteroduplex DNA |
Where it matters for labs
Two situations from the sources are worth planning around.
- Genotyping and mutation screening. In the Bio-Rad example, heterozygous samples were easy to read from the two heteroduplex bands above the homoduplex band.
- Genome-editing checks. Vouillot et al. compared T7E1 and Surveyor on zinc-finger-nuclease-induced mutations in frog embryos. In that system T7E1 was more sensitive with deletion substrates, Surveyor was better for single-nucleotide changes, and the authors concluded T7E1 was preferred for nuclease-triggered mutations. This is one study in one system, and mutation efficiency varies by locus, so the hit rate at your own locus must be evaluated. A sequencing readout such as Sanger sequencing reports the sequence directly rather than inferring a mismatch.
Common misunderstandings
“A homozygous mutant will always show a different band.”
Not necessarily. In the Bio-Rad example, a homozygous DF508 mutant showed no heteroduplex bands, and its homoduplex migrated like wild type. It became detectable only when mutant DNA was mixed with wild-type DNA, denatured and renatured. Because a single-sequence sample cannot form a heteroduplex, the analyst has to supply the second sequence deliberately.
“Extra bands after PCR mean contamination.”
They can instead be heteroduplexes. Nagamine and colleagues (1989) described the generation of heteroduplexes as a PCR artifact when two allelic versions are amplified together.
“Enzyme cleavage and gel mobility are interchangeable.”
Both depend on a mismatch, but they report it differently: one as a mobility shift, the other as fragments whose sizes correlate with mutation location. Neither source claims the methods give identical results on every sample.
Sources
- Bio-Rad Laboratories, Tech Note 2105 Rev C, “Heteroduplex Analysis of Cystic Fibrosis Samples on the DCode System” (Zoller, Redila-Flores). bio-rad.com. Read 2026-11-08.
- Mashal RD, Koontz J, Sklar J. Detection of mutations by cleavage of DNA heteroduplexes with bacteriophage resolvases. Nat Genet 1995;9(2):177-83. PMID 7719346. Read 2026-11-08.
- Vouillot L, Thelie A, Pollet N. Comparison of T7E1 and Surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. G3 2015;5(3):407-15. PMID 25566793. Read 2026-11-08.
