Just different words for exactly the same thing...both hemi and semi mean half

The difference between hemi-nested PCR and semi-nested PCR lies in the specific arrangement and combination of primers used during the second round of amplification.

In semi-nested PCR, two rounds of amplification are performed: in the first round, a set of two external primers (forward and reverse) amplifies a target region. In the second round, one of the primers (either forward or reverse) from the first round is reused, while the other primer is replaced by an internal primer that binds within the initial amplicon. This setup increases specificity because the internal primer ensures that only correctly amplified products from the first PCR are further amplified in the second PCR. Thus, semi-nested PCR uses one primer in common between the two rounds, and one different, internal primer.

On the other hand, hemi-nested PCR is sometimes used interchangeably with semi-nested PCR, but technically it refers to a situation where the internal primer is located very close to the original external primer, often within a few bases, but not entirely nested within the product. In some definitions, hemi-nested refers to using an internal primer that is not fully nested, meaning it may still bind partially to sequences outside the core of the first amplicon, or differs by only a few bases to improve specificity or avoid secondary structures. In practical terms, hemi-nested PCR is a subtype of nested PCR, but the term is less strictly defined and often overlaps with semi-nested PCR in usage.

In contrast, fully nested PCR requires both primers in the second round to be located entirely within the sequence amplified by the first PCR, increasing specificity even more, but also increasing the risk of missing low-abundance templates due to shorter binding regions.

In summary: ➡️ Semi-nested PCR: one primer reused + one internal primer. ➡️ Hemi-nested PCR: generally similar, but may involve an internal primer partially overlapping or located close to the original binding site, sometimes defined more loosely.

Due to the overlap in terminology, it is always important to specify exactly how primers are designed and positioned when describing PCR strategies.

Semi-Nested PCR: Two rounds of PCR are used. In the second round, one external primer from the first round is used with a new internal primer.

Hemi-Nested PCR: This term is often used interchangeably with semi-nested PCR. Technically, it may refer to a situation where the new internal primer is very close to or partially overlaps with the original external primer, but the general principle remains similar to semi-nested PCR in that one primer from the first round is reused with a new internal primer.

Simply put, both aim to amplify a smaller, more specific fragment of DNA after an initial amplification.