Type II restriction enzymes in practice: BsmI, isoschizomers and incubation temperature

Getting the digest right

1. Use the supplier's stated temperature. Several type II enzymes including this one have an elevated optimum, and a digest set at the conventional temperature will run slowly or incompletely. Read the data sheet rather than assuming, and check the block or bath actually holds the set point.
2. Check thermal inactivation separately. Whether an enzyme can be heat inactivated, and at what temperature, is a separate property from its incubation optimum. An enzyme that cannot be inactivated by heat needs a column clean-up or a spin before the next step, and that changes the protocol timing.
3. Methylation can block the site. Overlapping host methylation blocks some sites entirely, and whether yours is affected depends on the strain the DNA came from. Where it is, growing the plasmid in a methylation deficient strain is usually simpler than changing the strategy.
4. Isoschizomers and neoschizomers. Another enzyme recognising the same sequence may cut where this one cannot, or work in a buffer that suits your double digest better. A neoschizomer cuts the same site at a different position, which changes the overhang, so check the cut position and not only the recognition sequence.
5. Confirm the site before you order. Run the sequence against a restriction analysis tool and confirm the number and position of sites in the actual construct, including the vector. Ordering an enzyme for a site that the vector also carries is a wasted week.

When to substitute rather than troubleshoot

If a site is methylation blocked, the buffer is incompatible with the partner enzyme, or the incubation temperature is inconvenient for the workflow, substituting an isoschizomer is usually faster than optimising. Suppliers list them, and the substitution is a data sheet comparison rather than an experiment.

Check the overhang the substitute leaves. A neoschizomer that cuts at a different position within the same recognition sequence produces an end that will not ligate with what you planned.

Buying sensibly

Enzymes used occasionally should be bought in the smallest useful size, because activity declines with storage and handling and a large tube used twice a year is mostly waste. Enzymes used constantly are worth buying in bulk and aliquoting once.

Keep the lot and the data sheet with the protocol. Supplier buffer formulations and recommended conditions do change between catalogue revisions, and a protocol that cites a buffer by name without a date can quietly become wrong.

Cut site, overhang and what the design needs

An enzyme leaving a staggered cut gives an overhang that ligates directionally with a matching one, which is what makes classical cloning work. One cutting straight across gives blunt ends that ligate in either orientation and to anything, which is convenient and directionless.

Recognition site length decides frequency: a four-base site cuts often and suits digestion fingerprinting, while a six-base site cuts rarely enough to be useful for cloning. Choose on frequency and overhang rather than on availability.

Polymerases chosen for a property: Klenow and phi29 DNA polymerase

A polymerase lacking the exonuclease that chews back from an end is used for filling in overhangs and for labelling. A highly processive one from a phage displaces strands and copies a circular template many times over, which is why it underlies whole-genome amplification from tiny inputs.

Fidelity, processivity, strand displacement and template preference are the four properties that matter, and no enzyme is best on all of them. Pick the one whose weakness does not matter to your application.

Enzymes that modify rather than copy, from polyA polymerase to SUMO protease

Adding a homopolymer tail to a transcript is done enzymatically where an in vitro transcript needs one, and the tail's length affects stability and translation. Attaching a small tag enzymatically at a single site gives a defined labelling position no chemical method matches.

For proteolysis, a highly specific protease that cleaves a fusion tag without leaving a scar is what makes a tagged construct usable, and one that cleaves after a specific residue is the standard tool for generating peptides for mass spectrometry. Specificity is the product in both cases.

Methylation sensitivity, and where dpn1 comes in

Most enzymes in routine use are blocked by methylation at or near their site, which is why a digest that works on a PCR product fails on the same sequence taken from a plasmid grown in a strain that methylates it. The datasheet states the sensitivity and it is worth reading before blaming the enzyme: the fix is a different strain, a different isoschizomer, or an enzyme that does not care.

One enzyme is useful for the opposite reason. It cuts ONLY when its site is methylated, which makes it the standard way to destroy a bacterially produced template after a site directed mutagenesis reaction while leaving the newly synthesised, unmethylated product intact. That is why it appears in mutagenesis kits rather than in cloning protocols, and why it does nothing at all to a PCR product.

type iis restriction enzymes and the scarless assembly they allow

type iis restriction enzymes cut outside their recognition site, so the site can be placed where it will be removed by the cut, which is what allows multi fragment assemblies with no scar and one enzyme. Overhang design is the whole of the work, since unique four base overhangs decide the assembly order, and internal sites in a part have to be removed first.

ecori and the enzyme most protocols start with

ecori cuts a six base palindromic site leaving a four base overhang, and it is the enzyme most cloning protocols name first because it is cheap, active and well characterised. Its site occurs often enough to be useful and rarely enough to be specific, which is what makes a six base recogniser the workhorse class.

types of restriction enzymes, and what separates them

The types of restriction enzymes are separated by how they recognise and cut: the type two enzymes cut at or near a defined site and are the ones used for cloning, while the others cut at a distance or require methylation. Within type two, the site length, whether the ends are blunt or overhanging and the methylation sensitivity are the practical axes.

Common questions

What temperature should a BsmI digest run at?

Above the conventional digest temperature: check the supplier's data sheet for the stated optimum and set the block to it. Running it at the habitual temperature is the most common cause of an incomplete digest with this enzyme.

What is an isoschizomer?

A different enzyme recognising the same sequence. It may cut where the original is blocked by methylation or work in a more convenient buffer. A neoschizomer recognises the same sequence but cuts at a different position, changing the overhang.

Can BsmI be heat inactivated?

Check the data sheet, because inactivation temperature is a separate property from incubation temperature and not every enzyme can be inactivated by heat. Where it cannot, clean up before the next enzymatic step.

Why does my construct have unexpected sites?

The vector usually carries sites too. Run a restriction analysis over the complete construct including the backbone before ordering, rather than over the insert alone.

Blunt or sticky ends?

Sticky ends ligate directionally with a matching overhang, which is what classical cloning depends on. Blunt ends ligate in either orientation and to anything, which is convenient and loses directionality.

Which polymerase should I use?

Choose on fidelity, processivity, strand displacement and template preference, because no enzyme is best on all four. Pick the one whose weakness does not matter to your application.

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