14
votes
Accepted
Restriction enzymes, how are the recognition sequences determined?
This paper describes a simple method to determine restriction sites, which was used to determine the restriction sequence of the previously uncharacterised enzyme from Haemophilus gallinarum.
In ...
- 9,314
12
votes
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What if target DNA doesn’t have restriction sites
If you're synthesizing the insert then you just design it with restriction sites that are compatible to the vector. Otherwise, you can design PCR primers that have restriction sites in them. That way ...
- 181
10
votes
What makes DNA sticky-ends sticky?
The sticky ends are sticky because they have complementary bases. Typically used restriction enzymes cut the two complementary DNA strands at different spots, generating 'overhang', or sticky ends:
...
- 51.1k
10
votes
What makes DNA sticky-ends sticky?
What are sticky ends.
There is no substance that is attached making the DNA ends "sticky". What has actually happened is an overhang of at least a few nucleotides. Blunt ends are another kind of cut, ...
- 11.2k
7
votes
What does 'H' in BamHI stand for?
These enzymes are named by the bacteria and the strain from which they are isolated. For BamHI this is Bacillus amyloliquefaciens strain H. In the beginning the enzyme was named BamI, which was later ...
- 49.2k
7
votes
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How was Restriction Site of EcoRI sequenced?
The first determination of a recognition site for a restriction endonulease was reported in:
Kelly & Smith (1970) A restriction enzyme from Hemophilus influenzae II. Base sequence of the ...
- 22.7k
6
votes
What makes DNA sticky-ends sticky?
The "substance" is hydrogen bonds (H-bonds), or rather the potential to form them. Each of the unpaired A/T bases in the sticky ends have the potential to form 2 H-bonds with a complementary T/A, and ...
- 3,741
6
votes
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Can restriction enzymes (Type II) displace single-stranded binding proteins (SSB)?
This is an interesting point. In the case of typical restriction enzyme digestion, it is double stranded DNA that is being digested. So in this case the SSBs are presumably binding to the sticky ends ...
5
votes
Why do Type II Restriction Endonucleases cleave at palindromic sequences?
Short Answer
The palindromic symmetry of the restriction site allows a dimeric
enzyme to bind the DNA in a manner that bends the double helix in a
way that facilitates the endonuclease reaction....
- 22.4k
5
votes
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what's the difference between traditional genetic engineering and and CRISPR?
Bacteria and archaea evolved CRISPR as part of their adaptive immune system to protect themselves from invading viruses and foreign plasmids. The defence system relies on small, non-coding RNA ...
- 215
5
votes
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DNA Design for Multi-Site Restriction Enzymes
In its active form, SacII (also called Cfr42I) is a homotetramer that must bind two copies of its recognition sequence in order to efficiently hydrolyze its substrate.1 Upon binding two sites on a ...
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4
votes
How many Type II restrictions enzymes are currently available (commercially)
The Restriction Enzymes section at Biocompare is likely not comprehensive, but short of contacting every single molecular biology company and merging all their inventories into a master list, it's ...
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4
votes
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Why is EcoRI supplied with a unique buffer when it is allegedly 100% active in universal buffers?
Activity toward the desired substrate sequence is not the only concern in evaluating a restriction enzyme buffer system. In addition, you need to be concerned about so-called "star activity", or ...
- 1,534
4
votes
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Why don't restriction endonucleases digest transformed plasmids?
Laboratory strains used for the purposes of cloning have been genetically engineered to address this issue, typically by deleting genes of the various restriction-modification systems. There are four ...
- 17.5k
4
votes
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How to replace intron region in a plasmid?
Your strategy would work, but if possible you might be better off using XmaI rather than SmaI, as the latter is a blunt-end cutting enzyme.
Furthermore, your strategy would not replace the entire gpdA ...
- 608
3
votes
Are restriction enzymes active at −20 °C?
Enzymes have temperature optima based on the organism they were isolated from. So I would predict the there is virtually no activity at −20 °C. Another consideration is that the reactions are likely ...
- 3,407
3
votes
Accepted
Does mung bean nuclease cleave a phosphate group when it's chewing off 5' or 3' ssDNA ends?
According to Kroeker WD, Kowalski W, Laskowski M. 1976. Mung Bean Nuclease I. Terminally Directed Hydrolysis of Native DNA. Biochemistry 15(20):4463-4467
It was concluded that the products of the ...
- 17.5k
3
votes
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What happen if we inject restriction enzyme into the blood
The appearance of any foreign antigens (e.g., proteins like restriction enzymes fall n this category) in the circulatory system should trigger an immune response.
There are actually two membrane ...
- 3,407
3
votes
Accepted
How do Type I restriction enzymes work?
To answer your last question first: as long as a restriction enzyme recognises a specific sequence then goes on to cut the DNA it really doesn't matter where the cut takes place, as long as the ...
- 22.7k
3
votes
Where is cleavage site located?
The cleavage site is always located on the substrate. It is specific sequence of bases (if we speak about DNA or RNA) or amino acids (proteins) that is recognized by the particular enzyme and cut.
- 94
3
votes
Double Digestion with Restriction Enzymes Using Different Buffers
NEB double digest planner is suggesting to use 2.1 buffer for your combination of restriction enzymes (XmaI 50% and KpnI 75% activity). I would be more worried about the star activity of KpnI in ...
- 562
3
votes
Double Digestion with Restriction Enzymes Using Different Buffers
If you haven’t already ordered your enzyme, you could get high fidelity KpnI, which has been engineered for use in CutSmart.
You could also consider doing a sequential digest rather than a double ...
- 17.5k
3
votes
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Double Digestion with Restriction Enzymes Using Different Buffers
In my experience, a double digestion in CutSmart buffer will work perfectly well. The reaction may proceed slower, but incubate it a little longer and run a gel after the digestion - you'll see ...
- 5,610
2
votes
Accepted
Restriction endonucleases are found in?
There are many proposals for the ecological role of restriction-modification (RM) systems, and why they would exist on mobile genetic elements (e.g. plasmids and viruses). In this case, I am ...
- 262
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votes
Restriction endonucleases are found in?
The familiar restriction enzyme EcoRI is plasmid encoded.
Betlach et al. (1976) A restriction endonuclease analysis of the bacterial plasmid controlling the EcoRI restriction and modification of ...
- 22.7k
2
votes
Restriction endonucleases are found in?
If you have a dsDNA genome and replicate it by rolling circle like most bacteriophages and many plasmids do, an endonuclease is pretty much a necessity.
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2
votes
Accepted
Restriction sites
Most restriction enzymes have a 6bp restriction site (some have 8bp site also). So two restriction sites generally have to span 12bp. However some restriction sites can overlap. For example BamHI and ...
- 35.1k
2
votes
Cleavage of RNA by restriction enzymes?
As to why they don't recognize RNA-DNA heteroduplexes (which are present during transcription, for example), I suspect that the methylation which protects bacterial genomic dsDNA (see the DNA ...
- 3,264
2
votes
Accepted
Cleavage of RNA by restriction enzymes?
This paper found that these enzymes recognize RNA:DNA heteroduplexes. Such duplexes are unlikely to be encountered in vivo. They are present when DNA is primed for replication, but these duplexes are ...
- 17.5k
2
votes
Do restriction enzymes on read 3' to 5'?
Whatever sequence (plus direction) is target, so enzyme will cut. In your example "correct" and "antiparallel" sequences are two different sites.
Actually, your example of 5'-CTTAAG-3' is cut by ...
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