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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 short, a known sequence of DNA (from the phage $\phi \text{X174}$) is partially digested with the restriction enzyme, and the various digested fragments can be ...
11
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 when you amplify your insert you'll attach the restriction sites to it. EMBL, Addgene, and NEB all offer somewhat more detailed explanations of this on their ...
10
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:
These overhangs allow for perfect base pairing (C with G, A with T), which is the result of hydrogen bonding. Just like water molecules show strong affinity to ...
10
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, but have no overhanging residues.
Why are sticky ends sticky?
Restriction enzymes usually cut these ends deliberately so that a four nucleotides are ...
7
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 changed to BamHI.
See these two references:
Isolation of a sequence-specific endonuclease (BamI) from Bacillus
amyloliquefaciens H.
Recognition sequence of ...
7
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 recognition site. J Mol. Biol. 51: 393-409
The enzyme was then called endonuclease R, but is now known as HindII (or HincII).
The method used was to cut DNA with ...
6
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 each of the unpaired G/C bases have the potential to form 3 H-bonds with a complementary C/G.
From the perspective of a biophysicist, H-bonds are often thought ...
5
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.
More Detailed Answer
The ‘palindromic’ nature of the recognition/cleavage site of restriction endonucleases, such as EcoRV (illustrated here) results in a ...
5
Such universal restriction enzymes would be very dangerous to leave lying around even if contained within a vacuole. Remember that bacteria are prokaryotes and have no nuclear membrane to protect their DNA.
The analogy would be having a burglar alarm capable of burning down the house in order to protect it against thieves.
5
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 molecules (CRISPR RNAs/guide RNA) that in association with a CRISPR associated (Cas) protein silence foreign sequences by means of cleavage. Twenty nucleotides at the ...
4
First, not all restriction enzymes cut at palindromic sequences.
A lot of them do though, simply because it is more effective. Recognising a palindromic sequence enables them to cut both strands of DNA at the "same" site, because the strand will have the same sequence only in different directions at that site.
See Wikipedia for example.
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Notice that viral DNA is not the only foreign DNA that a bacterial can meet during its bacterial adventures. Plasmids, conjugated DNA and DNA inserted by transformation exist, and it may confer ecological advantages. Because of that, even if the enzymes would recognize only non genomic DNA and destroy it effectively, it's still more useful to have some ...
4
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 probably the best you can do on short notice. They currently list 289 uniquely-named enzymes, but I'm not enough of an expert to say how many are similar in ...
4
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 broad classes of restriction modification systems, which I will discuss individually. Unless individually referenced, most information below is based on the ...
4
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 after digestion, so there is no competition with the restriction enzyme.
If you are interested in a specific case where you are digesting ssDNA, and have ...
3
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 to be frozen solid, so that would limit diffusion, and also slow it down. But the real question is: what are you afraid of? Just the star activity? Even if there ...
3
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 invading DNA is destroyed.
The WP page on restriction enzymes provides a useful summary of the various classes of restriction enzyme, and led me to a review about ...
3
Just in case you are having difficulty visualising what happens in the answer from @Chris, here are the steps with the linker shown in lower case
...NNNNNGAATTCNNNNN... ds DNA
...NNNNNCTTAAGNNNNN...
|
V
...NNNNG AATTCNNNNN... after EcoRI digestion
...NNNNCTTAA GNNNNN...
|
V
...NNNNG ...
3
The recognition site for EcoRI is GAATTC, and the enzyme cuts after the first base. See this picture from NEB:
The overhang is: AATT, which is supplied by your oligo and fits into the overhang. If the next nucleotide whould be a C than the site would be recreated, since its a G its not. The new sequence of the old EcoRI site is GGATTG which is not ...
3
Note that a given bacterium will probably have more than one restriction enzyme, so the viral genome probably won't ever run out of targets. Even if it did, restriction enzymes could suffer mutations that may change the specificity. When you talk about microorganisms, it's more accurate to think in populations rather than individuals, since the colony would ...
3
I guess you are talking about the restriction modification system, not restriction enzymes in general (which are used a lot in the lab, for example).
If so, this paper might help answer your question.
I have not read through it totally, but at least some viruses trigger the downregulation of restriction enzymes to make them cleaving their DNA less likely. ...
3
Could be insert polymerization. If you have your stretch of DNA like this:
(5')-AATTagctagcatcgtgatcgacg-(3')
||||||||||||||||||||
(3')-tcgatcgtagcactagcagcGGCC-(5')
And you take that, flip it around, it will ligate onto itself, like this:
(5')-AATTagctagcatcgtgatcgacg-(3')(5')-CCGGcgacgatcacgatgctagct-(3')
|||||||||||||||||||| ...
3
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 terminally directed hydrolysis of native DNA possess 5’-phosphoryl groups because: (1) greater than 99% of acid-soluble activity applied to the column was ...
3
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 barriers that the enzymes have to traverse to gain access to the nuclear genome: the cell membrane, and the nuclear membrane. Proteins are typically too large and ...
3
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 activity of the enzyme toward other, non-desired nucleotide sequences.
For example, NEB supplies an enzyme-specific buffer for it's standard EcoRI, even though it ...
3
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.
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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 CutSmart buffer than the lower activity. The latter can be overcomed by prolonging reaction time or using more enzyme.
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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 digest (ie digest with one enzyme in its optimal buffer then change the buffer for digestion with the second enzyme). It is more work but benefits from easier ...
3
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 whether it has worked. The other answerers unfortunately did not mention that the best way is to check the restriction products yourself on a gel. Cut out the ...
2
I am not sure of a case where Taq doesn't add 3' A overhangs.
You could use TA cloning to clone your PCR product. The basic principle is to use a vector with 3' T overhangs. If you have a vector without these overhangs, you can use a terminal transferase + dTTPs to create them (or even Taq, but this is not as great because it adds a purification step to ...
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