How are restriction and DNA ligase enzymes used to insert a foreign gene into a plasmid?

Question

This is with the topic of Biotechnology and the bacterial transformation.

Answer 1

Those two are responsible for breaking down the bonds between the strands of DNA so that mRNA can attach to one of the strands and take the code for a protein to be synthesised. Then these two are used to join back the two strands again. Something similar happens and with the plasmid. The foreign gene is entered into the plasmid and those two help to join it together with the rest of the plasmid. They also speed up the process since they are enzymes.

Answer 2

Restriction enzymes recognize certain short sequences of nucleotides and cut the two strands of DNA at that point. This often leaves an uneven edge, with one strand longer than the other by one or sometimes 2 bases. The circular plasmid is then linearized (or opened up, if you want to think of it that way). If you have a gene that you want to insert, you can make it so that it has a little bit extra at each end that matches the uneven edges of the plasmid where the enzyme cut. Put the gene insert and the cut plasmid together in the presence of a ligase and the ligase will fasten together the matching ends to create a complete circular construct made from the plasmid and the insert. There are a few little details to take care of, such as making sure that the ligase doesn't just reconnect the plasmid without the insert, and that the insert 'reads' the right way, but that's the basic idea. General purpose plasmids are built so that they have what's called a 'multiple cloning site'; a region consisting of a half-dozen or more recognition sites one after the other, so you can pick and cjoose the restriction enzyme that works best for your application (or so that you can use 2 different enzymes to put your piece in facing in one particular direction). The circularized plasmid can then be used to transform a bacterium to either clone lots of copies of the gene or to express lots of the protein of the gene for you to collect.

Answer 3

Restriction enzymes will find a specific sequence in the DNA and cut it. There are two types of these, ones that cut through both strands at the same spot so that there is no overhang, and ones that cut on different spots and leave an overhang. The latter is more specific and more useful in splicing. You use the same restriction enzyme on both the DNA strand and the plasmid and basically hope that the gene of interest will insert into the DNA on some of the strands. Since the overhangs between the two will be the same (you used the same enzyme), they will pair up and attach.

The ligase seals where the two attached and the insertion is complete. You then have to test to see if insertion of the gene of interest was successful. Since rescriction enzymes recognize a 2-12 long base pair, some of the cuts will likely not include the gene of interest, but testing for that is another story :)

Since ligase is pretty simple, I'll attach a more comprehensive article on how the restriction enzymes work:

http://en.wikipedia.org/wiki/Restriction_endonuclease


In response to Mark S, usually, you'll sequence around the gene of interest (and usually the gene, too) to make sure it has sites that the restriction enyzme will cut. Otherwise, you can use several enyzmes and test later, although that is usually just for classroom experimentation purposes.

Answer 4

OK, restriction enzymes cut DNA at specific sequences. In chromosomal DNA there will be many in the entire genome but, if you're lucky, the gene you want will only have these sites on either side of it. Most restriction endonucleases recognize a six-base sequence, so the chances of this sequence occurring randomly are one in 4^6, or about one in 4096 base pairs. Many, but not all, of the possible six-base sequences have restriction endonucleases which have been discovered and which are commercially available.

OK, so you add restriction endonuclease into some DNA and it makes these specific cuts. Sometimes the cuts leave blunt ends on the DNA, sometimes there is an overhang. Now you cut the plasmid with the same DNA--most plasmids are engineered to have only one site for a particular restriction endonuclease. Assuming it's an overhanging site, you now have two ends of the site on the plasmid, and the corresponding two ends on the chromosomal DNA (think about two staggered rows of Lego's or bricks). The two pieces will fit together owing to these overhanging ends. If you now add DNA ligase, the enzyme seals up the ends by re-forming the phosphodiester bonds, and you have a complete plasmid, ready to go.

Notice that I mentioned overhanging ends. Restriction endonucleases sometimes leave blunt ends; DNA ligase can also seal blunt ends up, but the reaction is a little slower.

Oh, maybe a picture of overhanging ends would help. I've put a link below for the restriction enzyme EcoRI.

Answer 5

Restriction enzymes are bacterial enzymes used in molecular biology to cut DNA at specific sites. DNA ligase seals the nicks created by cutting with RE's. Both of these enzymes are the basic tools of molecular biology and biotechnology. In DNA recombinant technology, if one has a gene of interest, RE's are used to cut the gene and the vector plasmid (the carrier).

For example, HindIII restriction enzyme will be used to cut both gene and vector plasmid in separate reaction tubes. HindIII will create sticky ends once it cuts at the specific sites in both the plasmid and gene. The vector plasmid and the gene will be placed in a reaction tube with DNA ligase and necessary reagents, such as buffer. The DNA ligase will catalyze the sealing of the complementary sticky ends created by the restriction enzyme in both the plasmid and the gene . The result will be a circularized recombinant with the inserted gene into the plasmid.

Answer 6

Restriction enzymes cut DNA at EVERY specific point. If you use a restriction enzyme on your main bacterial DNA, it will chop it up into a million little pieces, and the DNA will be useless (which also kills the organism). Plasmids are "safer" because they are SMALLER pieces, so you can sometimes use one restriction enzyme to just make ONE cut -- which doesn't render the DNA useless. Plasmids are also not necessary to the survival of the bacteria, so it won't kill it. Yes, genetic engineers intentionally use restriction enzymes that are not found in particular species of DNA and use it on other species to cut and recombine the plasmid. I don't understand what you mean by the last sentence of your question, though.