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How does ligating the plasmid vector and P. putida DNA in the presence of a restriction enzyme increase recombination?
Ligating the plasmid vector and P. putida DNA in the presence of a restriction enzyme increases recombination by generating compatible ends on both the plasmid and the target DNA. The restriction enzyme cuts the DNA at specific sites, producing cohesive (sticky) or blunt ends that can easily anneal. When the plasmid vector and the P. putida DNA are mixed, these complementary ends facilitate the ligation process, allowing for more efficient insertion of the target DNA into the plasmid. This enhances the likelihood of successful recombination events, enabling the creation of recombinant DNA molecules.
Which Restriction enzyme are studied in Recombinant DNA Technology?
It's not the restriction enzymes that are studied, its the DNA. The enzyme cuts or "restricts" the DNA strand at a known sequence of nucleotides. Different enzyme, different sequence. For a Biomanufacturing application, where we want to insert foreign DNA, the gene of interest is cut and spliced with a restriction enzyme into a recombinant plasmid, transformed into a bacteria, and sent merrily on it's way to make Insulin, or whatever. With an unknown piece of DNA (a functional gene that makes a protein of interest or is being studied), the plasmid has "restriction sites" or nucleotide sequences, for several restriction enzymes, all of which I have mapped out. The unknown piece of DNA is cut at each end by a single restriction enzyme and inserted into the plasmid, which gives me some landmarks. I insert the plasmid into a bacteria, grow a culture so the bacteria makes many millions of copies of the plasmid, extract the plasmid, and run an experiment called a restriction digest. The restriction digests are a series of reaction with single enzyme and combinations of two and three enzymes, all cutting the plasmid at different nucleotide sequences. Then I run an agarose gel electrophoresis, which separates all the different pieces of DNA by size, and do an analysis called a Restriction Map. This counts the DNA fragments and their sizes, which enzyme and combination of enzymes produced which sizes and how many fragments, which enzyme cuts where, which cuts were definitely in the known part of the plasmid, which were probably in the unknown DNA, adding up nucleotide sequence numbers to make sure different mapping guesses agree, etcetera, etcetera, and so forth. Until at last, a map of the size and restriction sites of the unknown DNA insert into the known plasmid vector is deduced. This used to be done by hand, but there are computer programs that do it now. This is Research, the Technology is down the line a few steps when the gene has been characterized, the protein produced has been characterized, the trials are done, and the restriction enzyme to insert the gene into the bacteria for Bioman has been established
What is the restriction site of the restriction enzyme Hae III?
The restriction site of Hae III is GGCC. It cuts between the G and the C. This produces blunt ends.
Why must the donor DNA containing the desired gene and the plasmid DNA be cut with the same restriction enzyme?
If they aren't cut with the same restriction enzymes, they will not fit with each other. Say one r.enzyme cuts AA/GC CT and another cuts GA/TTT CC. If you try to fit them TT CG/GA CT AAA/GG together, one sticky end "GC" will not fit with the other sticky end "AAA". so you have to cut them with the same r.enzymes to let them fit.
What is the restriction site to restriction enzyme Hae III?
Hae III cuts at the site GGCC. It creates blunt ends - meaning a clean cut. This is found between the G and C.
How does ligating the plasmid vector and P. putida DNA in the presence of a restriction enzyme increase recombination?
Ligating the plasmid vector and P. putida DNA in the presence of a restriction enzyme increases recombination by generating compatible ends on both the plasmid and the target DNA. The restriction enzyme cuts the DNA at specific sites, producing cohesive (sticky) or blunt ends that can easily anneal. When the plasmid vector and the P. putida DNA are mixed, these complementary ends facilitate the ligation process, allowing for more efficient insertion of the target DNA into the plasmid. This enhances the likelihood of successful recombination events, enabling the creation of recombinant DNA molecules.
Hi does any one know if I want to put some restriction enzyme close to cDNA should I put some nucleotide befor this restriction enzyme?
Restriction enzyme cuts DNA strand at specific locations Restriction enzyme cuts DNA strand at specific locations
An enzyme that cuts double-stranded DNA at specific nucleotide sequences?
Such an enzyme is called a restriction endonuclease
Where does the restriction enzyme EcoRI cut in a DNA sequence?
The restriction enzyme EcoRI cuts DNA at a specific sequence of bases, which is GAATTC.
Which Restriction enzyme are studied in Recombinant DNA Technology?
It's not the restriction enzymes that are studied, its the DNA. The enzyme cuts or "restricts" the DNA strand at a known sequence of nucleotides. Different enzyme, different sequence. For a Biomanufacturing application, where we want to insert foreign DNA, the gene of interest is cut and spliced with a restriction enzyme into a recombinant plasmid, transformed into a bacteria, and sent merrily on it's way to make Insulin, or whatever. With an unknown piece of DNA (a functional gene that makes a protein of interest or is being studied), the plasmid has "restriction sites" or nucleotide sequences, for several restriction enzymes, all of which I have mapped out. The unknown piece of DNA is cut at each end by a single restriction enzyme and inserted into the plasmid, which gives me some landmarks. I insert the plasmid into a bacteria, grow a culture so the bacteria makes many millions of copies of the plasmid, extract the plasmid, and run an experiment called a restriction digest. The restriction digests are a series of reaction with single enzyme and combinations of two and three enzymes, all cutting the plasmid at different nucleotide sequences. Then I run an agarose gel electrophoresis, which separates all the different pieces of DNA by size, and do an analysis called a Restriction Map. This counts the DNA fragments and their sizes, which enzyme and combination of enzymes produced which sizes and how many fragments, which enzyme cuts where, which cuts were definitely in the known part of the plasmid, which were probably in the unknown DNA, adding up nucleotide sequence numbers to make sure different mapping guesses agree, etcetera, etcetera, and so forth. Until at last, a map of the size and restriction sites of the unknown DNA insert into the known plasmid vector is deduced. This used to be done by hand, but there are computer programs that do it now. This is Research, the Technology is down the line a few steps when the gene has been characterized, the protein produced has been characterized, the trials are done, and the restriction enzyme to insert the gene into the bacteria for Bioman has been established
Where in the DNA sequence does the restriction enzyme EcoR1 specifically cut?
The restriction enzyme EcoR1 specifically cuts the DNA sequence at the recognition site GAATTC.
Based on restriction maps of plasmid determine the number of DNA fragments and sizes of the fragments?
Plasmids are circular pieces of DNA, so the number of fragments equals the number of cuts from the restriction enzymes. You can easily see this if you start with one restriction enzyme that cuts the plasmid in only one place. Cutting the circle in one place yields you only one fragment. If the restriction cuts in two places, you end up with two fragments; with three places, three fragments, etc. With linear chromosomes, the situation is different. Cutting a linear chromosome in one place yields two fragments, cutting in two places yields three fragments, etc. So the number of fragments is always one more than the number of cuts. A restriction map of a plasmid will show all of the cuts the restriction enzymes made. Each cut is labeled with the enzyme that made it. One can count the spaces between cuts to determine the number of fragments that are produced. Restriction maps usually (but not always) also show the size of each fragment.
Why must you use an enzyme that will not cut anywhere within the gene that you are inserting into a plasmid?
If you are trying to take a gene from a DNA strand and put insert it into a plasmid, you wouldn't want a restriction enzyme to cut that gene up, or else it would be pretty useless. In other words, you need an enzyme or two that cuts outside that gene so that it can be functional after it's inserted into a plasmid. After your gene of interest is inserted into a plasmid, the plasmid can be put back into a bacterium, then you could genetically engineer plants with it or let the bacterium reproduce and produce many copies of a protein that you had wanted to make in the first place.
What is the restriction site of the restriction enzyme Hae III?
The restriction site of Hae III is GGCC. It cuts between the G and the C. This produces blunt ends.
A molecule that cuts DNA molecules at a specific sequence of nucleotides?
A restriction enzyme
What is a molecule that cuts DNA molecules at a specific sequece of nucleotides?
A restriction enzyme.
Why must the donor DNA containing the desired gene and the plasmid DNA be cut with the same restriction enzyme?
If they aren't cut with the same restriction enzymes, they will not fit with each other. Say one r.enzyme cuts AA/GC CT and another cuts GA/TTT CC. If you try to fit them TT CG/GA CT AAA/GG together, one sticky end "GC" will not fit with the other sticky end "AAA". so you have to cut them with the same r.enzymes to let them fit.
