How do restriction enzymes recognize a restriction site?
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The enzyme is built to attach itself to a particular sequence of nitorgenous bases.  Because the A, T, G, and C (if you know what restirction enzymes are, I'm assuming you'll know the abbreviations) are structured slightly differently, the enzyme won't properly fit on any of the other sequences, and without a proper fit, it would cut your DNA strand.
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I'll give some examples and some brief explanations right here:\n\nSubstrate = A substrate is a compound that binds to an enzyme and is converted to a product.  In other words, a substrate is something an enzyme digests.  Anything that is digested is the substrate.\n\nEnzyme = Enzymes are globular proteins, larger in size compared to their substrates.  In other words, they are made up of proteins and digests substrates.\n\nNow, enzymes are 'digesters' and one kind of enzyme can only bind to one kind of substrate.  (e.g. Enzyme A can only bind to Substrate A and not Substrate B)   \n\nLactose = substrate\nLactase = enzyme\n\nHow do I know???\n\nMost substrates have an 'O' to replace the 'A' in Enzymes.\n\nNow, what happens after the enzymes bind/digest to the substrate?\n\nIt converts the substrate into smaller products.  In the Maltose case, maltOse gets digested by maltAse into glucose.  So, glucose in the final product\n\nillustrated by:\n\n``````````(is digested by)\n``````````````Maltase\nMaltose    ---------------->  Glucose\n(substrate)``(enzyme)``(Product)\n\n\nAnother illustrated example:\n\nProtein as the substrate.\n\n````````(is digested by)\n```````````````Pepsin\nProtein      ------------------>  Peptones/Large Polypeptides\n(Substrate)  ```(Enzyme) ````````````````(Product)
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Transcription:\n\nSpecific parts of the DNA that make up the gene are unwound using the enzyme helicase. Then, RNA polymerase attaches complementary RNA nucleotides to the DNA strand in the 5' to 3' direction. RNA does not have thymine, so adenine pairs with uracil instead. For example, the TACGGTATC sequence in DNA would transcribe into a AUGCAUAG sequence in mRNA.
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AAUCGGACU\n\nBecause:\nG matches with C\nA matches with U\nBut when it's a T in DNA, it'll be an A in RNA. If it's an A in DNA, it'll be a U in RNA. That's the most confusing part about transcription.
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http://www.zymoresearch.com/protocols/protein/autol-instructions-4.pdf
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In terms of how many restriction endonucleases there are "in the world", the answer could theoretically be in the millions.  Different strains of the same bacterial species can make different RE, so you can imagine the potential for variety in nature.\n\nAs for commercially available: new england biolabs sells about 230 different enzymes.\n\nAs for what's commonly used, it depends on the lab and the target organism.  The lab I got my PhD in, we worked with a low gc bacteria, and we used very different enzymes than we use in my current lab, working with a high gc bacteria.  In either case, we used 20-30 different enzymes.
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1.  DNA ligase ligates the DNA to a solid plasmid\n\n2. Antiboitic resistance allows you to screen bacteria that contain the new plasmid.  Any bacteria without the antibotic resistance gene will die when plated out on a plate containing the proper antibiotic.  Without antibiotic on the plate, you would have a plate with a lawn of bacteria.\n\n3  Two reasons.  Blunt ends are difficult to ligate and that lowers the cloning efficiency.  Also if it does get ligated and some portion will,  a second round of analysis will be needed to determine the orientation of the gene. Which goes to the second point\n\nIf you use two restriction enzymes with different sticky ends you can clone the insulin gene in to the vector in a directional fashion.  ie. Its not cloned in bacwards.\n\nhope this helps\n\nStooleo
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1. Well, so far as I know enzymes are not changed by their reactions. \n2. There are also special molecules called "substrates" which specifically bind to an enzyme's "active site" (kinda like a key fitting into a hole) and is changed and then released by the enzyme.
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activator.\n\ncome on, think about it before you ask for help :P\n\ncould also be a repressor which represses a repressor of the operon in question.  but im sure the answer the asker is looking for is activator.
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DNA's most amazing characteristic is that it will bind to itself in a very specific manner, forming a helix. Figure 1 illustrates two DNA molecules binding. The variable groups of the polymer interact with eachother to mediate this binding. Adenine interacts with Thymine and Cytosine interacts with Guanine. No other bonds are energetically favorable, for example Cytosine cannot bond with itself. The bonds that these variable groups form are called are called base pairs and are illustrated in Figure 2. Thus, by knowing the sequence of bases of one strand of DNA we immediately know the sequence of the DNA strand which will bind to it, this strand is called the reverse complement or just the complementary strand. In Figure 1 the right hand strand is the reverse complement of the left hand side and vice-versa.\n\n\nFrom G. S. Sent, Molecular Biology of Bacterial Viruses. Copyright 1963 by W. H. Freeman and Company\n\nFigure Two:Illustration of DNA basepairing. Thermodynamically the interactions Adenine, Guanine, Cytosine, and Thymine are driven by both the hydrophobic stacking interactions hydrogen bond interactions. The specificity of the base pairing is driven mainly by hydrogen bonding and stearic interference.\nThis relationship to the complementary strand has many implications that make life as we know it possible, and allows all sorts of molecular biology techniques. In one of the greatest understatements of all time Watson and Crick said it best in their original paper on the structure of DNA:\n\n"It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.".
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an enzyme is a protein which lowers the free energy needed for it's target molecule or molecules to change their structure. for example - adding a phosphate group to another molecule. there are many ways in which the body regulates the enzymes capacity - some ways are by altering the substrate level - usually more substrate results in higher capacity - note that the change is NOT linear.\nanother way is by regulating the genes coding for these enzymes - inhibiting such a gene will result in lower enzyme levels and thus the overall capacity will be lower. note that in this case, each enzyme will work at it's usual capacity.\nwhat you are asking about is another way of controlling the enzymatic capacity. there are two general ways in which other molecules change the enzymes capacity - it can be competitive inhibition - a molecule which binds to the target site on the enzyme and lowers it's chance of binding to its' target molecule. the other method of inhibition is non-competitive inhibition - in this way a molecule binds to a site other that the target site, causing a conformational change in the enzyme and thus changes the affinity of the enzyme to it's target molecule.\na slightly different mechanism is used only in enzymes which have several sub-units (separate proteins that bind to each other to make one active enzyme). these make up many of the enzymes in our body. the mechanism used here is called allosteric change (note that all other regulation mechanisms are at work in these enzymes as well). allosteric change involves a binding of a molecule to the allosteric site which changes the conformation of the enzyme. this change can activate the enzyme and make it work more effectively, or it can inhibit the enzyme.\nso... an enzyme can be active, and by various mechanisms (involving not only other molecules but also "breaking" of the enzyme into active parts, changes in pH, temperature and more) it can change it's capacity - it can be lowered, inhibited almost completely or increased.
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emm...is this your homework??well..it seems that the question is not from you (individually). Hmm..for more understanding, check out this website:\n\nhttp://faculty.clintoncc.suny.edu/faculty/Michael.Gregory/files/Bio%20101/Bio%20101%20Lectures/Biochemistry/biochemi.htm\n\nI hope it solves your dillema, good luck :)
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1. Isolate the genome of organism on which the desired gene sits.\n2. Cut it with restriction enzyme with the desired recognition site.\n3. Perform polymerase chain reaction procedure with oligomer primers which have the suspected complimentary sequence with your gene\n4. Perform agarose gel electophoresis to isolate the multiplied sequences\n5. Separate the duplex structure of primer:DNA in alkaline medium.\n6. Perform Southern Blot Transfer (could be found in any molecular biology laboratory manual)\n7. Transform the target organism with the resulting gene fragments from the southern blot. (transformation procedure vary with the varying target organism)
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The function of DnaB helicase is to couple ATP hydrolysis with the unwinding of the DNA double helix at the replication fork, creating two antiparallel strands of ssDNA. DNA polymerase and DnaB helicase are complexed via the tao subunit of DNA polymerase which serves to stabilize the hexameric structure of the helicase. This now immobilized protein complex allows for DNA to pass through it. There are other helicases involved with replication but this is the only one found to be essential (knockout mutations are fatal) and the only one that can stimulate primase. If you need more info, please formulate a somewhat more coherent and specific question.
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restriction enzymes only work in proper salt and pH condtions.  The buffer is there to mimic the conditions the enzymes normall work in.
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It is an enzyme that make possible to transcribe DNA from RNA.\nThanks
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First of all make sure your template is good quality. Did you extract genomic DNA, do you have a library in plasmids...(?).\nTry re-purifying your template.\n\nMaybe your template is GC-rich or has some secondary structure.\nIs your template very long?If so make sure you let the polymerase is working at the elongating temperature for enough time (1min/kb)\n\nIf you are using plasmid template try not to use too much. Don't put more than 30-40ng DNA in a reaction. \n\nCheck your primers:\n-can they form a dimer?\n-can they anneal at more than one site on the template DNA with a only few mismatches?\n-is the difference in the Tm of the two primers more than 6 C (it shouldn't;ideally they should be the same and I try not to exceed a difference of 4)?\n-do your primers end in more than 3  c or g in a row?\n-what's their Tm and GC content?Tm should not be very low (I design my primers at 56-61) and GC 40-60%.\n\nThe most strigent conditions are using as annealing temperature 2 C below the lowest Tm of the two primers. Try lower temperatures.\n\nYou can try different enzymes Taq, Pfu, Pwo, etc, even mixtures. Not all enzymes have the same processivity.\n\nYou can try different Mg+2 concentrations.\n\nThere are a lot of control experiments you can do depending on your template and primers. Eg if you have a library in a  plasmid that has sites for universal primers you can use one such primer and the other respective primer you designed in order to check if there is something wrong with the quality of your primers. \n\nIf everything is perfectly designed and you've tried evreything but still get a very faint (but of correct size) band, gel purify it and use it as a template for a new PCR. In this case you'll have a very high chance of getting mutations so you'll need to use a high fidelity enzyme like Pwo or Pfu turbo and of course sequence a lot till you get a correct clone.