For example you could use a YAC (yeast artificial chromosome) for the dystrophin gene. These are specific to the type of organism in which you wish to grow the vector with insert.
The efficiency of ligation and transformation tends to decrease with extremely large inserts.įor large inserts there are different kinds of vectors (not plasmids) that can be used.įor an enormous insert like you are asking about you would need to use a type of vector known as an artificial chromosome. §Note: Polymerase chain reaction - you can learn more about this technique here:Ī typical plasmid can accommodate inserts of any size up to total size of around 50 kb, but plasmids that are more than 20 kb are very difficult to work with and may require special transformation techniques. †Note: There are hundreds of commercially available restriction enzymes recognizing many different sequences (many of which are palindromes, but not all).Īmong these the most commonly used are six-cutters (with 6 bp recognition sites - if you make a bunch of simplifying assumptions you can calculate that these enzymes on average will cut once every 4096 bp. There are many more tricks that have been developed, but adding sites at the ends of primers almost always works, so that is a very good one to know! This amplifies the insert you want and creates a copy of the insert DNA with whatever restriction sites you want added at the ends. If the regions flanking the sequence you want to clone don't contain any useful restriction sites you can use primers with restriction sites added to their 5' ends and then amplify the sequence using PCR§. Second, we often don't care if we clone a small amount of extra DNA, this means that we can search over a larger area than you might expect to find appropriate restriction enzymes. bacteria's innate immunity !)įirst, most vectors will have a region known as the "Multiple Cloning Site" (MCS) that can be cut with many different restriction enzymes† - this gives you more choices of enzyme and makes it more likely that you can find one that cuts near the ends of the region you wish to clone.
so host bacterium DNA is not cut by restriction but when new DNA is inserted by bacteriophage, it is not methylated and so it chopped by restriction enzyme and bacteria can survive (i.e. Now one more question arises that WHY (& HOW) THESE RESTRICTION ENZYMES CAN CUT ONLY FOREIGN DNA BUT NOT THE HOST BACTERIUM'S DNA?Įxplanation: if particular bacterium has restriction enzyme, it must have companion site specific DNA methylase which methylates DNA of host bacterium in site specific manner and methylated DNA is not the substrate for restriction enzyme. in this way it is the defensive enzyme that protects the host bacterial DNA from the DNA genome of foreign organism (bacteriophage) by specifically inactivating the invading bacteriophage DNA by digestion Restriction enzymes are found in bacteria and they have some biological role (explained below), but we are exploiting it in our way to use in experiment.īiological role of restriction enzymes in bacteria: when restriction enzyme is present in a given bacterium, such bacterium can prevent (restrict) the growth of certain bacterial viruses (bacteriophages) and this is the reason also to call it as RESTRICTION enzymes.
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