1. Fragments with non-complementary overhangs
Digestion with two different restriction enzymes can produce fragments with non-complementary overhangs, the knife is the most lizard-cloning fragment. Most commonly used plasmid vectors carry multiple cloning sites composed of several different restriction enzyme recognition sequences. Because the existing multiple cloning sites are so diverse, it is almost always possible to find a vector with restriction sites that do not match the outer DNA fragments. Therefore, the so-called directional cloning can be used to insert foreign DNA fragments into the vector. For example, the vector pUC19 is digested with BamHl and Hind-III, and then the large fragment of the vector is purified by gel electrophoresis or size exclusion gel chromatography to separate the small fragments of the polyclonal site that have been homogenized. Therefore, this vector can be connected to the exogenous DNA segment with sticky ends matched with the ends cut by BamHl and HindIII. Using the obtained circular heavy body E. coli, the ampicillin resistance was checked. Since the overhangs of HindIII and BomHI are not complementary, the vector fragment cannot be effectively circularized, so the efficiency of transforming E. coli is also extremely low. Therefore, most bacteria with ampicillin resistance contain quality DNA with exogenous DNA segments. The exogenous DNA segments serve as a bridge connecting Hind III and BamHI sites. Of course, the combination of restriction enzymes can be changed according to specific exogenous DNA segments.
If you want to make a directional cloning vector, it tries to avoid using most of the restriction sites directly adjacent to each other at the cloning site. After one of these sites is cut, the second site should be located within a few base pairs at the end of the linear DNA molecule, which is too close to the end and is not conducive to the effective cleavage of multiple restriction enzymes. Because of this, it is important to check whether the digestion of the carrier by the two restriction enzymes is complete. The following two inspection methods can be used:
1) Use one of the two restriction enzymes to digest the carrier. When the restriction buffer used has different ionic strength requirements, the enzyme with the lower salt concentration should be used. After digestion, a small amount of DNA should be checked by gel electrophoresis. If all plasmid DNA is changed from circular to linear molecules, adjust the salt concentration appropriately and add the second enzyme. At the same time, a pre-experiment was set up, which contains the circular plasmid DNA, and only the second of the two restriction enzymes was added. When all DNA in the pre-experiment is converted into a linear shape (as determined by gel electrophoresis), the large DNA fragment of the plasmid is purified by gel electrophoresis or ruled-exclusion gel chromatography.
2) Figure 1.7 shows a more rigorous method for checking the completeness of digestion. A small portion of DNA cut with the first restriction enzyme was end-labeled, purified by depletion column chromatography, and mixed with unlabeled linear DNA. It is then digested with a second enzyme. Complete digestion can release the DNA small fragments, which should have a 50% radioactivity, which can be detected by chromatography or by gel electrophoresis and autoradiography.
2. Fragments with the same end (blunt or sticky)
Exogenous DNA fragments with the same ends (blunt or sticky) must be cloned into a linear plasmid vector with matching ends. In the ligation reaction, both exogenous DNA and plasmid may be cyclized, and tandem oligomers may also be formed. Therefore, the concentration of the two DNAs in the ligation reaction must be carefully adjusted in order to achieve the optimal level of "correct" ligation products (see Section 3 of this section). In addition, alkaline phosphatase is often used to remove the 5 phosphate group to inhibit the self-ligation and circularization of plasmid DNA. In the in vitro ligation reaction, T4 phage DNA ligase can catalyze the formation of phosphodiester bonds between adjacent nucleosides only when one nucleotide contains 5 phosphate groups and the other contains 3 hydroxyl groups. Using bacterial alkaline phosphatase (BAP) or bovine intestinal alkaline phosphatase (CIP) to remove the 5 phosphate at both ends of the linear DNA can minimize the DNA segment of the plasmid, which can be effectively connected to the dephosphorylated plasmid DNA A ring-opening molecule with two cuts (Figure 1.8). Because the transformation efficiency of circular DNA (even nicked circular DNA) is much higher than linear DNA, most transformants contain recombinant plasmids.
3. Clips with blunt ends
When the exogenous DNA fragment has a flat end, there is also a trouble to cut the exogenous branch, which is that the connection efficiency of the nymph end is much lower than that of the DNA with the protruding end of the stocking. Therefore, the concentration of T4 phage DNA ligase and the concentration of exogenous and plasmid DNA required for the ligation reaction involving blunt-end molecules are much higher. It should also be noted that the addition of low concentrations of substances such as polyethylene glycol can often increase the efficiency of such reactions.
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