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Non-blunt ends are created by various overhangs. An overhang is a stretch of unpaired nucleotides in the end of a DNA molecule. These unpaired nucleotides can be in either strand, creating either 3' or 5' overhangs. [3] These overhangs are in most cases palindromic. The simplest case of an overhang is a single nucleotide.
Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA. It is called "non-homologous" because the break ends are directly ligated without the need for a homologous template, in contrast to homology directed repair (HDR), which requires a homologous sequence to guide repair.
Sticky ends of DNA however are more likely to successfully bind with the help of a DNA ligase because of the exposed and unpaired nucleotides. For example, a sticky end trailing with AATTG is more likely to bind with a ligase than a blunt end where both the 5' and 3' DNA strands are paired. In the case of the example the AATTG would have a ...
The classical restriction enzymes cut up, and hence render harmless, any unknown (non-cellular) DNA that enters a bacterial cell as a result of a viral infection. They recognize a specific DNA sequence, usually short (3 to 8 bp), and cut it, producing either blunt or overhung ends, either at or nearby the recognition site.
Since blunt-end does not have protruding ends, the ligation reaction depends on random collisions between the blunt-ends and is consequently much less efficient. To compensate for the lower efficiency, the concentration of ligase used is higher than sticky end ligation (10x or more). The concentration of DNA used in blunt-end ligation is also ...
DNA ligase can ligate complementary sticky and blunt ends, but blunt-end ligation is inefficient and requires a higher concentration of both DNA and DNA ligase than the ligation of sticky ends does. [6] For this reason, most restriction enzymes used in DNA cloning make staggered cuts in the DNA strands to create sticky ends.
Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3'-end of the PCR products. This characteristic is exploited in "sticky end" TOPO TA cloning. [1] For "blunt end" TOPO cloning, the recipient vector does not have overhangs and blunt-ended DNA fragments can be cloned.
Microhomology-mediated end joining (MMEJ), also known as alt-non-homologous end joining, is another pathway to repair DSBs. The process of MMEJ can be summarized in five steps: the 5' to 3' cutting of DNA ends, annealing of microhomology, removing heterologous flaps, and ligation and synthesis of gap filling DNA. [ 5 ]