Assume we are given a DNA fragment like
ACTTAGC....GCTCGGCA
TGAATCG...CGAGCCGT
so we have blunt ends. I am looking for a nuclease, that cleaves the end, such that we obtain sticky ends. Hence, the above fragment would look like
GC....GCTCGGCA
TGAATCG...CGA
(I hope the presentation makes clear, what I am looking for.)
Do you know an enzyme that does the job?
Also would it be possible the cut just one end of the DNA fragment, such that it looks like
ACTTAGC....GCTCGGCA
TGAATCG...CGA
Here if would be advantageous if I could slectively target the end sequence, just like with a CRISPR/Cas9 or CRISPR/Cpf1, but since the target sequence could also be present in the middle of the fragment, the cleavage should just occur at the end. Is that possible?
Q. I am looking for a nuclease, that cleaves the end, such that we obtain sticky ends
A. Are restriction endonucleases unsuitable for your purpose? Exonucleases are for blunting sticky ends so they are unsuitable for your purpose. If you are looking for some specific application then it might be beneficial if you gave more details regarding your purpose.
Q. Also would it be possible the cut just one end of the DNA fragment?
A. From your example, the recognition sites are different. So, considering you plan to use restriction endonucleases, just digest with one enzyme.
Q.Here if would be advantageous if I could slectively target the end sequence, just like with a CRISPR/Cas9 or CRISPR/Cpf1, but since the target sequence could also be present in the middle of the fragment, the cleavage should just occur at the end. Is that possible?
A. The Cas9 system uses a guide RNA that is ~20-25 bases long. This long sequence helps in reduction of off-target cuts. This specificity should be enough to solve your problem.
The problem with Cas9 is the requirement of the PAM motif for cutting, but I remember having seen some nucleases that can recognize alternate PAM motifs. I'm certain Google can help you in this case.
Subramaniyam
You can PCR your template with forward and reverse primers specific for your 5'- and 3'-ends in which you incorporate a "tail" with a convenient restriction site not already present in your template sequence.
For example,
Forward primer 5' acgcgtACTTAGC... (MulI restriction site in lower case letters)
Use a high-fidelity polymerase and low number of PCR cycles to minimize taq-introduced errors.
However, if the introduction of the restriction site is an inconvenience, try approaches suggested by others.
A restriction enzyme might work. But keep in mind that some restriction enzymes have lower efficiency at the end of the DNA. So if you get this DNA by PCR, add a few nucleotide at the end of the DNA when you design your primers.
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