End Repair and Blunting: As a result of dsDNA manipulations such as shearing, PCR, and restriction enzyme or fragmentase digestion, DNA fragments can contain 3’ or 5’ ends that are incompatible with downstream cloning or ligation protocols. DNA Polymerases are often used to eliminate undesired overhangs and enable either blunt-end or T/A ligation. To generate blunt fragments from unwanted 3’ overhangs, extra bases can be removed by using a polymerase that possesses 3’-5’ exonuclease activity (e.g., T4 DNA Polymerase). Unwanted 5’ overhangs can either be blunted using an enzyme with a 5’-3’ exonuclease activity (e.g., T4 DNA Polymerase) or alternatively, the recessed 3’ end can be extended via DNA polymerization in the presence of dNTPs. When blunting DNA that may contain a mixture of both 5’ and 3’ overhangs (as a result of shearing, fragmentase digestion, etc), it is important to use an enzyme containing both 3’-5’ and 5’-3’ exonuclease activities (e.g., T4 or DNA Polymerase I Large (Klenow) Fragment) in the presence of dNTPs.
For T/A cloning, Taq DNA Polymerase can be used to add a terminal 3’ adenine to blunt ended fragments that result from the blunting methods above or from Q5 or Phusion-based PCR.
For convenience, NEB provides Quick Blunting and End-Repair kits to simplify these options.
DNA Probe Generation: DNA probes can be generated to identify or isolate specific DNA sequences of interest by labeling (via radio- or fluorescently-labeled nucleotides) or affinity-tagging DNA that is complementary to a target DNA. The DNA probe is allowed to hybridize with the target sequence, confirming the location or presence of the target sequence. Affinity tags may allow isolation of the tagged DNA whereas fluorescence tags (as in fluorescence in situ hybridization) may allow the position of the sequence of interest to be identified along a chromosome, and copy number to be determined. When making DNA probes by adding a single labeled base on the 3’ end of a DNA sequence, it is important to use a polymerase without a 3’-5’ exonuclease activity (e.g., Taq or Therminator DNA Polymerase) to ensure that the label will not be removed after it is incorporated into the DNA.