Producing DNA samples by shearing, nebulization, restriction enzyme digestion or PCR amplification frequently leaves DNA molecules with ends incompatible for downstream experiments. Selective enzymatic treatment is used to prepare DNA for ligation.
Ligation, the subsequent step to DNA end modification in the cloning process, is the formation of covalent phosphodiester bonds between the 3′ hydroxyl and 5’ phosphate ends of DNA and the vector. Preparation of DNA for vector ligation can include different types of end treatments, such as end-blunting, phosphorylation and dephosphorylation. Some enzymes couple the removal of the 3’ phosphate to expose hydroxyl groups, and the addition of a phosphate group to the 5’ end. Ligation of DNA with other molecules can either be through complementary sequence base pairing or blunt-ended ligation. The coupled action of enzymes can be used to remove any terminal single-stranded overhangs that are left after DNA digestion by restriction endonuclease or added to DNA during PCR amplification.
These end treatments can also be used to alter the phosphorylation state of the 5’ end of DNA for detection, isolation and sequencing applications. Many investigations use the addition of a phosphate radioisotope for labeling applications.
Digested DNA typically possesses a 5’ phosphate group that is required for ligation. In order to prevent self-ligation, the 5′ phosphate can be removed prior to ligation. Dephosphorylation of the 5’ end prohibits self-ligation, enabling the researcher to manipulate the DNA as desired before re-ligating. Generally, it is a good idea to dephosphorylate the linearized vector to facilitate ligation of recombinant DNA molecules. In fact, vector self-ligation increases the background activity of the cloning process. Dephosphorylation can be accomplished using any of a number of phosphatases, including Shrimp Alkaline Phosphatase (rSAP) (NEB #M0371), Quick CIP Phosphatase (NEB #M0525) and Antarctic Phosphatase (NEB #M0289).
Blunting is the elimination of incompatible 3’ or 5’ overhangs for the promotion of blunt-end ligation. Several approaches may be used for DNA end blunting. Terminal unpaired nucleotides may be removed from DNA ends by using an enzyme with exonuclease activity, which hydrolyzes a terminal phosphodiester bond, thereby removing the overhang one base at a time. DNA fragments with 5’ overhangs may be blunted by filling in a recessed 3’ terminus with DNA polymerase in the presence of dNTPs. End removal or fill-in can be accomplished using a number of enzymes, including DNA Polymerase I Large (Klenow) Fragment (NEB #M0210), T4 DNA Polymerase (NEB #M0203) or Mung Bean Nuclease (NEB #M0250). Once blunted, DNA is universally compatible with other blunt-ended fragments and vectors.
Single- or double-stranded DNA with a 5′-hydroxyl terminus has to be phosphorylated prior to ligation as 5’ phosphate groups are required for ligation. Phosporylation of DNA ends with phosphate radioisotopes can also be used to label DNA in preparation of DNA for subsequent detection, isolation and sequencing applications. A number of polynucleotide kinases, including T4 PNK (NEB #M0201) and T4 PNK (3′ phosphatase minus) (NEB #M0236), can be used to transfer the γ-phosphate of ATP to a 5’ terminus of DNA.