Restriction Enzyme Digestion and DNA Modification

Restriction enzymes (REs) function by cutting double-stranded DNA at specific 4- to 8-base pair inverted repeat recognition sequences. The products of DNA cleavage are either blunt-ended or contain 5′ or 3′ overhangs. 

• Thermo Scientific Conventional Enzymes

Ligases

Regardless of the type of end generated by restriction digestion, cleavage of the DNA results in fragments with 3′-hydroxyl groups and 5′-phosphate groups at their termini. DNA ligase covalently connects 5′-phosphate and 3′-hydroxyl termini of duplex DNA (or RNA) in an ATP-dependent reaction.

• Anza T4 DNA Ligase Master Mix
• T4 DNA Ligase, 1 U⁄µl
• T4 DNA Ligase, 5 U⁄µl

Alkaline phosphatase hydrolyzes 3′ and 5′ phosphates from DNA and RNA. It is suitable for removing 5′ phosphates prior to end labeling and for dephosphorylating vectors prior to insert ligation. It can also be used to dephosphorylate proteins.

• Anza Alkaline Phosphatase

End repair allows DNA with 5′ or 3′ overhangs to be converted to 5′ phosphorylated blunt-end DNA for efficient ligation into blunt-end cloning vectors.

• Anza DNA End Repair Kit
  

Polynucleotide kinase is used to perform 5’-phosphorylation of DNA and oligonucleotides. When preparing DNA for the ligation step in cloning, either the insert DNA or the vector DNA should contain a 5’ phosphate. Often PCR-amplified DNA is treated with T4 PNK to phosphorylate the 5’ end for subsequent cloning ligation.

• Anza T4 PNK Kit

DNA blunting kits allow for conversion of DNA with cohesive ends to DNA with blunt ends for use in blunt-end ligation reactions. This is useful when the desired vector does not contain a recognition site that would produce blunt ends.

• Anza DNA Blunt End Kit

Polymerases are used to generate blunt ends and/or incorporate labeled DNA.

• DNA Polymerases
• See the complete list of DNA-modifying enzymes