Restriction Enzyme Digestion and DNA Modification
Cloning by restriction enzyme digestion and ligation is a simple and easy way of moving a fragment of double-stranded DNA from one plasmid to another.
- Choosing restriction enzymes whose recognition sequences flank your gene of interest
- Incubating the reaction for the recommended amount of time
- Purifying your fragment
- Ligating into your plasmid of interest
Invitrogen Anza Restriction Enzyme Cloning System
A complete system of restriction enzymes and DNA-modifying enzymes—for beautifully simple cloning.
- One buffer for all restriction enzymes
- One digestion protocol for all DNA types
- Complete digestion in 15 minutes
- Overnight digestion without star activity
A simple two-step protocol, regardless of the number of restriction enzymes in your reaction or the type of DNA you’re using—just prepare your reaction mixture and incubate at 37°C for 15 minutes.
Conventional Restriction Enzymes
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.
DNA Modification Enzymes
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.
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.
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.
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.
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.
Polymerases are used to generate blunt ends and/or incorporate labeled DNA.