Table of Contents
Restriction enzymes and ligases are two of the most basic “tools” of genetic engineering. Together they allow DNA to be cut at defined positions and then rejoined in new combinations. This chapter focuses on what makes these enzymes special and how they are used in practice.
Restriction Enzymes
Biological Origin and Function
Restriction enzymes (restriction endonucleases) are naturally occurring enzymes found primarily in bacteria and archaea. In their natural context they are part of a restriction–modification system:
- Restriction enzyme: cuts foreign DNA (e.g. from phages) at specific sequences.
- Modification enzyme (DNA methyltransferase): chemically modifies (methylates) the same sequences in the host DNA so that it is protected from cleavage.
This system functions as a primitive “immune system”: foreign DNA that is unmethylated is cut, while the bacterium’s own DNA is spared.
Genetic engineering makes use of the cutting ability of restriction enzymes, but usually without using the corresponding methyltransferases.
Recognition Sequences
Each restriction enzyme recognizes a specific short DNA sequence, usually:
- 4–8 base pairs long
- often a palindrome in double-stranded DNA (reading the same in 5'→3' direction on both strands)
Example: The sequence recognized by the enzyme EcoRI is
$$
5' \text{– GAATTC –} 3' \\
3' \text{– CTTAAG –} 5'
$$
Reading in the 5'→3' direction on each strand gives “GAATTC” on both, so it is palindromic.
The precision of recognition means:
- The same enzyme always cuts at exactly, and only, its own recognition sites.
- Different enzymes cut at different sequences, providing many “options” for manipulating DNA.
Types of Cuts: Sticky Ends and Blunt Ends
Restriction enzymes cut both strands of the DNA double helix within or near their recognition sequence. The exact position of the cut determines the type of end produced.
Sticky (Cohesive) Ends
Many enzymes cut in a staggered way, leaving short single-stranded overhangs:
- Overhang at the 5' end (5' overhang)
- Overhang at the 3' end (3' overhang)
Example: EcoRI cuts:
$$
5' \text{– G} \downarrow \text{AATTC –} 3' \\
3' \text{– CTTAA} \uparrow \text{G –} 5'
$$
Result:
- 5'–AATT overhangs on both DNA fragments.
- These overhangs are complementary, so two pieces cut with EcoRI can base-pair with each other.
Properties of sticky ends:
- They can hydrogen bond (anneal) with complementary overhangs.
- They greatly facilitate joining of DNA fragments by ligases.
- They can be designed to ensure that only fragments with matching overhangs join.
Blunt Ends
Some restriction enzymes cut both strands at the same position, leaving no overhang:
Example: SmaI recognition sequence and cut:
$$
5' \text{– CCC} \downarrow \text{GGG –} 3' \\
3' \text{– GGG} \uparrow \text{CCC –} 5'
$$
Result:
- Ends are completely paired; no single-stranded region.
- These are called blunt ends.
Properties of blunt ends:
- Any blunt end can, in principle, be joined to any other blunt end.
- Ligation is less efficient because there are no sticky overhangs to help align the fragments.
- Useful when you do not want to introduce a specific sequence or directionality.
Nomenclature of Restriction Enzymes
Names of restriction enzymes follow a convention based on their origin:
- 1st letter: genus (italicized in species name; capitalized)
- 2nd and 3rd letters: species (lowercase)
- Optional 4th letter: strain
- Roman numeral: order of discovery in that organism
Examples:
- EcoRI: Escherichia coli strain RY13, first enzyme (I)
- HindIII: Haemophilus influenzae strain d, third enzyme (III)
- BamHI: Bacillus amyloliquefaciens strain H, first enzyme (I)
Knowing the name gives you both the biological source and a unique identifier for its recognition sequence.
Isoschizomers and Neoschizomers
Because many organisms have evolved similar systems:
- Isoschizomers: different enzymes from different species that recognize and cut at the same sequence and in the same way.
- Neoschizomers: recognize the same sequence but cut at different positions within it, generating different ends.
These variants give flexibility in experimental design.
Practical Use of Restriction Enzymes
In genetic engineering, restriction enzymes are used to:
- Cut plasmid vectors and foreign DNA at defined positions.
- Create DNA fragments for cloning, mapping, or analysis.
- Analyze DNA by size pattern after digestion (restriction fragment length patterns).
Key practical aspects:
- Each enzyme has an optimal buffer, salt concentration, pH, and temperature (often 37 °C).
- Enzymes can be inactivated by heat after digestion, if needed.
- Overdigestion can sometimes cause “star activity” – non-specific cleavage under unfavorable conditions (e.g. wrong buffer, too high glycerol concentration).
DNA Ligases
Biological Role
DNA ligases are enzymes that join DNA fragments by forming a covalent bond in the sugar–phosphate backbone. They are essential in cells for processes such as:
- Sealing Okazaki fragments during DNA replication.
- Repairing DNA breaks.
Genetic engineering harnesses this natural function to join DNA fragments in vitro.
The Chemical Reaction
Ligases catalyze formation of a phosphodiester bond between:
- A 3' hydroxyl group (3'-OH) on one nucleotide
- A 5' phosphate group (5'-P) on the adjacent nucleotide
This can be summarized as:
$$
\text{DNA–3'–OH} + \text{DNA–5'–P} \xrightarrow{\text{ligase}} \text{DNA–3'–O–P–5'–DNA} + \text{H}_2\text{O}
$$
The reaction requires energy, provided by a cofactor:
- Most commonly used in vitro: T4 DNA ligase, which uses ATP.
- Some bacterial ligases use NAD⁺.
Ligation of Sticky Ends
Sticky ends with complementary single-stranded overhangs can spontaneously base-pair (anneal) due to hydrogen bonding. However, this annealing is not covalent and is reversible.
DNA ligase:
- Finds the annealed junction where a 5'-P is adjacent to a 3'-OH.
- Forms the covalent phosphodiester bond, making the joining permanent.
Properties:
- Ligation of compatible sticky ends is highly efficient.
- The specificity mostly comes from the restriction enzymes that created the overhangs – only matching overhangs anneal.
Common experimental strategies:
- Digest vector and insert DNA with the same restriction enzyme(s) so that their sticky ends match.
- Alternatively, use two different enzymes with incompatible ends to enforce directionality (see below).
Ligation of Blunt Ends
Blunt ends have no single-stranded overhangs, so they cannot anneal via base pairing before ligation. Ligation is possible, but:
- Ligase must bring the blunt ends together directly.
- The reaction is slower and less efficient than for sticky ends.
- Higher DNA concentrations, longer incubation times, and sometimes additives are used to improve ligation.
Because blunt ends are non-specific, any blunt-ended fragment can, in principle, ligate to any other blunt-ended fragment, which can be useful but may require more careful screening afterward.
Directional Cloning with Restriction Enzymes and Ligases
One frequent goal in genetic engineering is to insert a DNA fragment (the “insert”) into a vector in one specific orientation. Restriction enzymes and ligases are combined to achieve this.
Strategy:
- Cut the vector with two different enzymes that create different, incompatible sticky ends.
- Cut the insert with the same two enzymes, so that its ends match the vector ends.
- Mix vector and insert, allow sticky ends to anneal, then add ligase.
Consequences:
- The insert can ligate into the vector only in one orientation, because each end is unique.
- The vector is less likely to re-ligate without insert, because its two ends are not compatible with each other.
This approach is called directional cloning, and it relies entirely on the predictable sticky ends generated by restriction enzymes and the joining function of ligase.
Ligation Mixtures and Reaction Conditions
Key components of a typical ligation reaction:
- DNA vector and DNA insert with compatible ends.
- DNA ligase (often T4 DNA ligase).
- Buffer containing ATP and ions required by the enzyme.
Important parameters:
- Insert:vector ratio: adjusted to favor the desired product (e.g. 3:1 molar ratio of insert to vector).
- Temperature: sticky-end ligations often at ~16 °C or room temperature; blunt-end ligations may require lower temperatures and longer times to stabilize transient interactions.
- Time: from a few minutes (for very efficient sticky-end ligations) to overnight (especially for blunt ends).
The balance between efficiency and specificity is adjusted according to experimental needs.
Combining Restriction Enzymes and Ligases in Genetic Engineering
Together, restriction enzymes and ligases allow:
- Cutting DNA at precise, predictable sites (restriction digest).
- Creating ends (sticky or blunt) that determine which pieces can be joined.
- Joining DNA fragments to build recombinant DNA molecules (ligation).
Fundamental applications include:
- Constructing recombinant plasmids: cutting vector and insert DNA, then ligating.
- Swapping DNA fragments (e.g. replacing a gene or regulatory region).
- Inserting tags or markers by designing fragments with appropriate restriction sites at their ends.
Because of their specificity and reliability, restriction enzymes and ligases form the core of many classical genetic engineering procedures, and they also play supporting roles in newer methods that are covered elsewhere in the course.