Restriction Enzyme Mapping Explained

Restriction enzyme mapping is a technique used to determine the positions of specific restriction enzyme recognition sites within a DNA sequence. It is one of the oldest and most widely used methods in molecular biology, essential for cloning, plasmid verification, and genome analysis.

What Are Restriction Enzymes?

Restriction enzymes (also called restriction endonucleases) are bacterial proteins that cut double-stranded DNA at specific recognition sequences. They evolved as a defense mechanism against bacteriophages. In the lab, they are used as precise molecular scissors.

Most restriction enzymes recognize palindromic sequences, meaning the sequence reads the same on both strands in the 5' to 3' direction. For example, EcoRI recognizes:

The vertical bar shows where the enzyme cuts. EcoRI produces a 4-base 5' overhang, known as a sticky end.

Types of Cuts: Sticky Ends and Blunt Ends

Restriction enzymes produce two types of cut ends:

• Sticky ends (cohesive ends): The cut leaves a short single-stranded overhang. These overhangs can anneal with complementary sticky ends from other DNA fragments, making ligation more efficient. EcoRI, BamHI, and HindIII all produce sticky ends.
• Blunt ends: The enzyme cuts both strands at the same position, leaving no overhang. SmaI and EcoRV produce blunt ends. Blunt-end ligation is less efficient but more flexible since any two blunt ends can be joined.

Common Restriction Enzymes and Their Recognition Sites

How to Read a Restriction Map

A restriction map shows the positions of restriction sites along a linear or circular DNA molecule. The backbone represents the DNA, and vertical lines mark the cut positions. Each enzyme is usually shown in a different color or lane.

From a restriction map you can determine:

• The number of times each enzyme cuts the sequence
• The positions of each cut site in base pairs from the start
• The sizes of the fragments produced by a single or double digest

Using Restriction Maps for Cloning

When cloning a gene into a vector, you need compatible restriction sites on both the insert and the vector. The standard workflow is:

1. Identify restriction sites flanking your gene of interest
2. Choose enzymes that cut the vector at the correct location (the multiple cloning site)
3. Digest both the insert and vector with the same enzyme(s)
4. Ligate the compatible ends together
5. Transform into bacteria and screen colonies by restriction digest or PCR

Using two different enzymes (directional cloning) ensures the insert is inserted in the correct orientation and prevents self-ligation of the vector.

Verifying Constructs by Restriction Digest

After cloning, restriction digest is the standard method for verifying that the insert is present and in the correct orientation. You run the digest on an agarose gel and compare the band sizes to the expected pattern from your restriction map.

If the bands match the predicted sizes, the construct is correct. If not, the insert may be absent, in the wrong orientation, or the wrong size.

Frequency of Restriction Sites

A 6-base recognition site is expected to occur roughly once every 4096 bp (4^6) in random sequence. A 4-base cutter occurs about once every 256 bp. This means:

• 4-cutters are useful for generating many small fragments
• 6-cutters are better for generating larger, more manageable fragments
• 8-cutters (like NotI) cut very rarely and are used for large-scale genome mapping

Restriction mapping remains a core technique even in the era of next-generation sequencing. It is fast, inexpensive, and gives you direct physical information about your DNA construct that sequencing alone cannot always provide.