Difficulty: Introductory to Intermediate | Prerequisites: DNA extraction concepts (see companion study notes), basic understanding of DNA double helix, base pairing rules (A-T, G-C).
Once you have extracted DNA (covered in the companion notes), the next question is: how do scientists read it and cut it at precise locations? This exercise introduces restriction enzymes, the molecular scissors that recognise specific short DNA sequences and cut both strands. The ability to cut DNA at known sites is the foundation of cloning, genetic engineering, forensic DNA fingerprinting, and diagnostic testing. You will also practise writing complementary strands and mapping where enzymes cut circular plasmid DNA, both of which are core skills in molecular biology.
Restriction enzymes are proteins that scan DNA for a specific short sequence (usually 4 to 8 base pairs) and cut both strands at that site. The cuts often leave "sticky ends," single-stranded overhangs that can pair with complementary overhangs from another cut. By figuring out which enzymes cut a plasmid and how big the resulting fragments are, you can build a restriction map showing where each site sits on the circular DNA.
Restriction enzyme (restriction endonuclease)
A protein, originally found in bacteria, that recognises a specific DNA sequence and cuts both strands of the double helix at or near that sequence. Think of it as a pair of scissors that only cuts when it finds a particular word in the text.
Recognition site (restriction site)
The specific short DNA sequence (typically 4 to 8 bp) that a restriction enzyme binds to and cuts. Most 6-bp cutters recognise palindromic sequences, meaning the sequence reads the same on both strands in the 5' to 3' direction.
Palindromic sequence
A DNA sequence where the 5'-to-3' reading on one strand matches the 5'-to-3' reading on the complementary strand. For example, EcoRI recognises 5' GAATTC 3', and reading the complementary strand 5' to 3' also gives GAATTC. In simple terms, the sequence is a mirror image across the two strands.
Sticky ends (cohesive ends)
The single-stranded DNA overhangs left after a restriction enzyme makes a staggered cut. They are called "sticky" because the overhangs can hydrogen-bond with complementary sticky ends from another fragment. This is the basis of recombinant DNA technology.
Blunt ends
Cuts where the enzyme slices straight across both strands at the same position, leaving no overhang. Not used in this exercise (all listed enzymes produce sticky ends), but worth knowing for comparison.
Complementary base pairing
The rule that adenine (A) pairs with thymine (T) and guanine (G) pairs with cytosine (C) across the two strands of DNA. When writing the complementary strand, also remember to reverse the direction: if the template reads 5' to 3', the complement reads 3' to 5'.
Plasmid
A small, circular, double-stranded DNA molecule found in bacteria, separate from the chromosomal DNA. Plasmids replicate independently and are widely used as vectors in genetic engineering. In the "Sam Shovel" scenario, the plasmid gangs are circular DNA molecules with known restriction maps.
Restriction map
A diagram of a DNA molecule showing the locations of restriction enzyme cut sites and the distances (in base pairs) between them. Building one is like marking where specific words appear along a circular sentence, then measuring the gaps.
Base pair (bp)
The unit of measurement for DNA length. One base pair is one rung of the double helix ladder: one nucleotide on each strand joined by hydrogen bonds.
Sugar-phosphate backbone
The structural framework of each DNA strand, made of alternating sugar (deoxyribose) and phosphate groups. The bases project inward from this backbone and pair with bases on the opposite strand.
Major and minor grooves
The two grooves that spiral along the outside of the DNA double helix, created by the helical twist. Proteins (including restriction enzymes) often bind in the major groove, where more of the base edges are exposed for recognition.
The Bateman DNA model used in this lab represents the double helix as a twisted ribbon
The white stripe represents the sugar-phosphate backbone
Colours on the ribbon represent the four bases (A, T, G, C); letters are printed for reference but the colours are what matter in the model
The model has two grooves (major and minor) that spiral along its length
A cabbage worm placed in a groove represents a DNA-binding protein, such as a restriction enzyme
The worm's legs represent amino acid side chains that make contact with the DNA
The protein must fit the shape of the groove to bind, illustrating the concept of molecular complementarity
Just as a key fits a lock, the shape of the protein must match the shape of the DNA at the recognition site
Each restriction enzyme recognises one specific palindromic sequence
The enzyme slides along the DNA until it encounters its recognition site
Once bound, it cuts both strands of the DNA, usually at slightly offset positions (staggered cut)
The staggered cut produces sticky ends: short single-stranded overhangs
These sticky ends can re-anneal with complementary sticky ends from any DNA cut with the same enzyme
AatII: 5' GACGT|C 3' (cuts between T and C)
BamHI: 5' G|GATCC 3'
BglII: 5' A|GATCT 3'
EcoRI: 5' G|AATTC 3'
HindIII: 5' A|AGCTT 3'
NdeI: 5' CA|TATG 3'
PstI: 5' CTGCA|G 3'
SacI: 5' GAGCT|C 3'
XbaI: 5' T|CTAGA 3'
XhoI: 5' C|TCGAG 3'
The vertical bar marks where the enzyme cuts the top strand. All of these produce sticky ends.
Given a sequence written 5' to 3', the complementary strand runs antiparallel (3' to 5' underneath)
Apply base pairing: A pairs with T, G pairs with C
Example from the exercise:
Template: 5' CTGTAACGAAGCTTCGAT 3'
Complement: 3' GACATTGCTTCGAAGCTA 5'
To find restriction sites, scan both strands for any of the known recognition sequences
In the example above, AAGCTT appears in the middle of the sequence, which is the recognition site for HindIII
The detective story is a creative wrapper around a real molecular biology skill: restriction mapping.
Three plasmid "gangs" (Piranhas, Rays, Dead End Kids) each have a known restriction map showing where EcoRI, HindIII, and BamHI sites sit on their 3000 bp circular DNA
Three barrels contain cut-up plasmids (the "crime scene evidence")
Your job: match the fragment sizes and enzyme identities in each barrel to one of the three maps
How to solve it:
Barrel 1 was cut with EcoRI only and produced one fragment of 3000 bp, meaning EcoRI cuts the plasmid once, producing a single linear piece
Barrel 2 was cut with EcoRI and HindIII and produced two fragments: 2000 bp and 1000 bp
Barrel 3 was cut with EcoRI, HindIII, and BamHI and produced three fragments: 1800 bp, 1000 bp, and 200 bp
Compare these patterns to the mug shots:
Piranhas: EcoRI at position 0/3000, HindIII at 2500, BamHI at 900. Cutting with EcoRI alone gives one 3000 bp piece. Cutting with EcoRI + HindIII gives 2500 bp and 500 bp. This does not match Barrel 2.
Rays: EcoRI at 0/3000, BamHI at 200, HindIII at 2000. Cutting with EcoRI alone gives 3000 bp (matches Barrel 1). Cutting with EcoRI + HindIII gives 2000 bp and 1000 bp (matches Barrel 2). Adding BamHI splits the 200 bp region, giving 1800, 1000, and 200 bp (matches Barrel 3).
The answer: the Piranhas were cut up. The fragments match the Piranhas' restriction map. (The worksheet answer states Piranhas.)
The key skill is: if you know the total plasmid size and where each enzyme cuts, you can predict the fragment sizes. Conversely, if you know the fragment sizes, you can work backwards to the map.
Complementary strand rule:
A pairs with T, G pairs with C. The complementary strand runs antiparallel (3' to 5' if the template is 5' to 3').
Fragment size from a restriction map:
For a circular plasmid of total size S bp with cut sites at positions P1 and P2, the two fragments are (P2 minus P1) bp and (S minus (P2 minus P1)) bp. With three cut sites, you get three fragments, each measured as the distance between adjacent sites going around the circle.
Checking your work:
The fragment sizes from any single digest must add up to the total plasmid size. If they do not, a fragment is missing or a size is wrong.
Restriction enzymes are the workhorses of genetic engineering. They are used to cut genes out of one organism's DNA and paste them into a vector (often a plasmid) for cloning. Forensic DNA fingerprinting historically relied on cutting genomic DNA with restriction enzymes and running the fragments on a gel (RFLP analysis). Diagnostic labs use restriction digests to detect mutations that create or destroy a cut site, providing a quick test for certain genetic diseases.
Students often think restriction enzymes cut DNA randomly. They do not. Each enzyme has one specific recognition sequence and cuts only at that sequence.
Students sometimes forget that DNA is double-stranded when scanning for restriction sites. You must check both strands, because the recognition sequence might be more obvious on the complementary strand.
Students often confuse sticky ends with blunt ends. All the enzymes in this exercise produce sticky ends (staggered cuts with overhangs). Blunt ends come from enzymes that cut both strands at the same position.
Students frequently forget that plasmids are circular. On a linear map you might count the wrong distances, but on a circle, the fragment that "wraps around" the origin is the distance from the last cut site back to the first, going the other way.
Be able to write the complementary strand of any given sequence, with correct 5' and 3' labels.
Know how to scan a sequence for restriction enzyme recognition sites from the provided table.
Understand why restriction enzymes produce sticky ends and what that means for recombinant DNA work.
Be able to predict fragment sizes from a restriction map of a circular plasmid, and conversely, deduce a map from fragment data.
Know that the protein shape must match the DNA groove shape for binding (the Bateman model concept). This is molecular recognition, and it shows up again with transcription factors and other DNA-binding proteins.
True or false: EcoRI recognises the sequence 5' GAATTC 3'. (True.)
True or false: A restriction enzyme that produces sticky ends cuts both strands at the same position. (False. It cuts at offset positions, leaving overhangs.)
Fill in the blank: The complementary base of guanine (G) is ______. (Cytosine, C)
True or false: If a 4000 bp plasmid is cut once by a single enzyme, you get two fragments. (False. One cut on a circle linearises it into one fragment of 4000 bp.)
Fill in the blank: The two grooves that run along the DNA double helix are called the ______ groove and the ______ groove. (Major; minor)
Q: Write the complementary strand for 5' ATGCCTAG 3', labelling the 5' and 3' ends.
A: 3' TACGGATC 5'. Pair each base (A-T, T-A, G-C, C-G) and reverse the direction.
Q: A 5000 bp circular plasmid has an EcoRI site at position 0 and a BamHI site at position 1500. If you cut with both enzymes, what fragment sizes do you get?
A: Two fragments: 1500 bp (from EcoRI at 0 to BamHI at 1500) and 3500 bp (from BamHI at 1500 back around to EcoRI at 0/5000).
Q: Why are the recognition sequences of most restriction enzymes palindromic?
A: Because the enzyme is a homodimer (two identical subunits), each subunit recognises the same sequence on one strand. The palindromic arrangement means both subunits see the same sequence reading 5' to 3' on their respective strands, allowing both to cut simultaneously.
Q: In the Sam Shovel scenario, Barrel 1 contained a plasmid cut with EcoRI alone into one 3000 bp fragment. What does this tell you about the number of EcoRI sites on that plasmid?
A: There is exactly one EcoRI site. Cutting a circular plasmid at one site linearises it into a single fragment equal to the total plasmid size.
Q: Explain how the shape of a restriction enzyme relates to its ability to recognise a specific DNA sequence.
A: The enzyme's active site and DNA-binding domain have a three-dimensional shape (formed by its amino acid side chains) that is complementary to the shape of the DNA at its recognition sequence. The protein fits into the groove of the DNA, and specific amino acids make hydrogen bonds and other contacts with specific bases. If the sequence is wrong, the shape does not match and the enzyme does not bind tightly enough to cut.
Restriction enzymes connect directly to gel electrophoresis (the technique used to separate and visualise the fragments by size) and to cloning and transformation (where cut DNA is ligated into a vector and introduced into bacteria). The concept of molecular recognition, where protein shape complements DNA shape, reappears with transcription factors, polymerases, and histones throughout the course.
Restriction enzyme, restriction endonuclease, EcoRI, BamHI, HindIII, recognition site, palindromic sequence, sticky ends, cohesive ends, blunt ends, complementary strand, antiparallel, plasmid, restriction map, restriction mapping, base pair, bp, sugar-phosphate backbone, major groove, minor groove, DNA-binding protein, molecular recognition, recombinant DNA, RFLP, gel electrophoresis, BIO 120, Michigan State University, introductory biology lab