IMAT Biology · Microorganisms and Biotechnology
Recombinant DNA: Cutting, Joining and Cloning Genes
Restriction enzymes cut DNA at set sequences, ligase joins the pieces, and a vector carries the new combination into a host cell that is then selected and grown.
Why this matters
This is the most asked part of the chapter. Questions have asked for the fragments left after a plasmid is cut, the order of the steps, the bases of a sticky end, which bonds the enzymes break or make, the role of Agrobacterium, and (in 2026) which class of enzyme a restriction enzyme is.
Concept 1 of 3: Restriction enzymes, sticky ends and counting fragments
A restriction enzyme is molecular scissors that cut only at one short sequence. Bacteria make them to chop up the DNA of invading phages. Because the cut is always at the same sequence, every molecule cut by the same enzyme gets the same matching ends, and any two such ends can be joined.
Definition
- Restriction enzymes are endonucleases: they cut within a DNA molecule (exonucleases trim from the ends). By the enzyme classes they are hydrolases: they use water to break phosphodiester bonds in the sugar-phosphate backbone of both strands.
- Each recognises a recognition site of 4 to 8 base pairs, usually a palindrome: both strands read the same 5' to 3'. EcoRI recognises 5'-GAATTC-3' and cuts each strand between the G and the first A, leaving an AATT overhang.
- A staggered cut leaves short single-stranded overhangs, sticky ends, which pair with any complementary overhang by hydrogen bonds. A straight cut leaves blunt ends.
- The bacterium's own DNA is protected because its recognition sites are methylated.
- Counting pieces: each cut adds one piece to a line, but the first cut in a circle only opens it.
Fragments from n cuts
- number of cut sites (at least 1)
Worked example
An enzyme cuts at positions 400, 1100 and 2500 bp. Find the fragment sizes (a) in a linear DNA molecule of 3000 bp, and (b) for sites at 1000, 1800 and 4200 bp in a circular plasmid of 5000 bp.
Practice this conceptself-check · 4 quick reps
Restriction enzymes break phosphodiester bonds
The cut goes through the sugar-phosphate backbone, so the bonds broken are phosphodiester bonds. The few hydrogen bonds between the bases of a staggered cut simply come apart on their own. An enzyme that only broke hydrogen bonds would separate the strands without cutting anything.
Restriction enzymes are endonucleases, which are hydrolases
The six enzyme classes are oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases. A restriction enzyme hydrolyses bonds inside the DNA chain, so it is an endonuclease, one kind of hydrolase. Ligase is the enzyme that joins DNA, the opposite job.
A circle with n cuts gives n pieces
Do not add the extra piece you would get on a straight molecule, and do not forget the piece that crosses the plasmid's starting point. The sizes must add up to the plasmid's full length.
Concept 2 of 3: Ligase, reverse transcriptase and vectors
Cutting DNA is only half the job. The new piece must be sealed into a carrier, a vector, that can get into the host cell and be copied there. Each tool in the kit has one job, and IMAT likes to swap the jobs around in its options.
Definition
- DNA ligase seals the sugar-phosphate backbone by forming phosphodiester bonds (using ATP). The base pairing between sticky ends forms by itself; ligase makes it permanent.
- cDNA (complementary DNA) is made from mature mRNA by reverse transcriptase. It has no introns and no promoter, so a bacterium, which cannot remove introns, can express it once it is placed after a bacterial promoter.
- A vector carries DNA into a host cell. A good plasmid vector has an origin of replication, one or more marker genes (such as antibiotic resistance) and restriction sites where the gene can go.
- Agrobacterium tumefaciens carries the Ti plasmid. In nature it transfers a piece of this plasmid (T-DNA) into plant cells, where its genes make the cells divide and form a gall. Biologists use it to carry a new gene into plant cells.
| Tool | What it does | Point to remember |
|---|---|---|
| Restriction enzyme | Cuts DNA at a specific sequence | Breaks phosphodiester bonds; same enzyme gives matching sticky ends |
| DNA ligase | Joins DNA fragments | Forms phosphodiester bonds; uses ATP |
| Reverse transcriptase | Makes cDNA from an mRNA template | From retroviruses; the cDNA has no introns |
| Plasmid vector | Carries a gene into bacteria | Origin of replication, marker genes, restriction sites |
| Ti plasmid of Agrobacterium tumefaciens | Carries a gene into plant cells | The bacterium supplies the vector; in nature its genes make plant cells divide |
| Viral vector | Carries a gene into animal or human cells | Modified so that it cannot multiply |
| Liposome | Lipid sphere that fuses with the cell membrane | Non-viral vector, used in gene therapy |
Practice this conceptself-check · 4 quick reps
Ligase makes phosphodiester bonds, not hydrogen bonds
Complementary sticky ends find each other and pair by hydrogen bonds without any enzyme. Ligase then forms the phosphodiester bonds that close the backbone. Options saying ligase forms hydrogen bonds, or breaks phosphodiester bonds, swap the jobs.
Concept 3 of 3: Making and selecting a recombinant organism
The method follows the logic of the tools. Get the gene, open the vector with the same enzyme so the ends match, join them, put the result into a host, then find the few cells that took up the right plasmid. Only checked cells are grown on or used to modify a plant or animal.
Definition
- Transformation (here) is the uptake of a plasmid by the host cell. Bacteria are made to take up DNA by a heat shock in calcium chloride or by electroporation (a brief electric pulse).
- Only a small fraction of cells take up a plasmid, and some plasmids close up without the gene. So there are three kinds of cell: no plasmid, plasmid without the gene, and the wanted recombinant plasmid.
- Selection uses marker genes. If the gene is inserted inside a second marker, that marker is switched off (insertional inactivation). With lacZ as the second marker, on a plate with ampicillin and X-gal: no plasmid means no growth, an empty plasmid gives a blue colony, a recombinant plasmid gives a white colony.
- Fluorescent markers such as GFP (from a jellyfish) are also used: cells that glow carry the plasmid.
| Step | What is done | Tool |
|---|---|---|
| 1. Obtain the gene | Cut it out of DNA, make it as cDNA from mRNA, or copy it by PCR | Restriction enzyme, reverse transcriptase or PCR |
| 2. Open the vector | Cut the plasmid with the same restriction enzyme | Same enzyme, so the sticky ends match |
| 3. Join | Mix gene and plasmid; sticky ends pair; seal the backbone | DNA ligase |
| 4. Insert into the host | Bacteria take up the plasmid (transformation) | Heat shock in calcium chloride, or electroporation |
| 5. Select | Grow on a medium where only cells with the recombinant plasmid survive or show a colour | Marker genes |
| 6. Use | Grow the chosen bacteria in a fermenter, or deliver the checked plasmid to plant or animal cells | Fermenter; Agrobacterium or other vector |
Steps 1 and 2 can be done in either order; joining must come after both, and selection after insertion into the host.
Practice this conceptself-check · 4 quick reps
A transformed cell is not necessarily recombinant
A bacterium that has taken up a plasmid is transformed, but the plasmid may have closed up without the gene. That is why a second, screening step (such as blue and white colonies) is needed after the antibiotic selects for any plasmid.
Join before you insert
Ligation happens in the test tube, before the plasmid goes into the host, and the recombinant plasmid is checked before it is used to modify a plant or animal. An order that puts ligase after insertion into the host, or selection before the cutting, is wrong.
Summary — formulas & gotchas at a glance
A revision cheat-sheet for the formulas and gotchas above. Click any concept name to jump back to its full explanation.
Formulas (1)
- Restriction enzymes, sticky ends and counting fragments
Fragments from n cuts
Reference tables (2)
Ligase, reverse transcriptase and vectors7 rows
| Tool | What it does | Point to remember |
|---|---|---|
| Restriction enzyme | Cuts DNA at a specific sequence | Breaks phosphodiester bonds; same enzyme gives matching sticky ends |
| DNA ligase | Joins DNA fragments | Forms phosphodiester bonds; uses ATP |
| Reverse transcriptase | Makes cDNA from an mRNA template | From retroviruses; the cDNA has no introns |
| Plasmid vector | Carries a gene into bacteria | Origin of replication, marker genes, restriction sites |
| Ti plasmid of Agrobacterium tumefaciens | Carries a gene into plant cells | The bacterium supplies the vector; in nature its genes make plant cells divide |
| Viral vector | Carries a gene into animal or human cells | Modified so that it cannot multiply |
| Liposome | Lipid sphere that fuses with the cell membrane | Non-viral vector, used in gene therapy |
Making and selecting a recombinant organism6 rows
| Step | What is done | Tool |
|---|---|---|
| 1. Obtain the gene | Cut it out of DNA, make it as cDNA from mRNA, or copy it by PCR | Restriction enzyme, reverse transcriptase or PCR |
| 2. Open the vector | Cut the plasmid with the same restriction enzyme | Same enzyme, so the sticky ends match |
| 3. Join | Mix gene and plasmid; sticky ends pair; seal the backbone | DNA ligase |
| 4. Insert into the host | Bacteria take up the plasmid (transformation) | Heat shock in calcium chloride, or electroporation |
| 5. Select | Grow on a medium where only cells with the recombinant plasmid survive or show a colour | Marker genes |
| 6. Use | Grow the chosen bacteria in a fermenter, or deliver the checked plasmid to plant or animal cells | Fermenter; Agrobacterium or other vector |
Steps 1 and 2 can be done in either order; joining must come after both, and selection after insertion into the host.
Watch out for (6)
- Restriction enzymes break phosphodiester bonds→ Restriction enzymes, sticky ends and counting fragments
- Restriction enzymes are endonucleases, which are hydrolases→ Restriction enzymes, sticky ends and counting fragments
- A circle with n cuts gives n pieces→ Restriction enzymes, sticky ends and counting fragments
- Ligase makes phosphodiester bonds, not hydrogen bonds→ Ligase, reverse transcriptase and vectors
- A transformed cell is not necessarily recombinant→ Making and selecting a recombinant organism
- Join before you insert→ Making and selecting a recombinant organism
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