Recombinant DNA

Learning Targets

• I can define recombinant DNA.
• I can explain how DNA from different organisms can be combined.
• I can describe the steps used to create recombinant DNA.
• I can explain the role of plasmids in genetic engineering.
• I can identify examples of recombinant DNA technology.

Introduction

All living organisms use DNA to store genetic information. Although organisms may look very different, their DNA is constructed from the same four bases: adenine, thymine, cytosine, and guanine. The genetic code is also nearly universal, which means that a gene from one organism can sometimes be read and used by the cells of another organism.

Scientists can use enzymes to cut DNA from different sources and join the pieces together. The resulting DNA is called recombinant DNA. This technology allows useful genes to be transferred into microorganisms, plants, animals, or cultured cells.

Recombinant DNA technology is used to manufacture medicines, study genes, create genetically modified crops, and produce important substances such as human insulin.

What Is Recombinant DNA?

Recombinant DNA is DNA that has been created by joining genetic material from two or more different sources.

The word recombinant means formed by combining separate parts. In recombinant DNA, a section of DNA from one source is inserted into another DNA molecule.

For example, scientists can:

• Remove the human insulin gene from human DNA.
• Insert the gene into a bacterial plasmid.
• Place the recombinant plasmid inside a bacterial cell.
• Allow the bacterium to use the human gene to produce insulin.

The recombinant plasmid contains:

• Bacterial DNA from the original plasmid.
• An inserted gene originating from human DNA.

Because the final DNA molecule contains DNA from different sources, it is recombinant DNA.

Why DNA from Different Organisms Can Be Combined

DNA has the same basic chemical structure in almost all organisms. It consists of nucleotides containing the same four bases:

• Adenine, or A.
• Thymine, or T.
• Cytosine, or C.
• Guanine, or G.

The same complementary base-pairing rules also apply:

• A pairs with T.
• C pairs with G.

The genetic code is nearly universal. This means that a sequence of DNA bases usually provides the same basic instructions regardless of the organism in which it is found.

For example, if a human gene is inserted correctly into a bacterial cell, the bacterium may be able to read the gene’s instructions and produce the human protein.

However, the gene must be placed into the correct type of DNA molecule and may require suitable control sequences so that the host cell can express it.

Important Tools Used to Create Recombinant DNA

Scientists use several biological tools to create recombinant DNA.

Restriction Enzymes

Restriction enzymes cut DNA at specific base sequences called recognition sequences or restriction sites.

The same restriction enzyme is often used to cut:

• The DNA containing the desired gene.
• The vector that will carry the gene.

Using the same restriction enzyme produces matching ends that can join together.

DNA Ligase

DNA ligase is the enzyme that permanently joins DNA fragments.

Complementary sticky ends may first pair through hydrogen bonding. DNA ligase then seals the breaks in the sugar–phosphate backbone by forming phosphodiester bonds.

Restriction enzymes and DNA ligase have different roles:

• Restriction enzymes cut DNA.
• DNA ligase joins DNA.

Vectors

A vector is a DNA molecule used to carry genetic material into a host cell. Plasmids are commonly used as vectors in bacteria.

Other vectors include:

• Modified viruses.
• Artificial chromosomes.
• Small sections of DNA designed in laboratories.

Host Cells

A host cell is the cell that receives the recombinant DNA. Bacteria are commonly used because they grow quickly, reproduce rapidly, and are relatively easy to culture.

Yeast, plant cells, animal cells, and other microorganisms can also be used as host cells.

What Is a Plasmid?

A plasmid is a small, circular DNA molecule found naturally in many bacteria. It is separate from the bacterium’s main chromosome.

Plasmids can copy themselves inside bacterial cells. Some naturally contain genes that give bacteria useful characteristics, such as resistance to particular antibiotics.

Scientists use modified plasmids as vectors because plasmids:

• Are relatively small and easy to isolate.
• Can be cut open using restriction enzymes.
• Can carry an inserted gene.
• Can enter bacterial cells.
• Can be copied as bacteria reproduce.
• May contain marker genes that help identify transformed cells.

Important Parts of a Plasmid Vector

A plasmid used in genetic engineering usually contains several important regions.

Origin of Replication

The origin of replication is the site where the plasmid begins to be copied. It allows many copies of the plasmid to be produced inside a bacterial cell.

Restriction Sites

A plasmid contains one or more recognition sequences where restriction enzymes can cut it open. The desired gene can be inserted at one of these locations.

Selectable Marker

A selectable marker is a gene that helps scientists identify cells containing the plasmid. A commonly used marker may provide resistance to a particular antibiotic.

When bacteria are grown on a medium containing that antibiotic:

• Bacteria that received the plasmid may survive.
• Bacteria that did not receive the plasmid do not survive.

The use of antibiotic-resistance markers is carefully controlled in laboratories.

Promoter

A promoter is a DNA sequence that helps start the transcription of a gene. If scientists want the host cell to produce a protein, the inserted gene must usually be located near a promoter recognised by the host cell.

The Steps Used to Create Recombinant DNA

The exact procedure depends on the gene, vector, and host organism. A simplified process involves the following stages.

Step 1: Identify the Desired Gene

Scientists first identify a gene that produces a useful characteristic or protein.

Examples include genes that provide instructions for:

• Human insulin.
• Human growth hormone.
• Particular vaccine proteins.
• Useful agricultural characteristics.

The gene may be isolated from an organism, copied using PCR, or produced synthetically.

Step 2: Isolate or Copy the Gene

The desired gene must be obtained in sufficient quantity. Scientists may isolate it from DNA or use the polymerase chain reaction, or PCR, to make many copies.

Step 3: Remove a Plasmid from a Bacterium

Plasmids are separated from bacterial cells and purified. The selected plasmid will act as the vector.

Step 4: Cut the Gene and the Plasmid

The desired gene and plasmid are cut using the same restriction enzyme.

For example, EcoRI recognises the sequence GAATTC and produces sticky ends:

5′–G↓AATTC–3′
3′–CTTAA↑G–5′

Cutting both DNA molecules with EcoRI produces complementary AATT overhangs.

Step 5: Mix the DNA Fragments

The desired gene and opened plasmid are mixed. Complementary sticky ends attract one another and pair according to the base-pairing rules.

Step 6: Join the DNA with DNA Ligase

DNA ligase seals the sugar–phosphate backbone. The gene becomes a permanent part of the plasmid.

The resulting molecule is a recombinant plasmid.

Step 7: Insert the Recombinant Plasmid into a Host Cell

The recombinant plasmid is introduced into a bacterial cell. The uptake of DNA by a bacterial cell is called transformation.

Only some bacteria take up the plasmid successfully.

Step 8: Select the Transformed Cells

Scientists use marker genes to identify bacteria that received the plasmid.

For example, bacteria may be grown on a medium containing an antibiotic. Bacteria carrying a plasmid with the correct resistance gene survive, while bacteria without the plasmid do not.

Additional testing may be required to confirm that the plasmid contains the desired gene.

Step 9: Grow the Modified Cells

The successfully transformed bacteria are grown under controlled conditions. As they reproduce, they copy the recombinant plasmid.

A large population of genetically identical cells can be produced.

Step 10: Collect the Gene Product

If the inserted gene is active, the host cells produce the desired protein. The protein is then collected, separated from other cell materials, purified, and tested.

A Simplified Sequence

The entire process can be summarised as follows:

Desired gene identified
↓
Gene and plasmid cut with the same restriction enzyme
↓
Complementary sticky ends pair
↓
DNA ligase seals the DNA backbone
↓
Recombinant plasmid is formed
↓
Plasmid enters a host cell
↓
Modified cells are selected and grown
↓
Gene or protein is copied and collected

Worked Example: Recombinant Human Insulin

Insulin is a protein hormone that helps regulate blood glucose concentration. People with certain forms of diabetes may require insulin injections.

In the past, insulin for medical use was often collected from the pancreases of pigs or cattle. Recombinant DNA technology now allows microorganisms to produce human insulin.

A simplified production process is:

1. Scientists obtain the DNA sequence needed to produce human insulin.
2. A plasmid is isolated from a bacterial cell.
3. The insulin DNA and plasmid are cut using suitable restriction enzymes.
4. The insulin DNA is inserted into the plasmid.
5. DNA ligase joins the DNA fragments.
6. The recombinant plasmid is introduced into bacterial or yeast cells.
7. Cells containing the recombinant DNA are selected.
8. The cells are grown in large fermentation vessels.
9. The cells produce insulin.
10. The insulin is collected and purified for medical use.

The microorganisms do not naturally contain the human insulin gene. Recombinant DNA technology provides them with the instructions needed to produce the protein.

Gene Cloning and Protein Production

Recombinant DNA technology can have two related purposes.

Gene Cloning

Gene cloning means producing many identical copies of a gene.

When a recombinant plasmid is copied inside rapidly reproducing bacteria, the inserted gene is also copied. Scientists can then collect large quantities of the gene for research or further use.

Protein Production

If the inserted gene is placed beside suitable control sequences, the host cell may express the gene and produce its protein.

This process involves:

• Transcribing the DNA into messenger RNA.
• Translating the messenger RNA at ribosomes.
• Joining amino acids to form the protein.

Gene cloning produces copies of DNA. Gene expression produces the protein encoded by that DNA.

Examples of Recombinant DNA Technology

Human Insulin

Genetically modified microorganisms produce insulin used to treat diabetes.

Human Growth Hormone

Recombinant microorganisms can produce human growth hormone for people whose bodies do not produce enough of it.

Clotting Factors

Some people with haemophilia have difficulty forming blood clots. Recombinant cells can produce clotting proteins used in treatment.

Recombinant Vaccines

A gene for a protein found on the surface of a pathogen can be inserted into yeast or another host cell. The host produces the protein, which can be used as part of a vaccine.

The hepatitis B vaccine is an important example of a recombinant vaccine.

Chymosin Production

Chymosin is an enzyme used in cheese production. Genetically modified microorganisms can produce chymosin with the same function as the enzyme traditionally obtained from young calves.

Genetically Modified Crops

Genes can be inserted into crop plants to provide useful characteristics, including:

• Resistance to particular insect pests.
• Resistance to certain plant diseases.
• Tolerance of selected herbicides.
• Improved nutritional content.
• Increased tolerance of environmental stress.

Scientific Research

Scientists insert genes into cells to investigate:

• What a gene does.
• Where and when a gene is active.
• How a protein functions.
• How genetic changes affect cells.
• How diseases develop.

A gene may be joined to a reporter gene, such as one that produces a fluorescent protein. The fluorescence helps scientists observe where the gene is active.

Interpreting Recombinant DNA Diagrams

A recombinant DNA diagram often shows a plasmid, a desired gene, enzymes, and a host cell.

When reading such a diagram, look for the following features.

Identify the Donor DNA

The donor DNA contains the desired gene. It may come from a human, animal, plant, microorganism, or synthetic DNA source.

Identify the Vector

The vector is usually shown as a small circular plasmid. It carries the desired gene into the host cell.

Locate the Restriction Sites

Restriction sites show where the donor DNA and plasmid are cut. The same restriction enzyme should produce compatible ends.

Identify the Sticky or Blunt Ends

Staggered cuts produce sticky ends. Straight cuts produce blunt ends.

Check whether the ends on the gene and plasmid are compatible.

Locate DNA Ligase

If the diagram shows the gene being permanently joined to the plasmid, DNA ligase must be involved.

Identify the Recombinant Plasmid

The final circular plasmid should contain the inserted gene. It may be shown using a different colour or labelled as foreign DNA.

Follow the Plasmid into the Host Cell

The recombinant plasmid is inserted into a host cell. If bacteria are used, this step is called transformation.

Identify the Final Product

The final part of the diagram may show:

• Many copies of the gene.
• A population of modified cells.
• Production of a useful protein.
• A genetically modified organism.

Diagram Interpretation Example

A diagram shows the following sequence:

Human DNA → insulin gene removed
Bacterial plasmid → plasmid cut open
Insulin gene + opened plasmid → circular plasmid containing the insulin gene
Recombinant plasmid → bacterial cell
Bacterial cells → insulin production

The diagram can be interpreted as follows:

• The human DNA is the donor DNA.
• The insulin gene is the desired gene.
• The plasmid is the vector.
• A restriction enzyme cuts the gene and plasmid.
• Complementary ends allow the gene to fit into the plasmid.
• DNA ligase seals the DNA backbone.
• The completed circle is a recombinant plasmid.
• The bacterial cell is the host cell.
• The insulin is the recombinant protein product.

Advantages of Recombinant DNA Technology

Recombinant DNA technology can:

• Produce large amounts of useful proteins.
• Produce proteins that are very similar or identical to human proteins.
• Reduce dependence on animal tissues.
• Allow precise study of individual genes.
• Improve certain characteristics of crops.
• Support the development of medicines and vaccines.
• Produce useful enzymes for food and industry.

Limitations and Considerations

Creating recombinant DNA does not guarantee that the inserted gene will function correctly.

Scientists must consider:

• Whether the gene has been inserted in the correct position.
• Whether the gene is facing the correct direction.
• Whether the host cell can read the gene.
• Whether a suitable promoter is present.
• Whether the recombinant protein folds correctly.
• Whether unintended changes have occurred.
• Whether the modified organism could affect ecosystems.
• Whether the technology raises ethical, social, or economic concerns.

Recombinant products must be carefully tested for safety, purity, and effectiveness.

Did You Know?

The first medicine produced commercially using recombinant DNA technology was recombinant human insulin. It was approved for medical use in 1982. Its development demonstrated that microorganisms could be genetically engineered to manufacture a human protein on a large scale.

Key Terms

• Antibiotic-resistance marker: A gene that allows cells carrying a particular plasmid to survive exposure to a specific antibiotic.
• Complementary base pairing: The pairing of DNA bases in which A pairs with T and C pairs with G.
• DNA ligase: An enzyme that permanently joins DNA fragments by sealing the sugar–phosphate backbone.
• DNA methylation: The addition of methyl groups to DNA, often affecting how DNA is recognised or used.
• Donor DNA: DNA containing the desired gene that will be transferred or copied.
• Foreign DNA: DNA introduced into a cell or DNA molecule from another source.
• Gene cloning: The production of many identical copies of a particular gene.
• Gene expression: The use of information in a gene to produce RNA or a protein.
• Genetic engineering: The deliberate modification of an organism’s genetic material using biotechnology.
• Genetically modified organism: An organism whose DNA has been deliberately changed using genetic technology.
• Host cell: A cell that receives and maintains recombinant DNA.
• Origin of replication: A DNA sequence where the copying of a plasmid begins.
• Plasmid: A small, circular DNA molecule found in many bacteria and commonly used as a genetic vector.
• Polymerase chain reaction: A laboratory technique used to make many copies of a selected DNA sequence.
• Promoter: A DNA sequence that allows transcription of a nearby gene to begin.
• Recombinant DNA: DNA created by joining genetic material from two or more sources.
• Recombinant plasmid: A plasmid that contains an inserted piece of DNA.
• Recombinant protein: A protein produced when a host cell expresses an inserted gene.
• Restriction enzyme: An enzyme that cuts DNA at a specific recognition sequence.
• Restriction site: The DNA sequence recognised and cut by a particular restriction enzyme.
• Selectable marker: A gene used to identify cells that have received a vector.
• Sticky ends: Short, single-stranded DNA overhangs that can pair with complementary ends.
• Transformation: The uptake of external DNA, such as a plasmid, by a bacterial cell.
• Vector: A DNA molecule used to carry genetic material into a host cell.

Key Takeaways

• Recombinant DNA is formed by joining DNA from two or more different sources.
• DNA from different organisms can be combined because all DNA has the same basic structure and uses a nearly universal genetic code.
• Restriction enzymes cut the desired gene and vector at specific recognition sequences.
• Complementary sticky ends help DNA fragments pair.
• DNA ligase permanently joins the fragments.
• Plasmids are small circular DNA molecules commonly used as vectors.
• Recombinant plasmids can be introduced into bacterial host cells through transformation.
• Marker genes help scientists identify cells that received the plasmid.
• Modified cells can copy the inserted gene or produce the protein encoded by it.
• Recombinant DNA technology is used to make insulin, hormones, clotting factors, vaccines, industrial enzymes, and genetically modified crops.