Biology (9610) | Recombinant DNA technology

Welcome to Recombinant DNA Technology!

Hi! This chapter is all about one of the most exciting and important areas of modern Biology: how we can take a piece of DNA (a gene) from one organism and put it into another. This incredible technique, called Recombinant DNA Technology (RDT), is the cornerstone of biotechnology, allowing us to produce life-saving drugs like human insulin, create GM crops, and develop gene therapies.

Don't worry if the vocabulary seems technical at first. We will break down this complex process into four simple stages: finding the gene, cutting the DNA, copying the DNA, and finally, making the modified organism grow.

3.4.10.1 Principles of Recombinant DNA Technology

RDT fundamentally involves the transfer of DNA fragments from one organism or species to another.

The Key Principle: Universality of the Genetic Code

The most crucial idea that allows RDT to work is the universality of the genetic code.

• The genetic code is the sequence of base triplets (codons) that codes for specific amino acids.
• In almost all organisms—from bacteria to humans—a specific codon (e.g., AUG) codes for the same amino acid (Methionine).
• Therefore, if you transfer a human gene into a bacterial cell, the bacterium's transcription and translation mechanisms can still read the human gene and produce the corresponding human polypeptide (protein).

Analogy: Think of it like swapping out a motherboard (the DNA) in a computer (the cell). If both computers run on the same operating system (the universal genetic code), the new motherboard will work perfectly, even if it came from a different brand.

Key Takeaway: RDT works because the rules for reading DNA are the same for nearly all life forms.

3.4.10.2 Production of Fragments of DNA (Getting the Gene)

Before we can transfer a gene, we need to isolate the specific DNA fragment containing that gene. There are three main ways to do this:

Method 1: Using Reverse Transcriptase (mRNA to cDNA)

This method is often used when the desired gene comes from a eukaryotic organism (like a human).

1. An organism (like a human cell) that naturally produces the protein is identified (e.g., a pancreas cell that makes insulin).
2. The cell will contain many copies of the mRNA template for the desired protein, as mRNA is produced during transcription.
3. The enzyme Reverse Transcriptase is then used. This enzyme (originally found in retroviruses) catalyses the synthesis of a single strand of complementary DNA (cDNA) using the mRNA template.
4. The cDNA is then made into a double-stranded molecule.

Why is cDNA better? Eukaryotic genes contain non-coding sequences called introns. When the mRNA is made, these introns are spliced out. Since cDNA is made from the spliced mRNA, it contains only the necessary coding sequences (exons), making it much easier to express in a prokaryotic host cell (like bacteria) which cannot perform splicing.

Method 2: Using Restriction Endonucleases (The DNA Scissors)

Restriction endonucleases (also called restriction enzymes) are bacterial enzymes that cut DNA at very specific base sequences, called recognition sequences.

• They act like molecular scissors. Different enzymes cut at different specific sequences (e.g., EcoRI cuts at GAATTC).
• Often, the cut is staggered, leaving short, single-stranded overhangs on the fragment and the cut DNA molecule. These overhangs are called sticky ends.
• Importance of Sticky Ends: Sticky ends are crucial because they are complementary to each other. They can easily base-pair with other DNA fragments cut by the *same* restriction enzyme, allowing the new gene to be inserted into a vector easily.

Method 3: Artificial Gene Synthesis

If the sequence of the desired gene is known, short sequences can be built from scratch chemically using computer-controlled methods.

3.4.10.3 In vitro Amplification: The Polymerase Chain Reaction (PCR)

Once you have a tiny fragment of DNA, you often need millions of copies to work with. The fastest way to do this outside of a living cell is using the Polymerase Chain Reaction (PCR).

The Principles of PCR

PCR is an automated method used to amplify (copy) specific DNA fragments exponentially. It relies on temperature cycles to control DNA replication in a test tube.

PCR requires:

• The DNA fragment to be amplified (the template).
• Primers: Short, single-stranded DNA sequences complementary to the ends of the target fragment.
• Free DNA nucleotides (A, T, C, G).
• Taq Polymerase: A thermostable DNA polymerase (from the bacterium Thermus aquaticus). This enzyme can withstand very high temperatures without denaturing.

Step-by-Step PCR Process

The process happens in a thermal cycler and is repeated 20-30 times, doubling the amount of DNA each cycle.

1. Denaturation (90-95°C): The mixture is heated to break the hydrogen bonds between the complementary base pairs, separating the double-stranded DNA into two single strands.
2. Annealing (50-65°C): The mixture is cooled. The primers bind (anneal) to the complementary base sequences on the single DNA strands.
3. Extension/Elongation (72°C): The temperature is raised slightly to the optimal temperature for Taq polymerase. The polymerase binds to the primer and starts synthesising a new complementary strand of DNA, extending from the primer.

Did you know? PCR is essential in forensic science. A single hair follicle or a tiny spot of blood can provide enough DNA to be amplified and used for identification.

Key Takeaway: PCR is the fast, *in vitro* (in glass/test tube) method for making millions of copies of a DNA sequence using Taq polymerase and cycling temperatures.

3.4.10.4 In vivo Amplification (Genetic Engineering in Host Cells)

For large-scale, long-term production of a protein (like insulin), we often insert the DNA fragment into a host cell (usually a bacterium or yeast) and let the cell's machinery do the copying and translating. This is called in vivo amplification.

The Stages of In Vivo Cloning

Stage 1: Preparing the DNA Fragment

For the transferred DNA to be successfully transcribed and translated by the host cell, it needs instructions on when to start and stop.

• A promoter region is added to the start of the fragment. This is a binding site for RNA polymerase and tells the host cell's machinery to begin transcription.
• A terminator region is added to the end of the fragment, signalling the RNA polymerase to stop.

Stage 2: Inserting the Fragment into a Vector

A vector is a DNA molecule used to carry the genetic material into the host cell. The most common vector is a plasmid (a small, circular piece of DNA naturally found in bacteria).

The steps to create recombinant DNA:

1. The vector (plasmid) is cut open using the same restriction endonuclease that was used to create the DNA fragment. This ensures the sticky ends of the plasmid and the fragment are complementary.
2. The DNA fragment and the cut vector are mixed together. The sticky ends anneal (base-pair) together.
3. The enzyme DNA ligase seals the sugar-phosphate backbone, forming strong phosphodiester bonds, resulting in the completed recombinant plasmid.

Memory Aid: Restriction Enzymes cut (scissors), DNA Ligase joins (glue).

Stage 3: Transformation of Host Cells

Transformation is the process where host cells (e.g., bacteria) take up the recombinant plasmid vector.

Often, host cells are treated with calcium chloride solution and subjected to a brief heat shock to make their cell walls/membranes more permeable, encouraging them to take up the plasmid.

Stage 4: Identification using Marker Genes (Selection)

After transformation, you have a mixture of cells:

1. Cells that did not take up any plasmid (non-transformed).
2. Cells that took up the original, non-recombinant plasmid.
3. Cells that successfully took up the recombinant plasmid (these are the ones we want!).

To find the desired cells, marker genes are used. These are genes inserted into the vector (plasmid) along with the desired fragment.

• Marker genes usually code for resistance to a specific antibiotic (e.g., ampicillin resistance).
• When the host cells are cultured on an agar plate containing the antibiotic, only the cells that successfully incorporated the plasmid (and thus the antibiotic resistance gene) will survive and grow. All non-transformed cells are killed.
• This isolates the genetically modified (GM) cells, which are then cultured in large fermenters to produce massive quantities of the desired protein (e.g., insulin).

Key Takeaway: In vivo cloning uses plasmids as vectors and relies on restriction enzymes, DNA ligase, and marker genes to create and select transformed host cells for amplification.

3.4.10.5 Evaluation: Ethical, Financial, and Social Issues

Recombinant DNA technology is powerful, but its use brings important non-biological questions that you must be able to evaluate.

Ethical Issues (Is it right?)

• Moral Objections: Some people object to the manipulation of genetic material, viewing it as "playing God" or interfering with natural evolution.
• Safety Concerns: The possibility of accidentally creating harmful, antibiotic-resistant bacteria or genetically modified organisms (GMOs) that escape and affect natural ecosystems.
• Gene Therapy Risks: Ethical issues surrounding permanent alteration of human genes (germline therapy), and the long-term health risks involved.

Financial Issues (Who pays? Who benefits?)

• Patents and Ownership: Companies that develop specific GMOs or successful recombinant plasmids often patent them. This means they control the supply and price, making necessary treatments or high-yield seeds inaccessible to poorer communities or countries.
• High Development Cost: Developing RDT products, like new medicines or specialized enzymes, is very expensive, driving up the initial cost to the consumer.

Social Issues (Impact on Society)

• Accessibility of Drugs: RDT has lowered the cost of proteins like human insulin (which was previously harvested from pigs/cows) making essential medicine more accessible globally.
• Food Security: Genetically modified (GM) crops can offer higher yields, greater drought resistance, or resistance to pests, which is crucial for feeding a growing global population.
• Public Acceptance: There is often public concern and distrust regarding GM food products, leading to regulatory hurdles and market limitations in many countries.
• Long-term Effects: Worry that relying heavily on a few genetically identical GM crops might reduce genetic diversity, making food supply more vulnerable to a new, widespread disease.