Genome Projects

SPEC 3.8.3–3.8.4.1 - RECOMBINANT DNA TECHNOLOGY

Genome Projects & Recombinant DNA Technology

cDNA, Restriction Enzymes, Vectors, PCR, Transformation & Marker Genes

Sequencing projects have now read the complete genomes of a wide range of organisms, including humans. This has major scientific and medical consequences.

Simple Organisms

Determining the genome of simpler organisms allows the proteome (the complete set of proteins the organism can produce) to be determined.

Application: identification of potential antigens for vaccine production — by reading the genome of a pathogen, scientists can identify proteins to use as vaccine targets.

Complex Organisms

In more complex organisms, the presence of non-coding DNA and regulatory genes means that knowledge of the genome cannot easily be translated into the proteome.

Key Point

Sequencing methods are continuously updated and have become automated — modern next-generation sequencing is vastly faster and cheaper than early methods.

Recombinant DNA Technology

Key Definition

Recombinant DNA technology involves the transfer of fragments of DNA from one organism (or species) to another. The transferred DNA is expressed in the recipient (transgenic) organism.

Why Does This Work?

The genetic code is universal — the same codons code for the same amino acids in all organisms. Transcription and translation mechanisms are also universal. Therefore, DNA transferred from one species can be transcribed and translated by the cellular machinery of the recipient organism.

The overall workflow of recombinant DNA technology has three stages:

1

Obtain the target DNA fragment by one of three methods

2

Insert the fragment into a vector and transform host cells

3

Identify successfully transformed cells using marker genes

Three Methods: Obtaining a DNA Fragment

Method 1: cDNA from mRNA

1

Isolate mRNA from cells expressing the target protein

2

Reverse transcriptase synthesises single-stranded cDNA from the mRNA template

3

DNA polymerase makes it double-stranded

Advantage: cDNA has no introns — can be expressed in prokaryotes that lack splicing machinery

Method 2: Restriction Endonucleases

1

Cut genomic DNA with a restriction endonuclease

2

Enzyme cuts at a specific recognition sequence

3

Produces a fragment containing the desired gene with sticky ends

Note: Fragment includes introns — must be expressed in a eukaryotic cell

Method 3: Gene Machine

1

If the DNA or amino acid sequence is known, the gene can be chemically synthesised

2

Built nucleotide by nucleotide from scratch

Advantage: Fully customisable; no introns; can optimise codons for host organism

Exam Tip

You must be able to explain why cDNA is used when expressing a gene in bacteria: bacteria lack the spliceosome machinery to remove introns from pre-mRNA, so a gene with introns would produce a non-functional protein. cDNA (made from processed mRNA) contains only the coding sequence.

Restriction Endonucleases & Sticky Ends

Key Definitions

Restriction endonuclease: An enzyme that cuts double-stranded DNA at a specific recognition sequence, producing either blunt ends or sticky ends.

Sticky ends: Short, single-stranded overhangs produced when a restriction endonuclease cuts DNA in a staggered manner. Complementary sticky ends from different DNA molecules can base-pair (hydrogen bonds) and be permanently joined by DNA ligase.

Why the Same Enzyme is Essential

The target gene and the vector (plasmid) must be cut with the same restriction enzyme. This produces identical, complementary sticky ends on both. When mixed, the sticky ends base-pair together — the target gene can then be inserted into the vector.

Common Error

Students often forget that the same restriction enzyme must cut both the target DNA and the vector. Different enzymes would produce incompatible ends that cannot anneal together.

Inserting DNA into a Vector

Key Definition

Vector: A DNA molecule (e.g. a plasmid or bacteriophage) used to carry a foreign DNA fragment into a host cell.

Before inserting the gene into the vector, promoter and terminator sequences must be added to the DNA fragment so it can be transcribed and translated in the host cell.

1

Cut target gene and plasmid vector with the same restriction enzyme → complementary sticky ends produced on both

2

Mix together → complementary sticky ends anneal (hydrogen bonds form between complementary base pairs)

3

DNA ligase seals the gaps by forming phosphodiester bonds → recombinant plasmid created

4

Recombinant plasmid is introduced into a host cell (transformation)

Exam Tip

Know the two enzymes: restriction endonuclease cuts DNA; DNA ligase joins it. Both are essential — cutting produces the sticky ends, ligase permanently seals the recombinant molecule.

Transformation & Marker Genes

Key Definition

Transformation: The uptake of recombinant DNA (carried in a vector) by a host cell. Not all cells successfully take up the plasmid, so transformed cells must be identified.

For bacteria, transformation is achieved by heat shock — cells are briefly heated to ~42°C, which increases membrane permeability and allows the plasmid to enter.

Marker Genes

Marker genes are included in the vector to identify which host cells have been successfully transformed. Examples of marker gene types:

Antibiotic resistance genes: Transformed cells are grown on a medium containing that antibiotic — only transformed cells survive and grow

Fluorescent protein genes (e.g. GFP): Transformed cells fluoresce under UV light and can be identified visually

Spec Note

You will not be required to recall specific marker gene names in a written paper. Focus on understanding the principle: marker genes allow identification of successfully transformed cells.

PCR — Polymerase Chain Reaction

PCR is an in vitro method for amplifying specific DNA sequences. It requires: the target DNA, primers (short complementary oligonucleotides), heat-stable Taq polymerase, and free DNA nucleotides.

Step 1: Denaturation

95°C

Hydrogen bonds between complementary base pairs are broken → two single-stranded DNA templates produced

Step 2: Annealing

~55°C

Temperature lowered → short primers bind to complementary sequences at each end of the target region on each template strand

Step 3: Extension

72°C

Taq polymerase extends from the primers, adding free nucleotides to build a new complementary strand

Exponential Amplification

Each cycle doubles the number of copies of the target sequence. After n cycles: 2ⁿ copies. After 30 cycles: over one billion copies from a single starting molecule.

In Vivo Amplification & Applications

In Vivo Amplification

When recombinant plasmids are transformed into host bacteria, the bacteria replicate normally. Each time a bacterial cell divides, its DNA (including the recombinant plasmid) is replicated along with the host genome.

Culturing transformed cells in large numbers therefore amplifies the target gene in vivo.

Applications

Human insulin: bacteria containing the human insulin gene produce human insulin — no allergy risk, scalable production

Other proteins: human growth hormone, clotting factors (e.g. for haemophilia)

Vaccine antigens: identified via genome projects, produced recombinantly

GM crops: herbicide resistance, enhanced nutritional content

Gene therapy: delivering functional copies of genes to patients

Ethics & Evaluation

AQA requires you to evaluate ethical, financial and social issues around recombinant DNA technology, and to balance humanitarian benefits (life-saving medicines, crop yields) against concerns from environmentalists and anti-globalisation activists (gene escape, corporate ownership of genes).

Summary

Genome Projects

Genome sequencing → proteome determination (simpler organisms); non-coding DNA complicates this in complex organisms

Automated sequencing; applications include vaccine antigen identification

Three Methods to Obtain a DNA Fragment

cDNA: mRNA → reverse transcriptase → cDNA (no introns; for prokaryote expression)

Restriction endonuclease: cuts genomic DNA at recognition sequence → sticky ends (contains introns)

Gene machine: chemical synthesis from known sequence (no introns)

Inserting into Vector & Transformation

Same restriction enzyme cuts target + vector → complementary sticky ends → anneal → DNA ligase seals → recombinant plasmid

Add promoter and terminator sequences; transform host cells (heat shock for bacteria)

Marker genes (antibiotic resistance, GFP) identify successfully transformed cells

PCR (In Vitro Amplification)

Denaturation (95°C) → Annealing (~55°C, primers bind) → Extension (72°C, Taq polymerase) → 2ⁿ copies after n cycles

Quick-Check Practice

Q1. Explain why cDNA, rather than genomic DNA, is used when inserting a human gene into a bacterium for protein production. [3 marks]

Genomic DNA contains introns / non-coding sequences (1); bacteria do not have the (splicing) machinery to remove introns from pre-mRNA (1); cDNA is made from processed mRNA so it contains only the coding sequence / exons and can be translated correctly by bacterial ribosomes (1).

Q2. Describe how a target gene is inserted into a plasmid vector using restriction endonucleases and DNA ligase. [4 marks]

The target gene and the plasmid are both cut with the same restriction endonuclease (1); this produces complementary sticky ends on both (1); the sticky ends anneal / hydrogen bonds form between complementary bases (1); DNA ligase seals the gaps by forming phosphodiester bonds, producing a recombinant plasmid (1).

Q3. Describe the three stages of one PCR cycle. [3 marks]

Denaturation at ~95°C — hydrogen bonds between DNA strands broken, producing two single strands (1); annealing at ~55°C — primers bind to complementary sequences at each end of the target region (1); extension at 72°C — Taq polymerase extends from the primers, synthesising new complementary strands using free nucleotides (1).

Q4. State the role of marker genes in recombinant DNA technology. [2 marks]

To identify / select cells that have been successfully transformed / that have taken up the recombinant plasmid (1); e.g. antibiotic resistance gene — transformed cells survive on antibiotic medium; fluorescent protein gene — transformed cells fluoresce under UV (1).