Producing DNA Fragments (AQA A-Level Biology): Revision Notes
Producing DNA Fragments
Recombinant DNA technology represents one of the most significant scientific advances in recent decades. This technology enables genes to be manipulated, altered and transferred between organisms, creating recombinant DNA - genetic material that combines DNA from two different organisms. The resulting organisms are called transgenic or genetically modified organisms (GMOs).
The success of this technology relies on a fundamental principle: the genetic code is universal. All living organisms use the same genetic code, meaning that DNA transferred from one organism can be interpreted and expressed by another. Additionally, the mechanisms of transcription and translation are essentially identical across all life forms, allowing transferred genes to function normally in their new host.
Why produce DNA fragments?
Many human diseases result from the inability to produce specific metabolic chemicals, particularly proteins like insulin. Traditional treatments involved extracting these chemicals from human or animal donors, which presented several problems:
- Risk of immune system rejection
- Risk of infection transmission
- High costs
- Limited availability
Producing large quantities of pure proteins from other sources offers significant advantages, leading to the development of techniques to isolate, clone and transfer genes into microorganisms that can serve as biological factories.
Methods of producing DNA fragments
There are three main approaches to producing DNA fragments containing desired genes:
Using reverse transcriptase
Reverse transcriptase is an enzyme found in retroviruses (such as HIV) that catalyses the production of DNA from RNA - the reverse of normal transcription. This process is particularly useful when working with genes that are highly expressed in specific cell types.
Worked Example: Producing Insulin using Reverse Transcriptase
The process works as follows:
Step 1: Source selection - Cells that readily produce the desired protein are selected. For example, β-cells from the islets of Langerhans in the pancreas are used for insulin production, as these cells contain large quantities of insulin mRNA.
Step 2: mRNA extraction - The relevant mRNA is extracted from these specialised cells.
Step 3: cDNA synthesis - Reverse transcriptase uses the mRNA as a template to synthesise a single-stranded complementary copy of DNA. This DNA is called complementary DNA (cDNA) because its nucleotide sequence is complementary to the mRNA.
Step 4: Double-strand formation - The mRNA is then hydrolysed with an enzyme, and DNA polymerase is used to build complementary nucleotides on the cDNA template, creating a complete double-stranded copy of the gene.
This method is advantageous because the resulting cDNA contains only the coding sequence of the gene, without introns or other non-coding regions that would prevent expression in prokaryotic cells.
Using restriction endonucleases
Restriction endonucleases are enzymes that organisms use as defence mechanisms against pathogens. Bacteria produce these enzymes to cut up viral DNA that has been injected into them during infection.
Each restriction endonuclease cuts DNA at a specific sequence of bases called a recognition sequence. These sequences are typically 4-8 base pairs long and have a unique characteristic: they are palindromic, meaning they read the same on both strands when read in the same direction.
There are two main types of cuts that restriction endonucleases can make:
Example: Types of Restriction Enzyme Cuts
Blunt ends: Some restriction endonucleases cut straight across both strands of DNA between two opposite base pairs, leaving two straight edges. For example, the HpaI restriction endonuclease recognises the sequence GTTAAC and produces a straight cut.
Sticky ends: Other restriction endonucleases cut DNA in a staggered fashion, leaving an uneven cut where each strand has exposed, unpaired bases. For example, the HindIII restriction endonuclease recognises the six base pair sequence AAGCTT and produces a staggered cut, leaving sticky ends.
The advantage of sticky ends is that they can easily bind to other DNA fragments cut with the same enzyme, as the unpaired bases are complementary to each other.
The gene machine
Modern biotechnology allows genes to be manufactured artificially in the laboratory using automated processes. This method involves several sophisticated steps:
- Sequence determination: The desired amino acid sequence of the target protein is determined, and from this, the corresponding mRNA codons are identified and the complementary DNA triplets are worked out.
- Computer input: The desired nucleotide sequence for the gene is fed into a computer system.
- Safety and ethics checking: The sequence is checked for biosafety and biosecurity to ensure it meets international standards and ethical requirements.
- Oligonucleotide design: The computer designs a series of small, overlapping single strands of nucleotides called oligonucleotides, which can be assembled into the desired gene.
- Automated synthesis: Each oligonucleotide is assembled by adding one nucleotide at a time in the required sequence using automated machinery.
- Gene assembly: The oligonucleotides are joined together to create the complete gene using the polymerase chain reaction (PCR).
- Complementary strand synthesis: The polymerase chain reaction also constructs the complementary strand of nucleotides to create the required double-stranded gene.
- Replication: The process multiplies the gene many times to produce numerous copies.
- Vector insertion: Using sticky ends, the gene can then be inserted into a bacterial plasmid, which acts as a vector for storage, cloning or transfer to other organisms.
- Quality control: The genes are checked using standard sequencing techniques, and any with errors are rejected.
Advantages of the gene machine:
- Any nucleotide sequence can be produced
- Very rapid production (as little as 10 days)
- High accuracy
- Artificial genes are free of introns and non-coding DNA
- Can be transcribed and translated by prokaryotic cells
Applications and significance
The ability to produce DNA fragments has revolutionised biotechnology and medicine. These techniques enable:
- Production of human proteins in bacterial or yeast cells
- Development of gene therapies
- Creation of genetically modified crops
- Production of vaccines and other medical treatments
- Research into gene function and regulation
The universal nature of the genetic code means that these techniques work across all forms of life, providing strong evidence for evolution and the common ancestry of all living organisms.
Key Points to Remember:
- Recombinant DNA combines genetic material from two different organisms, creating transgenic or GMO organisms
- Reverse transcriptase produces DNA from RNA templates, particularly useful for highly expressed genes like insulin
- Restriction endonucleases cut DNA at specific palindromic recognition sequences, creating either blunt or sticky ends
- The gene machine allows artificial synthesis of any desired gene sequence using computer-designed oligonucleotides
- The universal genetic code enables successful gene transfer between any organisms