Genetic engineering techniques
Imagine a Garda forensic team in Dublin who find a speck of blood no bigger than a pinhead at a crime scene. Forty years ago, reading or modifying a gene was the work of years. Today a laboratory can copy a fragment of DNA a billion times in a single afternoon, cut it and paste it into another organism, or rewrite one specific letter of a genome with almost surgical precision. These four techniques (PCR, restriction enzymes, molecular cloning and CRISPR-Cas9) are the toolbox that made modern biotechnology possible, and PCR, gel electrophoresis, restriction enzymes and plasmid vectors are all part of your Leaving Certificate course.
The polymerase chain reaction (PCR)
At a crime scene, only a microscopic speck of blood containing a few picograms of DNA is recovered, an amount completely insufficient to analyse: how can you obtain millions of identical copies of that exact fragment in just a few hours without using living cells?
What is the polymerase chain reaction (PCR) and what is it used for? Name two practical situations in which amplifying DNA is essential.
What is the polymerase chain reaction (PCR) and what is it used for? Name two practical situations in which amplifying DNA is essential.
The PCR cycle: denaturation, annealing and extension
Restriction enzymes: molecular scissors
When a bacteriophage injects its genome into a resistant bacterium, the viral DNA is immediately fragmented and degraded while the bacterial DNA remains intact: what molecular mechanism makes it possible to recognise and cut invading sequences with pinpoint precision?
What are restriction enzymes, from which organisms are they obtained, and what is their function in genetic engineering?
What are restriction enzymes, from which organisms are they obtained, and what is their function in genetic engineering?
Molecular cloning: DNA ligase, vectors and transformed bacteria
In the past, people with diabetes in Ireland relied on insulin extracted from the pancreases of cattle and pigs, at high cost and with a risk of allergic reactions: how can an ordinary bacterium be made to produce an inexhaustible supply of an exactly human protein?
What is a transgenic organism? Give an example and outline, in general terms, how it is produced.
What is a transgenic organism? Give an example and outline, in general terms, how it is produced.
Put the steps required to clone a human gene in bacteria in order and justify the sequence: introduce the recombinant plasmid into the bacterium, cut the gene and the…
Put the steps required to clone a human gene in bacteria in order and justify the sequence: introduce the recombinant plasmid into the bacterium, cut the gene and the plasmid with the same restriction enzyme, join the gene to the plasmid using DNA ligase, select and multiply the transformed bacteria.
A research group aims to synthesize the peptide hormone insulin in the bacterium Escherichia coli. To do this, they directly isolate the insulin gene from the nuclear…
A research group aims to synthesize the peptide hormone insulin in the bacterium Escherichia coli. To do this, they directly isolate the insulin gene from the nuclear DNA of human pancreatic cells and insert it into a bacterial plasmid alongside an efficient E. coli promoter.
- a) After introducing the plasmid into the bacteria, they find that an aberrant, inactive polypeptide of incorrect size is synthesized. Explain why eukaryotic genomic DNA cannot be properly expressed in a bacterium, referencing gene structure and RNA processing machinery.
- b) Design the biotechnological strategy to solve this problem: indicate which molecule you should isolate as the initial template, what type of specific synthetic primer you would use taking advantage of a post-transcriptional modification of that molecule, and which enzyme of viral origin you will use to synthesize the first strand of complementary DNA (cDNA).
- c) When comparing the sequence of the obtained cDNA with that of the initial nuclear gene, it is observed that the cDNA is much shorter and lacks the original human promoter sequence. Explain why this happens and justify how the bacterium is made to transcribe this cDNA once cloned into the plasmid.
CRISPR-Cas9: precision gene editing
CRISPR-Cas9 allows genome editing with high precision. Explain simply what role guide RNA plays and what role the Cas9 protein plays in this technique.
CRISPR-Cas9 allows genome editing with high precision. Explain simply what role guide RNA plays and what role the Cas9 protein plays in this technique.