Enzyme involved in the transcription of DNA and the production of mRNA. The enzyme is involved in the breakdown of the hydrogen bonds between the two strands of DNA in the formation of mRNA, and the build up of the mRNA strand from 5-3 end.
DNA sequencing is the process of working out the precise sequence of nucleotides within a strand of DNA
The genetic material directly upstream of a gene (towards the 5’ region of the sense strand). This region of DNA initiates transcription.
The specially shaped site on an enzyme where the substrates of the reaction bind. It is formed by the folding of the amino acid chains which make up the protein.
A long chain of hundreds or even thousands of amino acids joined by peptide bonds.
The basic building blocks of proteins. There are twenty amino acids used, in different combinations, to make every protein required by the human body.
A polymer made up of amino acids joined by peptide bonds. The amino acids present and the order in which they occur vary from one protein to another.
Bases, sometimes called nitrogenous bases, are the parts of the DNA molecule that join the two helix strands. They are like rungs on a ladder. There are four bases: adenine (A), thymine (T), guanine (G) and cytosine (C). Each base can only join with one other base; i.e. they join together in pairs: A with T and G with C.
A short piece of DNA which is responsible for the inheritance of a particular characteristic. It codes for the production of a specific protein. Genes occupy a fixed position, called a locus, on a particular DNA molecule.
Mutations can occur randomly during DNA replication. Examples include addition, deletion and substitution of nucleotide bases.
If the mutation occurs in a gene, it can alter the amino acid sequence coded. Modifying the amino acid sequence of a polypeptide chain can have various consequences including modifying protein structure. Hydrogen, ionic and disulphide bonds hold the tertiary and quaternary structures of proteins together. Amino acid changes can disrupt these bonds- or even create new bonds. This can transform the structure of a protein. Structural modifications of a protein can change the shape of its active site, change its function, or stop it functioning.
If the mutation occurs within a non-coding gene promoter region, it can result in increased or decreased levels of protein expression. These mutations can influence the frequency of transcription by altering the affinity of RNA polymerase for the sequence.
Mutations confer antimicrobial resistance in exactly the ways described above. Microbes can be resistant to multiple antimicrobials, often due to multiple resistance mechanisms. Specific mechanisms are shown in the animations below.
Mutations are identified via DNA sequencing. When DNA sequencing first began, scientists did each step manually. Sanger sequencing was the first automated method for DNA sequencing. Sanger sequencing uses a chain termination technique. The target DNA is copied many times in different length fragments. Fluorescently labelled chain terminating nucleotides mark the end of each fragment. Subsequent fluorescence detection enables the sequence of the DNA to be determined.
Since the introduction of Sanger sequencing there have been many advancements in sequencing technologies. In 2014, Oxford Nanopore Technologies introduced a new strand sequencing technique. This technique differs from Sanger sequencing as it enables significantly longer fragments of DNA to be sequenced.
Central to the technique are nanopores, which are nano-scale protein holes in membranes. These nanopores have ionic currents passing through them. During sequencing, a single strand of DNA is passed through the nanopore. This changes the ionic current; changes are specific to the nucleotide passing through. These changes are measured and the sequence is subsequently determined.
An example of the nanopore technology is the MinION- which is a pocket sized device costing $1,000. This device is revolutionary as unlike previous sequencing techniques it can be used everywhere. In 2016, NASA astronaut Kate Rubins used the MinION on the International Space Station- she became the first person to sequence DNA in Space!
Kate Rubins background as a biology researcher enabled her to become a NASA astronaut. For more information on careers in science have a look on our careers website.
Answer the following questions:
a) What are some examples of mutations that can arise during DNA replication?
Addition, deletion and substitution of nucleotide bases.
b) What are the possible consequences of a mutation occurring in a gene?
Mutations within coding regions of DNA can alter the amino acid sequence. Modifying the amino acid sequence of a polypeptide chain can modify protein structure. This is because the bonds holding together the tertiary and quaternary structures of proteins can be disrupted. This can alter the structure of a protein. Structural modifications of a protein can change the shape of its active site, change its function, or stop it functioning.
c) How does nanopore sequencing technology work?
Nanopore sequencing is a strand sequencing technique. This technique involves nanopores (nano-scale protein holes in membranes). These nanopores have ionic currents passing through them. During sequencing, a single strand of DNA is passed through the nanopore. This changes the ionic current passing through the nanopore; changes are specific to the nucleotides passing through. These changes are measured and the sequence is subsequently determined.
d) What mechanisms of antimicrobial resistance occur in both bacteria and fungi?
The antimicrobial resistance mechanisms that are shared by both bacteria and fungi are efflux of medicines, changes to the cell wall preventing medicine entry and modified drug targets (which prevent antimicrobial recognition of its target).
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