Cancer is a life-threatening disease that is caused by abnormal cell growth, resulting in the production of tumours which could potentially spread throughout the entire body.
Given that much of these are the result of a mutation within the genes responsible for regulating growth, the key question we can ask ourselves is - what kind of factors can cause these mutations?
Contrary to the popular assumption that all tumours are sterile environments (i.e. they are free from microbes), it turns out that there are many intrinsic (within our body) and extrinsic (environmental) factors involved, with cancer-causing microorganisms would be an excellent example of an extrinsic factor.
With that in mind, scientists have invented a technique known as microbial DNA fingerprinting that could allow us to identify such microbes by detecting their unique DNA profiles.
This leads us to the next question - how does this technique work?
On that note, this article will introduce you to this new and possibly revolutionary idea which may transform cancer therapy by decreasing the time taken to diagnose a patient and identify the root cause of their illness.
The principle behind this technique
To give you a little bit of context first - a microbe is, essentially, an organism that is too small to be seen without a microscope. Common examples of these are bacteria, fungi, yeast, and protozoa. In essence, they can be found in many places, including the air, soil, and within the human body itself. And most importantly, a lot of these microbes can actually make us ill, some of which can cause severe diseases such as cancer.
Meanwhile, the microbiome is the genetic material (i.e. DNA and RNA) of all of the microbes that are found within and on our bodies.
Now that you are aware of those - the process of microbial DNA fingerprinting is fairly straightforward. As its name suggests, DNA fingerprinting is a method for us to identify and compare different DNA samples. To begin with, researchers would collect the DNA from the microbiome. After that, they will use that DNA sample to identify all the different microbes present within the microbiome. And if we’re lucky, we can gather some intel on the potential cancer-causing microbes which could be living inside our body.
A deep dive into the procedure
How is it done?
The DNA fragments, which have been separated from the microbe, will first need to be cloned and amplified. This basically means we generate multiple copies of DNA fragments so that we have plenty of samples to work with.
After that, these fragments can be visually analysed and compared to known DNA profiles that are saved in databases.
1. Getting the DNA fragments via enzymatic digestion
The microbial DNA has to be completely isolated and sliced using a type of enzyme known as restriction enzymes. The resulting product of this step will produce DNA fragments of known lengths, which can then be used for comparison against those in the database.
Next, the ligation (i.e. joining together) of each fragment into separate plasmids (known as cloning vectors) allows the introduction of the microbial DNA into a cell that has been subjected to optimal conditions for reproduction.
2. Amplification
Because bacterial and viral DNA are quite small, it needs to be amplified in order for us to analyse it effectively. With that in mind, researchers would use a technique known as the polymerase chain reaction (PCR) to exponentially increase the amount of DNA available for investigation.
This technique can be broken down into three major steps:
a. Heat denaturation (~90°C)
Heat denatures the intermolecular forces between the opposing strands of the double helix. Such intermolecular forces include the hydrogen bonds between the base pairs A=T as well as C≡G. Once that’s done, we would have two single strands of DNA ready for the next step.
b. Annealing using primers
To initiate DNA replication, a primer would be introduced into the mixture. In particular, the primer has to be complementary to the DNA strands so that it can bind to the single strand and determine where the extension starts.
c. Extension with DNA polymerase
Finally, DNA polymerase is introduced so that it can add the four deoxyribonucleotides - dATP (adenine), dGTP (guanine), dCTP(cytosine) and dTTP(thymine) - whenever appropriate to elongate the new strand of DNA. This ultimately results in two new double-stranded DNAs.
Let’s take a quick look at Figure 1 to give you a better idea of what is going on:
Figure 1: The process behind the polymerase chain reaction (PCR). Starting from the left, double-stranded DNA is denatured by heat to form two single-stranded DNA. At a lower temperature, two primers are attached to the single strands by complementary base pairing. Next, DNA polymerase binds to where the primers are added. Then, it travels along the DNA, adding the suitable deoxyribonucleotides whenever necessary to extend the DNA. In essence, steps one to three of the PCR protocol are repeated so that we can produce many copies of the DNA sample. Typically, performing 30 cycles of this reaction can result in the production of 1.07 billion copies of our target DNA! This figure has been adapted from yourgenome.org.
3. Separation and analysis via agarose gel electrophoresis
Once we have obtained this DNA, we need to analyse it. The way in which we do so is by a process called gel electrophoresis. This separates DNA fragments and other related macromolecules by their size and charge.
Figure 2: An illustration showing the Watson-Crick base pairing in a DNA ladder. No matter the size of the DNA fragment, the mass-to-charge ratio will always be the same; because for every base pair, there are two negatively charged phosphate groups on either side. The larger fragment on the left contains eight base pairs (and thus 16 phosphate groups), giving it an overall charge of -16. As a result, its mass-to-charge ratio would be -0.5. On the other hand, the smaller DNA fragment contains four base pairs (and so eight phosphate groups). Thus, its mass-to-charge ratio is also -0.5. It will always be in that proportion. This figure was adapted from Microbe Notes.
As shown in Figure 3, these samples are then loaded into the wells of an agarose gel, whereby the agarose gel essentially works as a “molecular sieve” through which the DNA fragments move at a rate proportional to their size. Smaller molecules, which naturally have a lower mass and size, will move faster across the gel. Therefore, they will be able to travel further in comparison to heavier and larger molecules.
The size of the pores is dependent on the percentage concentration of agarose in the gel. This can be altered to allow the movement of larger or smaller DNA samples, whereby there is an optimal concentration for large fragments (~0.5% agarose) and small fragments (~2%), respectively. In a typical experiment, you could expect to use a 1% concentration.
One important thing to note is that DNA fragments carry an overall negative charge (due to the phosphate group in the backbone). This, therefore, means that they will be loaded on the same side as the cathode (which is negatively charged), and once the electric current is applied, the DNA samples will then migrate across the gel towards the positively-charged anode.
Figure 3: This is the experimental set-up to perform gel electrophoresis, whereby the samples are loaded into the wells next to the cathode. When the power supply is turned on, the positively-charged anode will attract the negatively-charged DNA and as a result, these samples will migrate across the gel towards the anode. Other than that, the buffer solution is added in order to provide ions that keep the current as well as the pH at a constant level. This figure is courtesy of yourgenome.org.
At the end of this procedure, you would find multiple bands of DNA along the gel, and these bands represent the different sizes of DNA present in the sample. Usually, researchers would add an ethidium bromide dye to the samples before loading them into the gel and then observe these bands under an ultraviolet (UV) light source right after running the gel electrophoresis protocol.
Besides that, to help us figure out the actual size of each band fragment, scientists would also load a DNA marker (a.k.a. a DNA ladder) into one of the wells. As the length of the DNA fragments in the marker is already known, we can compare them to our actual samples, thereby allowing us to estimate the actual size of the DNA samples in each band.
Take a look at Figure 4 to picture this better.
Figure 4: An example of a gel electrophoresis experiment. In essence, there are different columns, starting with the DNA ladder on the far left followed by the DNA samples of interest in the wells on the right. To determine the size of the DNA in each band, you’ll just need to match each band to the ones on the DNA ladder. For example, based on this diagram, the size of DNA sample A would be 1,000 base pairs (bp), whereas sample B contains DNA samples that are of 200 bp and 800 bp in size, respectively. This figure is courtesy of yourgenome.org.
Since the various bands represent the different sizes of DNA present within the microbe, we can thus use this piece of information to identify its species as each of them will have its own unique DNA profile. Meanwhile, there are many databases such as Ensembl Bacteria containing the DNA profiles of cancer-causing microbes, so we can easily retrieve the critical information and compare them to the DNA profile of an unidentified microbe.
As a result, this experimental technique has demonstrated its usefulness in the clinical as well as forensic settings.
What has been done to improve this technique?
With the National Health Service (NHS) being understaffed, this creates long waiting lists which may lead to the delay in the process of diagnosing cancer.
To tackle this issue, a new method known as ‘liquid biopsy’ has been developed, whereby the patient’s blood is drawn directly and sent off to a lab to be screened. The screening involves using machine learning and artificial intelligence to compare the patient’s blood with a library filled with the genetic fingerprints of common cancer-causing bacteria and viruses.
Once the diagnosis report has been generated, the results can then be quickly communicated to the patients over the phone, thereby making the entire diagnostic process much faster and way more efficient!
All in all, liquid biopsies can serve as a screen for potential carcinogenesis from which a doctor can monitor a patient to detect their potential tumor as early as possible. Nonetheless, this test alone is not enough to diagnose someone with full confidence as we are still not able to identify the specific type of cancer the patient may have via this technique. Thus, further research would be necessary in order to enhance the applications of liquid biopsy.
Why should you care?
There are 367,000 new cases of cancer every year in the UK, in which 15 to 20% of all these cases are caused by microbes. This basically equates to between 55,050 and 73,400 microbe-associated cases for cancer each year!
Thus, it is imperative for us to learn about and develop ideas on how we could prevent this debilitating disease, or diagnose it as early as possible so that we can reduce the number of cancer-related deaths globally.
In an ideal world, cancer would be diagnosed almost right away so that the appropriate treatment protocols could be provided to the patient as soon as possible. Additionally, we can imagine the diagnostic tests to be minimally (or even not) invasive, with the entire process only needing a relatively short amount of time.
Having said that, genetic screening presents a great potential for the development of cutting-edge cancer diagnosis tools. As scientists are now developing genomic analysis tools to have a higher throughput (i.e. many samples can be tested at the same time) and can be done in smaller devices, it is not hard to imagine a future where we would soon be able to carry out a microbial DNA fingerprinting test in the comfort of your own home!
Author
Eve Littlejohns
BSc Biomedical Science
University of Bristol