Snapshot: What is Polymerase Chain Reaction (PCR)?

Polymerase chain reaction, or PCR, is a commonly used laboratory technique that was invented in the 1980s. The method has many applications in different fields, ranging from identifying individuals in forensic science, detecting pathogens in water supply, and genetic testing in medicine.

PCR works by first obtaining a sample that contains genetic information (DNA) such as blood. Then using a specialized enzyme called “DNA polymerase”, researchs can amplify and make billions of copies of a specific segment within the DNA that they are interested in (the region of interest). Because of this amplifying capacity, PCR is a very sensitive test and can be useful to detect even small trace amounts of DNA in a sample.

A DNA double helix rests on a print-out illustration of the DNA letters A, T, C and G.
PCR can help us read the genetic code of specific segments or regions of interest. Photo Credit: Darryl Leja, NHGRI.

By producing billions of copies of DNA, it also makes it possible for scientists to analyze the region of interest. To amplify only the region of interest within the original DNA sample, scientists design and use “primers”. Primers are very short single-stranded DNA sequences specifically designed to match up with a specific region of interest within the DNA. Two primers are used because one is for the beginning of the sequence, and the other is for the end.

How is a PCR test done in the lab?

To perform the PCR reaction in the laboratory, scientists mix the genetic sample, the primers, some nucleotides (the building blocks of DNA), minerals (such as magnesium), and the important enzyme DNA polymerase in a small test tube. The test tube is put into a thermocycler, a device that can quickly and accurately change temperatures. In order for the DNA replication process proceed, there needs to be mutiple suddent changes in tempurature.

A visual depicition of the PCR process, it is described in a step-by-step process in the text.
A diagram depicting the steps during Polymerase Chain Reaction (PCR). Image by Nola Begeja.

The first step of the cycle is called “denaturation”. This is where the genomic DNA from the sample is melted to a very high temperature of 96 °C. This makes the double-stranded DNA becomes single-stranded.

The next step is called “annealing”, where the temperature is reduced to 55 °C. This lets the primers can bind to the region of interest within the single-stranded genomic DNA.

Then, “elongation” occurs where the temperature is raised to 72 °C. This allows the DNA polymerase to effectively replicate the DNA bound by primers to synthesize double-stranded DNA by adding nucleotides. This process cycles from 20-40 times, and each cycle exponentially increases the quantity of DNA.

How is PCR used in ataxia research and treatment?

Certain types of ataxia have specific mutations to DNA that cause disease. For example, some types of spinocerebellar ataxia are caused by a type of mutation called polyglutamine expansion. The tests used to diagnose these types of ataxia use PCR to check to see if the mutations are there. Researchers also use PCR to confirm that cells, animals, or humans have the DNA mutation that researchers want to study. By checking this, researchers know what samples they are working with before starting their experiments.

If you would like to learn more about polymerase chain reaction, take a look at these resources by the Science Learning Hub and Your Geonome.

Snapshot written by Nola Begeja and edited by Larissa Nitschke

Snapshot: What is an action potential?

You may have heard that nerve cells (or neurons) in the brain use electrical activity to communicate with one another. The proteins responsible for creating these electrical signals are called ion channels. How do neurons use these electrical signals to communicate with one another in a meaningful way?

A good way to think about the brain is that it is wired into circuits. These brain circuits are not unlike the electrical circuits that power our home, computers, and cell phones. This means that within these circuits, an individual neuron must be able to send and receive electrical signals to and from its neighbors. The electrical signals used to transmit information down the length of a neuron are called action potentials.

cartoon of blue neurons sening small electrical signals to eachother
An artist’s drawing of action potentials (white) being sent out by neurons (blue) to communicate with eachother. Image courtesy of Flickr.

Brain communication: the action potential

Neurons use the electricity harnessed from the opening of ion channels to generate an action potential. Normally, when a neuron is not active, it rests at a negative resting state (or, a hyperpolarized membrane potential). This means that ion channels are closed and the neuron is not actively sending any signals to other neurons. Don’t worry, these voltages aren’t large enough to zap you! Neurons operate in a range between -90 millivolts and +40 millivolts, which is thousands of times smaller than the voltages that power circuits in our homes!

A neuron can become activated with the opening of certain ion channels, particularly ones that allow sodium ions into the neuron. Each time a sodium ion flows into the neuron, the membrane potential becomes slightly more positively charged, or depolarized. Once a specific threshold of depolarization is reached, a huge number of sodium channels open all at once and the cell’s membrane potential moves up to +20 mV.

Interestingly, another major type of ion channel, called a potassium channel, becomes activated on a slight delay compared to when sodium channels open. When potassium channels open, a large amount of positively-charged potassium quickly exits the neuron. This exit of potassium causes the membrane potential returns to its negative resting state. This allows the membrane to become hyperpolarized back to where it began. Now the cycle can start all over when another signal tells the neuron it is time to act. This whole cycle, from -70 mV to +20mV and back again, is the definition of an action potential. Action potentials quickly travel down the neuron in a single direction. Once they reach the end, they help generate a different type of chemical signal that tells the next neuron to generate an action potential of its own. And thus, the information continues to travel through the circuit.

An action potential is an all-or-none response. This means that if the threshold voltage is not reached, the neuron will remain silent and no action potential will be fired. In most neurons, a signal from a neighboring neuron causes the opening of sodium channels to help this signal initiate. However, in certain cell types (such as Purkinje neurons), action potentials can happen spontaneously, and all the time – sometimes even hundreds of times per second!

Approximate plot of a typical action potential shows its various phases as the action potential passes a point on a cell membrane. The membrane potential starts out at approximately −70 mV at time zero. A stimulus is applied at time = 1 ms, which raises the membrane potential above −55 mV (the threshold potential). After the stimulus is applied, the membrane potential rapidly rises to a peak potential of +40 mV at time = 2 ms. Just as quickly, the potential then drops and overshoots to −90 mV at time = 3 ms, and finally the resting potential of −70 mV is reestablished at time = 5 ms.
A diagram of how researchers plot a typical action potential. Image courtesy of WikiMedia.

Why do neuronal action potentials matter for cerebellar ataxia?

Researchers who study mouse models of ataxia have noticed that Purkinje neuron action potentials can undergo big changes during disease. Depending on the type of cerebellar ataxia, Purkinje neuron action potentials may take on a different shape or even disappear completely. Either of these situations could make it difficult, or even impossible, for a neuron to send proper signals to its neighbors. Some researchers suggest that improving action potential firing might be one way to improve ataxia symptoms. For this reason, identifying drugs that improve action potential firing is a major area of therapeutic research in ataxia.

If you would like to learn more about action potential, take a look at these resources by Khan Academy and Wikipedia.

Key Definitions

Membrane potential: the electrical voltage of a cell’s outer membrane. Changes in membrane potential are controlled by the opening and closing of many different types of ion channels.

Hyperpolarized: a negatively-charged membrane potential. A neuron usually rests at -70 mV when it is silent. It returns to that voltage after an action potential is completed.

Depolarized: a positively-charged membrane potential. This usually occurs when sodium channels open during the early part of an action potential. This cuases the cell quickly jumps up to +20 mV.

Snapshot written by David Bushart and edited by Celeste Suart.

Snapshot: What is poly-ADP-ribose (PAR)?

DNA repair is an important topic when talking about of neurodegenerative disorders. The amount of biochemical stress the brain experiences increases naturally as we age. Some connections have been made between the amount of stresses on the brain and the age people develop neurodegenerative disorders.

Many of these natural stresses can damage DNA. For this reason, many researchers are trying to find ways of helping or fixing DNA repair. Chemicals that effect DNA repair could be used as new drugs. Here, we will focus on just one part of the DNA damage response that has been a great success in cancer drug discovery.

PAR is like a net that pulls in proteins that repair DNA

Poly-ADP-ribose, also called PAR, are long molecules in the cell. They are made of of the same building blocks cells use to store enegry. PARylation is when these long chains of PAR are made and attached to different parts of the cell. This happens in response to many different types of stress. For example, a stress could be if a cell’s DNA is damaged or it is infected with a virus.

When DNA damage happens, PAR molecules are attached on the surface of proteins and can act as a basket to trap other proteins. PAR is made and woven together by PAR polymerase proteins (called PARPs). PARPs add PAR chains all around a site of damage to let other parts of the cell know that damage has happened. This attracts DNA repair proteins to DNA damage by binding to PAR and performing their role to fix the damage.

a lage black fishing net on a white background, it is worn in some placed.
PAR can act like a fishing net that “catches” and pulls in proteins to help fix DNA damage. Image of a fishing net by Nikodem Nijaki on Wikimedia.
To much PAR causes cells to run out of energy

Even though PAR does a good job of signalling that DNA damage has happened, it takes a lot of energy to make. If the damage can not be fixed, the cell will keep trying to make PAR until runs out of energy. This can lead to PAR molecules causing cell death. This effect of too much PAR can be seen in multiple types of neurodegenerative diseases.

A type of cerebellar ataxia called AOA-XRCC1 is known for having higher levels of PAR due to DNA damage. When researchers reduced the amount of PAR in a mouse model of AOA-XRCC1, the mouse had fewer ataxia symptoms and lost fewer neurons. This type of ataxia is caused by a mutation in a protein called XRCC1, which normally helps fix DNA and binds to PAR chains. But in the disease, the XRCC1 gets stuck at DNA along with the long chains of PAR.

These findings may be applicable to other types of ataxia and neurodegenerative disorders because of their link to higher levels of DNA damage. A lot more work to be done on PARylation and its role in neurodegeneration. But a lot of research has been done on PAR in cancer. Many drugs have been FDA approved for cancer patients as safe and effective. Cancer and ataxia are very different diseases. But all the work that has previously been done has laid the groundwork for new research in neurodegeneration.

If you would like to learn more about poly-ADP-ribose , take a look at these resources by the National Cancer Institute and Cancer Research UK.

Snapshot written by Carlos Barba-Bazan and edited by Dr. Ray Truant

Continue reading “Snapshot: What is poly-ADP-ribose (PAR)?”

Snapshot: What is Non-Homologous End Joining?

What is DNA damage?

As we go through life, our DNA undergoes a lot of stress, which can ultimately lead to DNA damage. The different stressors that can cause DNA damage are environmental factors such as UV light, radiation, and certain toxins. Additionally, DNA damage can be caused by metabolic processes that occur naturally in our body. DNA damage can manifest in multiple forms. One form of DNA damage are DNA breaks, which arise when either one or both of the DNA strands break, creating a physical separation between the DNA bases. Every day, each cell will experience about 50,000 single-stranded breaks, and every cell division will lead to about ten double-stranded breaks.

A blue DNA molecule that is broken in half, like it has been cut with a pair of scissors
An artist’s drawing of a double-stranded DNA break. Image part of the public domain.

How is the DNA repaired?

Thankfully, although DNA damage is a common occurrence in our cells, there are many ways our body can repair the damage. The type of repair is thereby determined by the type of DNA damage and the growth stage of the cell. A commonly used repair pathway to fix double-strand breaks is non-homologous end joining (NHEJ).

How NHEJ can repair DNA.  DNA damage cuses either a single stranded or double stranded break. If it is a double stranded break, then NHEJ can "correct" the damage, but dome genetic information will be lost.
How NHEJ can repair DNA. Damage to DNA can cause Single Strand (SS) or Double Strand (DS) breaks. Non-Homologous End Joining can repair double stranded break, but also causes small deletions of DNA. Image by Eder Xhako, Created with BioRender

NHEJ occurs mainly when the cell is actively growing. Double-stranded breaks are very harmful to the cell. If left unrepaired, the break can make the whole DNA chromosome unstable, and result in death of the cell. In order to minimize the damage caused by the breaks, NHEJ is a very fast, although often imprecise, method of DNA repair. When DNA undergoes a double-stranded break, NHEJ will act like glue and stick the two broken DNA ends together. Depending on where the break occurred and how much damage happened, the repair can then either lead to a complete repair or the loss of some DNA base pairs at the junctions resulting in permanent deletions. It is more common for NHEJ to lead to small deletions rather than a precise repair. Depending on the location of the deletion, it can have either no consequence or be potentially damaging to a gene

How do scientists use NHEJ to their advantage?

Outside of the context of natural DNA damage, scientists have learned how to utilize the naturally occurring NHEJ repair pathway to inactivate genes of interest. As such, scientists have developed methods, such as the CRISPR-Cas9 system, to artificially introduce double-stranded breaks in essential and functional parts of the gene. The double-strand break then causes the cell to repair the double-stranded break using NHEJ. As NHEJ often leads to small deletions, which will damage the essential part of the gene, the gene will often be rendered non-functional. In this way, scientists can study what happens in the absence of the gene.

If you would like to learn more about Non-Homologous End Joining, take a look at this video by Oxford University Press.

Snapshot written by Eder Xhako and edited by Larissa Nitschke

Snapshot: What is RNA-seq?

RNA-seq is a technology that has been used more and more in recent years to study both basic biology and disease. It’s a powerful tool has enabled scientific discovery at an unprecedented rate. But what exactly is RNA-seq? And, more importantly, what can it tell us?

RNA-seq is short for “RNA sequencing.” In essence, it’s quite similar to a technique you may have heard of before: whole genome sequencing. Our genome (i.e., our genetic code) is made up of DNA, which consists of 4 building blocks – chemicals abbreviated as A, T, C, and G – that are strung together in a code. Just like how different sequences of letters in the alphabet make up different words and sentences, different sequences of DNA building blocks make up the many different genes in our genome.

In whole genome sequencing, researchers determine the DNA code for every gene in an organism’s body. RNA sequencing, on the other hand, provides the sequence of a related chemical code: RNA.

What is RNA?

Picture a library full of shelves upon shelves of books. Together, the books contain all the instructions to make every piece of our bodies – everything from the smallest molecule in a cell to a whole organ. In this example, the books are our genes, and every cell in our body contains the whole library.

a densely densely back library shelf with an assortment of books
A library full of shelves of books, much like a human cell full of DNA. Photo by Alfons Morales on Unsplash.

Say a cell needs to make protein X . Instead of checking out the book, a copy of the book is made. That copy is called RNA, which will then be used to make protein X. This copy-making process, known as “transcription,” gives the cell more flexibility when it comes to how much of protein X to produce: in general, the more protein X the cell needs, the more copies of RNA are made. The total amount of protein X that is made from its gene is called “gene expression.”

What can we learn from RNA?

Because each of our cells has a specific role, they do not express every one of our genes; like us, they only read the books that are relevant to the topic at hand. If a gene is not expressed, even when we have it on the shelf, it is not functional. So, if we could have a readout of what genes are being expressed in a certain tissue, we could better understand what the cells in that tissue are doing. The easiest way to do that is by sequencing RNA.

How can we use RNA-seq for research?

One of the great things about RNA-seq is that it provides information about not only which genes are being expressed, but how much each gene is being expressed. For instance, as we age, the amount of growth factor produced by our body drops. If you perform RNA-seq on tissue samples from both a child and an adult, you can expect increased gene expression of growth factors in the child (indicated by an increased amount of growth factor RNA in the child’s tissue sample).

Gene expression is affected by a number of other factors, as well. In the case of illness – even when the disease is caused by a mutation in a single gene – a number of genes are likely to be differentially expressed as the result of your body’s attempt to compensate. If we compare an ataxic patient to a healthy individual, for example, we can expect to find hundreds of differentially expressed genes.

By comparing healthy individuals and patients using RNA-seq, we can learn what gene expression patterns are altered in disease. Tapping into this information helps scientists determine what went wrong in a specific disorder, which then informs them about what to do next. Whether this leads them to identify biomarkers, honing diagnostic strategies, or developing new treatments, RNA-seq acts as an important preliminary step in their research.

To learn more about the process of gene expression, check out this animation.

If you would like to learn more about RNA-seq, take a look at these resources by Thermo Fischer Scientific and Bite Size Bio.

Snapshot written by Sophia Leung and edited by Maxime Rousseaux.