On the track of genes

Every living creature on earth possesses its own specific genetic information. There has been huge progress in deciphering genetic information through gene sequencing during the last decades. Accordingly, the increasing knowledge of DNA-analysis enabled humankind to identify victims of crimes, accidents and convict culprits. Furthermore, a better understanding of genetic diseases has been developed. The potential for their cure is still improving. Despite these past successes, there is still a lot of unknown territory concerning the impact of DNA on genetic diseases.//By Hanna Kaschke

Since gene research is a very hot topic, a new research institute has been founded in Germany that is entitled the Institute for Functional Gene-Analysis. This institute was created by eight professors on 6. June 2018, and is based at the University of Applied Science Bonn-Rhein-Sieg. This foundation was necessary to carry out research that is unattached to funded projects. It empowers them to move their work forward with their own institutional financial support route.
Richard Jäger, professor for forensics at the University of Applied Science Bonn-Rein-Sieg and institute member put it this way: "We would like to find mutated DNA-sequences and understand their impact on the function of genes. This understanding is critical for a better understanding of genetic diseases." The institute is an interdisciplinary scientific facility that unifies professors of bioinformatics, biochemistry, biology, and immunology. Since the institute was very recently founded, the laboratories are expected to be ready for use in spring 2019. Until then their research continues through projects like the FunForGen project.

Project FunForGen

A research team at the University of Applied Science Bonn-Rein-Sieg investigates how correctly encoded genomes are functioning. Through their research physicians gain a better understanding of genetic diseases and develop more suitable therapies for patients, including Parkinson-disease and metabolic disorder. This might provide a better understanding of genetic diseases in general.
The so-called project "FunForGen" an acronym for Functional and Forensic Genomic through Next-Generation-Sequencing (NGS), is divided into four smaller subprojects. Each subproject makes use of the same NGS technology; there is no other overlap between them.
Professor Jörn Oliver Sass's research is on rare hereditary metabolic diseases. Metabolic diseases cause neurodegenerative symptoms and are incurable. Debilitating conditions are the result in the progressive degeneration of nerve cells, leading to a patient's deaths. This progressive degeneration of nerve cells causes problems with movement (called ataxias), or mental functioning (called dementias). Despite the fact that affected genes are often known, the exact location and the effects of mutated DNA are unknown. For this reason, Sass wants to resolve the function of those mutations using NGS technology.
Professor Christopher Volk performs similar research on the Parkinson disease. Up to now applied therapies often don't help patients, and he wants to discover the responsible mutations with the goal of finding specialized treatments for each mutation. Richard Jäger's research project specializes in the identification probes the possibility of identifying persons through NGS.
Professor Ralf Thiele creates bioinformatic tools to analyze the received data of over one million bases or more. It should enable the university research team to prognosticate the molecular effects of mutated sequences reclining on comparing databases and creating molecular models.Puzzle DNA-sequences, Quelle: Pixabay - CC0 - Freie kommerzielle Nutzung. Kein Bildnachweis nötig

Puzzle DNA-Sequenz, Quelle: Pixabay - CC0 - Freie kommerzielle Nutzung Kein Bildnachweis nötig

Puzzle DNA-Sequenz, Quelle: Pixabay - CC0 - Freie kommerzielle Nutzung, Kein Bildnachweis nötig

Applications of NGS

NGS can be used in broad areas of biology. Comparing the genome sequences of different types of animals and organisms, such as chimpanzees and yeast, can provide insights into the biology of development and evolution. In forensic it allows identifying the culprit or victim with only a hair. By using NGS, researchers are now able to compare vast stretches of DNA (one million bases or more) from different individuals very quickly and cheaply. Those comparisons yield an enormous amount of information about the role of inheritance in susceptibility to disease. Researchers go even further and are using NGS to precisely discover which genome is responsible for disease and how it's working. For instance, physicians are increasingly able to use sequence data to identify the particular type of cancer within a patient. This will help them to offer better choices regarding the best therapeutic approach for these patients.
"The rate of progress is stunning. As costs continue to come down, we are entering a period where we are going to be able to get the complete catalog of disease genes. This will allow us to look at thousands of people and see the differences among them, to discover critical genes that cause cancer, autism, heart disease, or schizophrenia", says Eric S. Lander, a professor at MIT and Harvard Medical School as well as director of the Broad Institute in Cambridge.

A summary of genetic biology

DNA is the construction manual that contains the information for building bodies and cells. It is composed of tiny chemical components (A, C, G, T). These so-called nucleotides form a repeating double helix structure. For a scientist, it is quite interesting to get to the bottom of how exactly DNA is composed of and in which ways does it influence genetic diseases. Next-Generation-Sequencing is a process where scientists decrypt genetic material and take a closer look at sequences of DNA (genomes). To be more precise, they investigate the series of repeating nucleotides (polymorphism). By doing this, scientists are capable of understanding the function of individual genomes and their collaboration. For instance, scientists now possess the possibility to find out exactly which genomes are responsible for genetic diseases like a metabolic disease. Therefore, they can offer more suitable therapies for patients with a better chance of success.

Sequencing technology

DNA sequencing has a rich history. Starting with Watson and Crick in 1953 building a conceptual framework for DNA replication and encoding proteins in nucleic acids, the first generation of DNA sequencing arose. Over the second generation of DNA sequencing the luminescent method with PCR through to nanopore sequencing the technic of NGS advanced.

Next-Generation-Sequencing

NGS stands for Next-Generation-Sequencing and is an umbrella term for the most recent set of DNA sequencing technologies. There are a variety of next-generation sequencing techniques using different technologies. However, most share a standard set of features that distinguish them from Sanger sequencing. They are highly parallel, meaning many sequencing reactions can take place at the same time. By taking advantage of parallelism, NGS offers the benefit of being a lot faster and cheaper than the Sanger sequencing. Furthermore, reactions are tiny and can be performed at once on a chip. Last but not least, low-cost sequencing technology can be used for personalized medicine and is more comprehensive than Sanger sequencing.

Sanger sequencing

This sequencing method also called the chain termination method, and is well known as one of the first methods of DNA sequencing - developed by the British biochemist Fred Sanger and his colleagues in 1977. It was used to determine the sequences of many relatively small fragments of human DNA.
The method compromises five steps. First, the DNA to be sequenced must be denatured and converted from double-stranded DNA into single-stranded DNA. This denaturing is done through the application of heat at a temperature of 94 degrees Celsius for about one minute. The DNA splits into a template strand that's complementary strand.
In the next step, a primer is annealed to the template strand with a temperature between 50 to 60 degree Celsius for 15 seconds to allow the addition of nucleotides in the next step called extension.
In the third step extension, the temperature is heated up to 74 degrees Celsius to allow the DNA-Polymerase for mooring on the primer. The nucleotides serve as link material. Every cycle only one nucleotide is added. Once the nucleotide connected with the DNA-Polymerase, the process stops.
These three steps are repeated over and over again. At that, the four nucleotides (A, C, G, T) are rotating with the result of having rotating DNA strand endings. That way various DNA strands with different lengths emerge.

Gel electrophoresis

Essential for the evaluation of results is the technic gel electrophoresis. Gel electrophoresis allows separating DNA fragments according to their size.
A gel is placed in a gel box with two electrode parts and is temporally exposed to an electric gradient. Then DNA samples are loaded into wells (indentations) at one end of a gel, and an electric current is applied to pull them through the gel. Since DNA fragments are charged negatively, they move towards the positive electrode. Small pieces move faster through the gel than large ones because all DNA fragments have the same amount of charge per mass.
When a gel is stained with a DNA-binding color, the DNA fragments can be seen under UV light as bands, each representing a group of same-sized DNA fragments. By comparing the bands in a sample to the DNA ladder, one can determine their approximate sizes. At the end of this process, the DNA fragments lie sorted by size and nucleotides. Scientists can read the results and make their own conclusions.

Sanger Sequencing today

Although genomes are now typically sequenced using other methods that are faster and less expensive, Sanger sequencing is still in extensive use for the sequencing of individual pieces of DNA, such as fragments used in DNA cloning or generated through polymerase chain reaction (PCR).

Overview of the different types of analyzing DNA

Polymerase chain reaction (PCR): A technique to make many copies of a specific DNA region in vitro (in a test tube rather than an organism)

Gel electrophoresis: A method used to separate DNA fragments according to their size

Recombinant DNA: DNA that is assembled out of pieces from multiple sources

DNA cloning: A molecular biology technique that makes many identical copies of a part of DNA, such as a gene

DNA sequencing: the process of determining the sequence of nucleotides (As, Ts, Cs, and Gs) in a piece of DNA.

External links:

Article at HBRS-Onlinemagazine Dopppelpunkt: "Spurensuche in menschlicher DNA" by Vanessa Wüsthoff

Read the story of DNA and learn more about the historical discoveries of DNA.

Interview about NGS at Onlinemagazine "The Atlantic": "When will genomics cure cancer?"

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