Pyrosequencing is a technique to sequence DNA, thus to identify the base pairs of DNA, based on the synthesis of a complementary strand. In contrast to the well-known Sanger sequencing approach not the determination of the complementary strand synthesis leads to information about the bases, but the synthesis itself coupled to the amount of free pyrophosphate. The method was developed by Pål Nyrén and his student Mostafa Ronaghi in Stockholm in 1996.

As with Sanger sequencing a DNA polymerase and a primer molecule are used in order to build up a new DNA strand based on the already existing complementary strand. This process is “watched” and consequently used to sequence the present DNA molecule. In addition one of the four existing nucleoside triphosphates (NTPs) is added, followed by another one (after usage and/or degradation of the first one) and so on. The synthesis process of the newly formed strand, thus the integration of the fitting NTP, can be made visible with the help of a complex enzyme system and a light detector because a small light flash is emitted through an enzymatic cascade when the fitting nucleotide is inserted on the strand. If a non-fitting nucleotide is added, there is no enzymatic reaction and no detectable light flash occurs. After this reaction the remaining NTPs, which were not used, are degraded by enzymes. The following NTP is added. If it fits the measurable light flash occurs again. The light flash occurs at the latest after the fourth addition of NTPs because then all possible pairing options have been used. The intensity of the occurring light flash accounts for the amount of NTPs of one sort that were utilized. That means, for example, that the flash of three used adenine NTPs is linearly more intense than the use of one adenine NTPs. Knowledge of the intensity of the emitted light (displayed on the Y-axis) and the used NTP (displayed on the X-axis) per run consequently leads to the sequence of the DNA.

The light emitting enzymatic reaction is based on the following principles: When a complementary NTP is inserted on the DNA strand a pyrophosphate (PPi) is released from the used NTP. PPi then reacts to ATP (the biologically most important energy containing molecule) with help of the enzyme ATP-sulfurylase. The ATP molecule is then used to power the luciferase-reaction. Within this reaction the luciferin molecule is transformed into oxyluciferin. The intensity of the detectable emitted light flash during this chemical reaction is proportional to the first produced and then used amount of ATP molecules.

Pyrosequencing is a process that can be highly automated and is therefore very useful for the frequent and fast analysis of DNA samples. This is especially of high value when unique gene mutations within the DNA, called Single Nucleotide Polymorphisms (SNPs) (which are not necessarily negative), have to be analyzed. For example genetically inherited diseases can be detected using this sequencing approach.

Figure displaying the principles behind the technique of pyrosequencing 1.

An excellent page about the technique of pyrosequencing you can find at http://www.pyrosequencing.com/DynPage.aspx?id=7454.

1. Quaigen, “The principle of Pyrosequencing technology,” 2010, http://www.pyrosequencing.com/DynPage.aspx?id=7454.

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The Neandertals fascinate us ever since they were first discovered in 1856 in the Neander valley near Düsseldorf, Germany. Because Neandertals and the presently living humans (Homo sapiens) occurred in the same geographic area at about the same time from about 400,000 to 30,000 years ago, some researchers considered it likely that individuals from types could have interbred. The resulting gene transfer would be measurable. Partial identification of the Neandertal DNA in the form of mitochondrial DNA (mtDNA) a couple of years ago, led to the conclusion that no gene transfer between Neandertals and Homo sapiens took place 1. Since the mtDNA is only a part of the whole genome, no absolute conclusion could be drawn from these conclusions. In order to obtain a better view of the relationship between the Neandertal and out own genome, the analysis of the whole amount of (nuclear) DNA became necessary. On May 7th an international team of researchers published their success in this endeavour 2. The possibility of comparing our own genome to the Neandertal genome can potentially answer some interesting questions. First of all if gene transfer really occurred, but more important the results of the study lay the foundations for further research into the direction of “Which genes make us unique as humans?”. The first draft identification of the Neandertal genome helps to answer this question because it narrows the amount of genes in question. Previously only the comparison between us and our closest kin, the chimpanzee, was possible.

The approach the researchers took was rather special and in the following I want to try to explain why.

The main difficulty of analyzing the Neandertal genome was of technical nature because of the poor condition the sample DNA was in. During the course of time the molecular structure of the DNA had changed and about 95% of the amount of sample DNA consisted of foreign DNA, meaning microorganism or contamination through present day humans. Some of the involved sequences, most about 200 base pairs (bp) long, could be identified as genes trough certain methods of analyzing ancient DNA making use of the polymerase chain reaction (PCR). Among those genes were the MC1R gene involved in skin pigmentation parts of the FOXP2 gene which plays a role in speech and language, fragments of the ABO blood group locus and a gene involved in taste perception.

However the PCR method is not useful for analysing genomes on a large-scale as the researchers aimed for in the present study. The new technology of high-throughput DNA sequencing allowed a better analysis of the whole genome. All these steps were undertaken in a so-called clean-room laboratory of the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany, in order to prevent the sample DNA from further contamination. The following sequencing process can be subdivided into several steps unique steps that were special for this research project.

In order to tackle the challenges the ancient DNA imposed on the researchers they made use of certain techniques. Among those were:

  • The already mentioned “clean-room” working in order to prevent further contamination of the DNA.
  • The use of “unique sequence tags” and primers in order to be able to distinguish ancient DNA from modern one.
  • Making use of restriction (cutting) enzymes that preferably cut microbial DNA so that the proportion of ancient DNA was gradually increased over time.

Subsequently the technique of pyrosequencing (will be explained later) was used to determine the sequence of short pieces of DNA. The problem with this faster and cheaper technique, when compared to the Sanger approach, is that the length of DNA that can be read in any single sequencing run is much shorter and therefore difficult to analyze. In order to purify and glue the puzzle pieces of the now sequenced Neandertal DNA together bioinformatic tools were used. Complex alignment algorithms were used to assemble the molecular pieces. In order to have an orientation on the DNA during this process the Neandertal DNA was aligned along chimpanzee, rhesus, and mouse genomes as well as all nucleotide sequences in GenBank, a gene database. Sequences with significantly better matches to the primate genomes than to other sequences within the database were further analyzed. Because DNA samples from three individuals were used all the efforts described above finally yielded a 1.3 fold coverage of the Neandertal genome, which is an impressive result considering the large-scale damages and contamination of the original sample.

One of the most important aspects of this work was the possibility to analyze the genes that make us uniquely human by comparing our present day genome to the now known Neandertal genome.  Since Neandertals are our closest kin such a comparison is of greatest value. Through the use of microarray technology and the previous data a different team of researchers 3 succeeded in doing so. By analyzing 14,000 potentially interesting protein coding regions of the Neandertal DNA and alignment with human data is was possible to identify 88 amino acid substitutions that uniquely fixed in humans. Among others, genes that were affected by those fixed changes are for example ABC transporters, potassium channels, G-protein coupled receptors and certain transcription factors. Further research is essential to determine their importance for the development of the present day human species.

In addition further conclusion on the question whether Neandertals and humans ever have interbred could be made. This became possible by testing if Neandertals are more related to certain presently living human populations. Because modern humans most likely originated from Africa and if Neandertals split from the presently living human species before those began to develop towards their present form, it would be probable that non-Africans and Africans would be related to Neandertals to the same extent. Surprisingly this was not the case. Furthermore, when Green et al. compared the Neandertal genome to five different human populations they observed that Europeans, Chinese and Papua New Guineans are equally related to Neandertals, whereas Africans are not at all. Since no Neandertal fossils have been found in Asia so far the researchers concluded that the admixture of Neandertals and humans most have occurred before the divergence of Asian populations from the European ones. Computational modelling led tot the conclusion that between 1 and 4 % of all human genes are of Neandertal origin.

This article was written with the help of the ScienceMag’s special on the first draft of the Neandertal genome 4 as published on May 7th.

1.  Krings, M. et al. Neandertal DNA Sequences and the Origin of Modern Humans. Cell 90, 19-30 (1997).

2.  Green, R.E. et al. A Draft Sequence of the Neandertal Genome. Science 328, 710-722 (2010).

3.  Burbano, H.A. et al. Targeted Investigation of the Neandertal Genome by Array-Based Sequence Capture. Science 328, 723-725 (2010).

4.  Marathe, T. Science/AAAS | Methodology – The Neandertal Genome. ScienceMag Special (2010).at <http://www.sciencemag.org/special/neandertal/feature/methodology.html&gt;

Some more

May 12, 2010

Here I’m planing to publish things I just happen to like… or that have moved my thoughts.

During the last weeks research groups of among others a research group from the Max Planck Institute for Evolutionary Anthropology (Leipzig, Germany) published interesting articles over new insights of human evolution. In the following I want to present some ideas and results of the researchers. In the first place to understand them myself…

Welcome!

May 12, 2010

Hi there, and welcome to my blog.

In future I want to establish a growing overview over (molecular) biology research themes that I personally find interesting or I’m personally engaged in as a student. This idea grew because I’d like to share and spread knowledge about the small things. First of  all academically, thus first and second year stuff, but also based on my fascination for biology as a whole.

Hopefully I’ll see you back.