Vermeidung von Informationsüberlauf und Rauschen bei der Identifizierung von komplexen DNA-Mischungen durch einen reduktionistischen Ansatz.

Genetic information transfer, which is the transfer of parental traits to offspring’s, completely depends on specific molecular recognition. This information is stored in stretches of DNA known as genes. Various gene expression profiling studies are used to investigate the activity of specific genes at specific instant. Many studies have successfully shown that molecular recognition is dependent on the complexity of biological systems. Increase in complexity often results in unspecific hybridization. Subsequent quantification of this data results in reduced signal to noise ratio. Often, the information of interest, especially when it is in small amounts, is clouded by larger amounts of unspecific information. These problems need to be addressed for profiling studies to be more reliable. Often large amounts of unspecific information may present a significant challenge, as it increases the time and effort needed to retrieve specific information. This dissertation proposes a novel and simple method to identify and reduce hybridization noise in gene expression profiling techniques. First part of this dissertation deals with the development of a biomolecular model system. Second part deals with identification, retrieval and subsequent verification of the specific information from model system with noise. Finally, the retrieved information is analysed for fidelity and specificity by various gene expression profiling techniques.Genetische Informationsübertragung ist die Übertragung der elterlichen Eigenschaften auf die Nachkommen, sie hängt vollständig von spezifischer molekularer Erkennung ab. Die Information ist in bestimmten DNA Abschnitten (Gene) gespeichert. Verschiedene Genexpressionsanalyse-Studien werden zur Identifizierung und Quantifizierung der Aktivität bestimmter Gene durchgeführt. Viele Studien konnten erfolgreich zeigen, dass die "molekulare Erkennung“ von der Komplexität der biologischen Systeme abhängig ist. Eine Erhöhung der Komplexität führt häufig zu unspezifischer Hybridisierung. Die anschließende Quantifizierung dieser Daten führt zu Ergebnissen verringertem Signal-Rausch-Verhältnis. Oft sind die angestrebten Informationen in größeren Mengen an unspezifischen Informationen verborgen, insbesondere wenn es sich bei den gesuchten Informationen um kleine Datenmengen handelt. Oft stellen aber große Mengen von unspezifischen Informationen eine bedeutende Herausforderung dar, indem sie die Aufwand zur Informationsgewinnung erheblich erhöhen. Der erste Teil der vorliegenden Dissertation beschäftigt sich mit der Entwicklung eines molekularbiologischen Modellsystem. Der zweite Teil befasst sich mit der Identifizierung, dem Abruf und der Überprüfung der spezifischen Informationen aus dem Modellsystem durch Hybridisierung. Schließlich werden die gewonnenen Informationen mittels verschiedener Genexpressionanalysetechniken auf ihre Genauigkeit und Spezifität hin untersucht

Information Limited Oligonucleotide Amplification Assay for Affinity-Based, Parallel Detection Studies.

Molecular communication systems encounter similar constraints as telecommunications. In either case, channel crosstalk at the receiver end will result in information loss that statistical analysis cannot compensate. This is because in any communication channel there is a physical limit to the amount of information that can be transmitted. We present a novel and simple modified end amplification (MEA) technique to generate reduced and defined amounts of specific information in form of short fragments from an oligonucleotide source that also contains unrelated and redundant information. Our method can be a valuable tool to investigate information overflow and channel capacity in biomolecular recognition systems

Specific molecular recognition.

<p>The figure illustrates the sequence specific temperature dependent nucleic acid hybridization of a DNA. The strand (TGACATGCTAATC) is complementary to ssDNA strand (ACTGTCGATTAG).</p

Experimental approach for development of a model system with specific information clouded by noise.

<p>Specific information of interest (orange) sandwiched by redundant information (green) is gradually clouded by vector DNA (turquoise) and cellular DNA (blue). Vector DNA and cellular DNA represent potential noise sources of increasing complexity.</p

Modified end amplification (MEA) technique.

<p>The DNA template (1) requires a well defined end point of the template (2) that can be produced for instance by means of a restriction enzyme (red). Denaturation (3) at 95°C and annealing (4) at 50°C are similar to conventional PCR. Taq polymerase (blue) generates a complementary ssDNA oligonucleotide fragment from a single primer (red) during the extension step (5). The complementary ssDNA oligonucleotide fragment is extended until the end of the source strand (6). Use of a single primer results in linear amplification (7). There are n copies of complementary ssDNA oligonucleotide fragments after n cycles.</p

Application of modified end amplification (MEA) approach, identification, retrieval and verification of specific information.

<p>Oligonucleotide sequences are extracted with the MEA technique and purified using standard nucleic acid purification and extraction procedures. They are immobilized on a southern blot membrane by hybridizing to complementary oligonucleotide sequences with a marker. The presence of small fragments of defined length from MEA technique results in highly specific hybridization, and noise is minimized.</p

Southern blot for 40 nt sequences.

<p>The complementary sequence is labelled with a marker. This marker enables detection of the hybridized sequence through X-ray radiography. Lanes 1, 2 and 3, unpurified MEA 40 nt product in three different amounts (20, 10, 5 uL), lane 4 and 5, the ssDNA product from modified isothermal amplification method with a single primer. Lane 6–8, purified MEA products. Lanes 9–10, reference ssDNA sequences of 40 nt length, of oligonucleotide composition as the intended 40 nt sequence.</p

Different macro- and micro-rheological properties of native porcine respiratory and intestinal mucus.

Aim of this study was to investigate the similarities and differences at macro- and microscale in the viscoelastic properties of mucus that covers the epithelia of the intestinal and respiratory tract. Natural mucus was collected from pulmonary and intestinal regions of healthy pigs. Macro-rheological investigations were carried out through conventional plate-plate rheometry. Microrheology was investigated using optical tweezers. Our data revealed significant differences both in macro- and micro-rheological properties between respiratory and intestinal mucus