Executive Project Summaries
- Real-Time PCR Analysis of Heliobacter pylori in Patients
- Real-Time PCR Analysis of Cow & Pig DNA in Heparin
- GMO Analysis of Plants (Using Real-Time PCR)
- Mice Genotyping Using Real-Time PCR Analysis
- Eradication of BVD (Bovine Virus Diarrhoe)
- Detection of Sequence Variants (SNP Analysis by Sanger Sequencing)
- De novo Sequencing of Enterococcus hirae and Mycoplasma conjunctivae
- Targeted Sequencing of a Plant Genome
- RNA Isolation, Reverse Transcription and Measurement of Gene Expression
- Metagenomic Analysis of Gut Microbiota
- Analytical Study to Assess the Quality of Oligonucleotides for Diagnostic Applications
- GMP-like production of Oligonucleotides as Key Raw Material for Diagnostic Kits
- Synthesis of an Oligonucleotide Library (also degenerate Codons) with a Controlled Number of Sequence Variations
|Real-Time PCR Analysis of Heliobacter pylori in Patients
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Helicobacter pylori is a Gram-negative bacterium which is found to be present in the stomach-lining of ~3 billion people around the world. Although the majority of individuals infected with the bacterium experience no symptoms, it is linked with chronic gastritis, gastric ulcers and can promote stomach cancer. The spread of H. pylori is not known, but person-to-person transmission by either the oral-oral or fecal-oral route are believed to be most likely. In the present project, Microsynth was asked to develop a real-time PCR assay for unambiguous diagnosis of H. pylori from oral biopsies and perform the testing of the biopsies. A major project challenge was to avoid the occurrence of false-negative results. For more information please see the document "Project Note_H_Pylori" under Related Downloads.
|Real-Time PCR Analysis of Cow & Pig DNA in Heparin||>>to the top|
Heparin is widely used as an injectable anticoagulant and as anticoagulant coating on various experimental and medical devices. Pharmaceutical grade heparin is derived from mucosal tissues of pig intestines or bovine lungs. Microsynth was inquired by an industrial customer to develop a sensitive multiplex real-time PCR assay, which is suitable to quantify residual traces of pig and cow DNA in heparin and conduct routine testing on heparin samples. Since heparin is known to be a strong inhibitor in PCR/real-time PCR assays, a major challenge of the project was to develop a robust protocol, which includes the specific digestion of heparin. For more information please see the document "Project Note_Heparin" under Related Downloads.
|GMO Analysis of Plants (Using Real-Time PCR)||>>to the top|
The goal of this project was to first develop a reliable and robust DNA isolation procedure for a specific plant type. Second, a sensitive, selective and cost-efficient real-time PCR system had to be established and validated. Following these initial developmental activities, Microsynth was assigned to perform GMO (genetically modified organism) screening on a fee-for-service basis. More than 55’000 samples have been successfully assessed over a time period of 11 years.
|Mice Genotyping Using Real-Time PCR Analysis||>>to the top|
The specific genetic identification of genetically engineered animals is critical to the efficient pursuit of research and in reducing the number of animals involved in a research project. Microsynth has genotyped hundreds of rat tissues and blood for the detection of single nucleotide polymorphisms. For this purpose the genomic DNA was isolated from tissue or blood and analyzed by real-time PCR.
|Eradication of BVD (Bovine Virus Diarrhoe)||>>to the top|
BVD is one of the most widespread and economically important cattle diseases, causing annual losses of > 10 M in Switzerland. Therefore, the Swiss government set-up an eradication program which included the testing of the complete cattle livestock (first phase) and subsequently monitored all newborn calves during several years (second phase). Microsynth was successful to become one of a few laboratories which were accredited by the Swiss government to qualify for BVD testing. Further, Microsynth signed exclusive contracts with 5 Swiss cantons. For more information please see the document "Project Note_BVD" under Related Downloads.
|Detection of Sequence Variants (SNP Analysis by Sanger Sequencing)||>>to the top|
Microsynth has performed many exon sequencing projects over the last years. All projects have undergone the following three phases:
- Establishment of robust and at the same time economic PCR and sequencing systems
- PCR amplification of all samples and subsequent double-stranded DNA sequencing
- Data analysis (sequence alignment of the analyzed samples against reference samples utilizing a specific bioinformatics program to identify the SNPs)
|De Novo Sequencing of Enterococcus Hirae and Mycoplasma Conjunctivae||>>to the top|
On behalf of the Institute of Clinical Research, University of Bern, we accomplished the de novo sequencing of the 3 Mb genome of Enterococcus hirae. This sequencing project was accomplished by combining next-generation sequencing technologies (GS FLXTM / 454TM from Roche Diagnostics) with traditional Sanger sequencing. The roughly 100 primary contigs obtained from 454 high-throughput sequencing were scaffolded with 3 kb and 8 kb paired-end sequencing runs from the GS FLXTM system. The remaining gaps of the assembly were closed “in silico” or by using PCR.
On behalf of the Institute for Veterinary Bacteriology (University of Bern) and in collaboration with the Wellcome Trust Sanger Institute, we performed de novo sequencing of the 1 Mb genome of Mycoplasma Conjunctivae. This sequencing project was addressed by combining next-generation sequencing technology (GS FLXTM / 454TM from Roche Diagnostics) with traditional Sanger Sequencing technology. The reads from GS FLXTM System (shotgun reads) were assembled together with Sanger paired-end reads of different insert sizes. All remaining gaps were closed “in silico” or using PCR.
|Targeted Sequencing of a Plant Genome||>>to the top|
The objective of this project was to screen a BAC library containing 322’000 clones for sequences of interest and then to sequence the identified clones. We approached this project by first establishing a special pooling system to allow efficient screening. Based on this pooling system, the library was screened by >2000 PCRs. More than 1000 identified BACs were sequenced using the GS FLXTM System from Roche Diagnostics. For more information, please see the document "Project Note BACSeq" under Related Downloads.
|RNA Isolation, Reverse Transcription and Measurement of Gene Expression||>>to the top|
The objective of this project was to establish and validate real-time PCR systems that allow the quantification of the mRNA expression of 150 candidate genes. For this purpose, 150 PCR primer/probe sets were designed, synthesized and validated using a specific positive control. In parallel, a protocol for RNA isolation and reverse transcription was established. Gene expression of these 150 genes was measured using the validated assays.
|Metagenomic Analysis of Gut Microbiota||>>to the top|
The analysis of 16S rDNA amplicons from microbial communities by means of 454 sequencing has become an essential toolkit to evaluate the microbial diversities. An industrial customer wanted us to perform comprehensive analysis of the gut microbiota. Microsynth was faced with four major challenges that had to be overcome: (i) high-throughput isolation of high-quality DNA from samples (stool is one of the most difficult matrices in this context!), (ii) the selection of suitable variable 16S rDNA regions for 454 sequencing, (iii) design and evaluation of a robust PCR system, and (iv) set-up of an analysis pipeline for proper processing and evaluation of next generation sequence data to obtain high-quality data delivered in form of a user-friendly report. For more information, please see the document "Project Note_BactCommunities" under Related Downloads.
|Analytical Study to Assess the Quality of Oligonucleotides for Diagnostic Applications||>>to the top|
Fluorescently labeled oligonucleotides for diagnostic applications have to meet high requirements in terms of quality (e.g. purity, stability, documentation etc.). The objective of this project was to compare and assess for a biomedical company the overall quality of fluorescently labeled oligonucleotides from 3 different suppliers using multiple analytic methods (HPLC, MALDI-MS).
|GMP-like Production of Oligonucleotides as Key Raw Material for Diagnostic Kits
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Fluorescently labeled oligonucleotides for diagnostic applications have to meet high requirements in terms of quality (e.g. purity, stability, documentation etc.). The objective of this project was to develop and validate manufacturing protocols for fluorescently labeled oligonucleotides to be used in diagnostic kits in the forensic field. This involved special aseptic treatment of labware and the development of outstanding purification protocols yielded in the synthesis of high quality oligonucleotides tagged with a fluorescence marker. Generally known challenges such as the contamination with extrinsic DNA and the interference with dye blobs could be successfully circumvented.
|Synthesis of an Oligonucleotide Library (also degenerate Codons) with a Controlled Number of Sequence Variations||>>to the top|
The objective of this project was to produce an oligonucleotide library1 containing a mixture of oligonucleotides with a pre-defined number of sequence variations for an DNA-templated combinatorial display. After receiving the desired library sequences from our customer, we planned the route of synthesis. Afterwards, all designed oligonucleotide sequences were synthesized and handed over to the customer.
- Daguer J.P., Ciobanu M., Alvarez S., Barluenga S., Winssinger N., DNA-templated combinatorial assembly of small molecule fragments amenable to selection/amplification cycles. Chemical Science. 2011. online DOI: 10.1039/c0sc00574f
- Ellington, A.D. and J.W. Szostak, In vitro selection of RNA molecules that bind specific ligands. Nature.1990. 346(6287): p. 818-22.
1 Background information:
Oligonucleotide libraries are needed for various techniques. One of the most well-known techniques is SELEX (systematic evolution of ligands by exponential enrichment) . Usually oligonucleotide libraries are produced by means of wobbling the bases on the synthesizer in a random manner. The disadvantage of such approach is that the sequence variation of the library cannot be managed, thus lowering the quality of the library. This is especially true for libraries where longer sequence areas have to be wobbled (e.g. a library supposed to cover a sequence area of 25 N wobble bases where N can be A, T, C or G would require an oligonucleotide production scale of several kilograms to obtain all sequence variations!). Thus, in practice, the length of the randomized regions is often shortened, leading to libraries with less structural complexity and less opportunities for the identification of ligands in the SELEX process. To overcome the disadvantages of the current synthesis technique for longer randomized regions in oligonucleotides libraries, we have implemented a particular synthesis technique during which the sequence variation can be controlled and therefore the production scale can be lowered dramatically by exclusion of unwanted sequence combinations. Furthermore, the synthesis technique can be used to replace the trimer-synthesis technique. During this technique the DNA strand is synthesized codon-wise.