Project Examples
We’ve successfully performed many different projects (small to large projects) over the last decade, ranging from one-month projects to projects lasting several years. Here are some selected examples to illustrate our know-how and experience in the area of molecular biology.
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.
BVD is one of the most widespread and costly infectious cattle diseases. Microsynth AG is one of a few laboratories accredited by the Swiss government to perform tests for BVD. The company has signed exclusive contracts with several Swiss cantons accounting for about one-third of the Swiss cattle population to be tested. Therefore, a high-throughput real-time PCR System (including RNA isolation) has been developed and validated to allow the screening of up to 10’000 samples per day. To date, we have tested >800’000 ear samples (initial test) and 5’000 blood samples (confirmatory testing in case of positive initial testing).
Microsynth has performed many exon sequencing projects over the last years. All projects have undergone the following three phases:
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.
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.
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.
The analysis of 16S rDNA (16S) genes by means of 454 sequencing of bar-coded 16S rDNA has become an essential component of the microbial ecologist’s toolkit to evaluate the microbial composition of diverse habitats such as soils, oceans and our own bodies. The 16S rDNA gene sequences are highly conserved within organisms of the same genus or species but differ between organisms of a different genus or species. This fact allows a phylogenetic analysis of organisms and the identification of new taxa.
Microsynth has performed many projects in this area over the last years. All projects have undergone the following five phases:
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.
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).
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.
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[1]. 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.
1 Background information:
- GMO Analysis of Plants (Using Real-Time PCR)
- Eradication of BVD (Bovine Virus Diarrhoe)
- Detection of Sequence Variants (SNP Analysis by Sanger Sequencing)
- De novo Sequencing of Enterococcus hirae and Mycoplasma conjunctivae
- PCR Screening of a BAC Library and Subsequent Sequencing of Identified Clones
- RNA Isolation, Reverse Transcription and Measurement of Gene Expression
- 454 Sequencing of Bar-Coded 16S rDNA Gene Amplicons
- Mice Genotyping Using Real-Time PCR Analysis
- 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
| 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.
| Eradication of BVD (Bovine Virus Diarrhoe) | >>to the top |
BVD is one of the most widespread and costly infectious cattle diseases. Microsynth AG is one of a few laboratories accredited by the Swiss government to perform tests for BVD. The company has signed exclusive contracts with several Swiss cantons accounting for about one-third of the Swiss cattle population to be tested. Therefore, a high-throughput real-time PCR System (including RNA isolation) has been developed and validated to allow the screening of up to 10’000 samples per day. To date, we have tested >800’000 ear samples (initial test) and 5’000 blood samples (confirmatory testing in case of positive initial testing).
| 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.
| PCR Screening of a BAC Library and Subsequent 454 Sequencing of Identified Clones | >>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.
| 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.
| 454 Sequencing of Bar-Coded 16S rDNA Gene Amplicons | >>to the top |
The analysis of 16S rDNA (16S) genes by means of 454 sequencing of bar-coded 16S rDNA has become an essential component of the microbial ecologist’s toolkit to evaluate the microbial composition of diverse habitats such as soils, oceans and our own bodies. The 16S rDNA gene sequences are highly conserved within organisms of the same genus or species but differ between organisms of a different genus or species. This fact allows a phylogenetic analysis of organisms and the identification of new taxa.
Microsynth has performed many projects in this area over the last years. All projects have undergone the following five phases:
- DNA extraction from hundreds of samples
- Synthesis of high-quality, bar-coded PCR primers (in our own production facility)
- Hundreds of PCRs run simultaneously including purification, quantification and equimolar pooling
- 454 sequencing with the GS FLXTM System from Roche Diagnostics
- Bioinformatical sorting of the reads according to their barcode
| 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.
| 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 |
>>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 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[1]. 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.
References:
- 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) [2]. 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.