Biochemical and genetic test methods

Our dedicated team ensures the highest quality standards applied for each analysis and for results you can rely on

Our genetic expertise and state-of-the-art testing methodology is at the heart of the services we provide.

 

 

  1. Genetic Test Methods

Biochemical and genetic testing methods and workflows

CENTOGENE´s in-depth medical expertise is supported by the application of cutting-edge technologies. We utilize almost all of the existing testing methods in our workflows, processing every sample in-house. We adhere for each testing method to the highest international standards (CAP, CLIA, ISO) for establishment and validation.

Genetic and biochemical testing methods at CENTOGENE

Sanger sequencing

The term DNA sequencing refers to methods for determining the order of the nucleotide bases adenine, guanine, cytosine and thymine – in a molecule of DNA using a capillary sequencer.

Hotspot analysis:

Investigates mutations that show an increased frequency in the population. There are different hotspot mutations based on the ethnicity/ancestry of each patient (as provided by physician).

Full gene sequencing:

Analysis of the entire protein-coding regions of a gene (associated with a specific disorder ) including exon-intron boundaries.


Fragment length analysis

Some diseases are caused by insertions/deletions which cannot be detected via classical Sanger sequencing. Moreover, in some genes, there are repeats of nucleotide motives CAG. The size of the repeat regions consisting of e.g. CAGs varies between individuals and is polymorphic in normal individuals. However, when the number of repeats exceeds a certain threshold, neurological symptoms may be caused (pathological repeat expansion). In these cases, fragment length analysis (FLA) and/or repeat primed assays are utilized to detect the extent of the expanded repeats using a capillary sequencer.

Downloads for biochemical and genetic testing methods

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Deletion/duplication testing

It identifies large deletions or duplications using MLPA, qPCR or ddPCR.

MPLA:

MLPA (multiplex ligation-dependent probe amplification) is a semiquantitative multiplex PCR method detecting abnormal copy numbers of up to 50 different genomic DNA or RNA sequences, which is able to distinguish sequences differing in only one nucleotide. Probes bind to the DNA, are ligated and amplified by PCR. Results are obtained using a capillary sequencer.

Although for most hereditary conditions (partial) gene deletions or duplications account for less than 10% of all disease-causing mutations, for many other disorders this is 10 to 30 % or even still higher. The inclusion of MLPA in clinical settings can therefore significantly increase the detection rate of many genetic disorders.

qPCR:

The polymerase chain reaction (PCR) is one of the most powerful technologies in molecular biology. Using PCR, specific sequences within a DNA template can be copied, or “amplified,” many thousand-fold to a million-fold using sequence-specific oligonucleotides, heat-stable DNA polymerase, and thermal cycling. In real-time PCR (qPCR), the amount of DNA is measured after each cycle via fluorescent dyes that yield increasing fluorescent signals in direct proportion to the number of PCR product molecules (amplicons) generated. Data collected in the exponential phase of the reaction yield quantitative information on the starting quantity of the amplification target.

ddPCR:

In digital droplet PCR (ddPCR) is somewhat similar to a qPCR approach, however a water oil emulsion technique is used to divide the PCR solution into smaller reactions. The sample is partitioned into nanoliter-size samples and encapsulated into oil droplets. In every droplet, the amplification process (40 cycles) takes place independently. Only in droplets containing the target DNA sequence can amplification occur resulting in a fluorescent signal based on probes or DNA intercalating dye. Thus, there are only two possibilities: “positive/negative” –> digital: “on/off.” The number of positive droplets correlates to the starting amount of DNA thus allowing for absolute quantification.

Latest scientific articles - Deletion/duplication testing

  • Clinical and genetic characteristics of sporadic adult-onset degenerative ataxia

    The objective of this study is to define the clinical phenotype and natural history of sporadic adult-onset degenerative ataxia and to identify putative disease-causing mutations. The primary measure of disease severity was the Scale for the Assessment and Rating of Ataxia (SARA). DNA samples were screened for mutations using a high-coverage ataxia-specific gene panel in combination with next-generation sequencing. The analysis was performed on 249 participants. Our study provides quantitative data on the clinical phenotype and progression of sporadic ataxia with adult onset.

  • AMPA-receptor specific biogenesis complexes control synaptic transmission and intellectual ability

    AMPA-type glutamate receptors (AMPARs), key elements in excitatory neurotransmission in the brain, are macromolecular complexes whose properties and cellular functions are determined by the co-assembled constituents of their proteome. Here we identify AMPAR complexes that transiently form in the endoplasmic reticulum (ER) and lack the core-subunits typical for AMPARs in the plasma membrane. Central components of these ER AMPARs are the proteome constituents FRRS1l (C9orf4) and CPT1c that specifically and cooperatively bind to the pore-forming GluA1-4 proteins of AMPARs. Our results provide insight into the early biogenesis of AMPARs and demonstrate its pronounced impact on synaptic transmission and brain function.

  • NGS and beyond: Pioneering the new genome-based panel generation

    Tired of spending significant money and time only to end up with negative results? Join this webinar to discover a complete and unique test with increased diagnostic accuracy and significant time and cost savings.

Biochemical/biomarker analysis

Enzyme assay:

Measurement of enzyme activities for lysosomal storage disorders (LSDs): enzymes extracted from dried blood spots (DBS) or leucocytes convert artificial substrates into products which can be quantified using tandem mass spectrometry or via fluorimetric/spectrophotometric methods.

Biomarker analysis:

Measurement of biomarker concentrations in dried blood spots via tandem mass spectrometry.

Latest scientific articles - Biomarker and biochemical analysis

  • Reductions in glucosylsphingosine (lyso-Gb1) in treatment-naïve and previously treated patients receiving velaglucerase alfa for type 1 Gaucher disease

    Gaucher disease (GD), an autosomal recessive lipid storage disorder, arises from mutations in the GBA1 (β-glucocerebrosidase) gene, resulting in glucosylceramide accumulation in tissue macrophages. Lyso-Gb1 (glucosylsphingosine,lyso-GL1), a downstream metabolic product of glucosylceramide, has been identified as a promising biomarker for the diagnosis and onitoring of patients with GD. This retrospective, exploratory analysis of data from phase 3 clinical trials of velaglucerase alfa in patients with type 1 GD evaluated the potential of lyso-Gb1 as a specific and sensitive biomarker for GD.

  • C26-Ceramide as highly sensitive biomarker for the diagnosis of Farber Disease

    Farber disease (FD) is a rare autosomal recessive disease caused by mutations in the acid ceramidase gene (ASAH1). Low ceramidase activity results in the accumulation of fatty substances, mainly ceramides. Hallmark symptoms at clinical level are periarticular nodules, lipogranulomas, swollen and painful joints and a hoarse voice. FD phenotypes are heterogeneous varying from mild to very severe cases, with the patients not surviving past their first year of life. This study has the highest number of enrolled Farber patients and carriers reported to present. Liquid chromatography multiple reaction mass spectrometry (LC/MRM-MS) studies revealed that the ceramide C26:0 and especially its isoform 1 is a highly sensitive and specific biomarker for FD (p < 0.0001). The new biomarker can be determined directly in the dried blood spot extracts with low sample consumption. This allows for easy sample preparation, high reproducibility and use in high throughput screenings.

  • CentoAcademy® - High-throughput genetic and biochemical analyses of lysosomal storage disorders master course October 16-18, 2017

    Gaining deep insights about using genetics and targeted mass spectrometry to detect patients suffering from lysosomal storage diseases and benefit from experiences via hands-on courses supervised by our experts.

Next generation sequencing

Next generation sequencing (NGS) enables us to generate a large amount of sequencing data in a massively parallel manner (millions of sequencing reactions at the same time). This makes sequencing much faster and much more cost-effective, and thus allows us to sequence large panels, whole exomes (WES) and even whole human genomes (WGS).

Whole exome sequencing

Identifies the molecular basis of a genetic disorder in an affected person by analyzing the total number of exons (= exome), the complete protein-coding region of the genome. These regions make up in total approx. 1% of the genome.

Whole genome sequencing

Reveals comprehensive information about the genetic composition of an individual by exhaustively covering the whole genome, thus both protein-coding and non-coding regions.

Latest scientific articles - Next generation sequencing

  • Clinical and genetic characteristics of sporadic adult-onset degenerative ataxia

    The objective of this study is to define the clinical phenotype and natural history of sporadic adult-onset degenerative ataxia and to identify putative disease-causing mutations. The primary measure of disease severity was the Scale for the Assessment and Rating of Ataxia (SARA). DNA samples were screened for mutations using a high-coverage ataxia-specific gene panel in combination with next-generation sequencing. The analysis was performed on 249 participants. Our study provides quantitative data on the clinical phenotype and progression of sporadic ataxia with adult onset.

  • NGS and beyond: Pioneering the new genome-based panel generation

    Tired of spending significant money and time only to end up with negative results? Join this webinar to discover a complete and unique test with increased diagnostic accuracy and significant time and cost savings.

  • Clinical exome sequencing - Results from 2819 samples reflecting 1000 families

    A study was conducted using whole exome sequencing (WES) to identify underlying pathogenic variants, or likely pathogenic variants, in 1,000 diagnostic cases from 54 different countries. Patients selected displayed a wide variety in the number, nature and severity of symptoms. Clinical information given by the requesting physicians was translated to HPO terms and WES was performed on patient samples according to standardized settings.

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