Methods

Gene diagnostics make use of commercially available reagents and instruments that performance have been tested and validated in our laboratory. Performance of the instruments and analysis methods is monitored continuously.

DNA extraction

Genomic DNA is extracted from EDTA whole blood using Qiagen's QIAamp® DNA Blood kit. Purity, quality and quantity is analyzed using phometric and/or fluorometric method before genetic analysis.

TaqMan®-method

Known variants are analyzed by using qPCR method (TaqMan chemisty). TaqMan chemistry is based on allele specific probes and and primers. TaqMan assay design take into account e.g. other known nearby variants and repeat regions. TaqMan chemistry is suitable for detection of previously known single nucleotide and short indel variants.

Sanger sequencing method

Coding regions of the target genes are amplified using PCR method and subsequently analyzed using Sanger sequnencing method. All the know sequence variations (e.g. SNPs, repeats and GC rich regions) are taken into account in during target specific PCR primer designing proces. Sanger sequencing is appropriate analysis method for single nucleotide variants and short insertions and deltions.

Next Generation Sequencing

Illumina's instruments are used in next generation sequencing (NGS) . NGS sequencing take advantage of Sequencing by Synthesis (SBS) chemistry. It is based on massive parallel sequencing, where millions of short DNA fragments are simultaneously amplified. Sequenced fragments are aligned to the human reference sequence and alternative alleles are identified.  NGS sequencing is targeted to exonsregions of the genes. The method is suitable for analyzing single nucleotide variants as well as short insertions and deletions.  

MLPA method

Deleetioanalyysit tehdään kapillaarisekvensointilaitteella kaupallisella Multiplex-PCR-menetelmään pohjautuvalla kitillä (Multiplex Ligation-dependent Probe Amplification, MLPA®). MLPA-menetelmän käyttö deleetioanalytiikassa perustuu tutkittavan geenin eksonien kopioluvun määrittämiseen.

Deletion analysis make use of MLPA (Multiplex Ligation-dependent Probe Amplification) method. MLPA analysis reveals gene exon copy number changes (large insertions and deletions). 

Data analysis

Sekvensoinnissa ja deleetioanalytiikassa havaittuja sekvenssimuutoksia verrataan kirjallisuudesta löytyviin aikaisemmin raportoituihin sekvenssimuutoksiin niiden kausaalisuuden arvioimiseksi. Neutraaleiksi arvioituja havaittuja sekvenssimuutoksia ei raportoida. TaqMan-analyysillä tutkittavat mutaatiot ovat aiemmin kirjallisuudessa todettu kausaalisiksi. Tutkimustuloksesta toimitetaan lausunto hoitavalle lääkärille.