DNA Analysis Laboratory

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DNA Analysis Laboratory

DNA analysis laboratory is equipped with modern machines that provide a comprehensive analysis of nucleic acids.

Our services include DNA and RNA analysis in a wide range of biological samples, including human, microbiological, animal or plant material.

High-throughput rapid nucleic acid isolation

DNA and RNA isolation using QIASymphony robot (min. 24 samples; 96 samples in 7 hours)

DNA isolation from different sources:

  • blood
  • tissues (tissue homogenisers – mechanical or cryohomogenisation upon request)
  • cell cultures
  • human and animal excrections and secretions
  • bacteria (bacterial genomes)
  • viruses (viral NA isolation from blood or culture)
  • biotraces (minimum 96 samples) from buccal swabs, Guthrie cards and other sources

Plasmid isolation (mini, midi, maxiprep) is done only manually!

Protein purification

Purification of 6xHis-tagged proteins

Genotyping of single known mutations using many molecular techniques differing with cost, sensitivity and throughput

  • ARMS-PCR (amplification refractory mutation system)
  • RFLP (restriction fragment length polymorphism) – only in case of presence of site recognised by restrictases
  • HA (heteroduplex analysis) capillary electrophoresis, possible for ~ 80% of mutations
  • HRM (high-resolution melting curve) using real-time PCR, Qiagen Rotor Gene Q or Roche LightCycler

All aforementioned techniques require careful optimisation. For this purpose, providing a valid (Sanger- or pyrosequencing verified) positive control is required. For some amplicons, no indirect techniques are available and only direct sequencing is possible.

Genotyping of unknown mutations using many molecular techniques differing with cost, sensitivity and throughput

  • Pyrosequencing: High sensitivity sequencing (95%) for analysis of known mutations (eg. KRAS mutations in tumor samples; length of analysed sequence ~80 bp) using PyroMark Q24 sequencer (Qiagen)
  • NGS (next-generation sequencing) allows for simultenaous sequencing of many amplicons in many samples using MiSeq sequencer (Illumina)

Optimised Illumina kits for known disorder-related genes (MiSeq)

  • TruSight Tumor 15 – 15 genes involved in malignancies
  • TruSight Tumor 26 – 26 genes involved in malignancies
  • TruSight Cancer – cancer predisposition panel,  >1700 exons, 94 genes (gene lists available on manufacturer’s website and upon request)
  • TruSight Cardio – 174 genes involved in hereditary heart diseases
  • TruSight Inherited Disease – selected regions of 552 genes involved in hereditary diseases
  • TruSight Myeloid – 54 genes involved in myeloid malignancies
  • TruSight Autism – 101 genes involved in autism spectrum disorders
  • TruSight One  – 4813 genes associated with known clinical phenotypes

miRNA NG sequencing  and targeted RNA sequencing

  • microRNAs from different species and tissues
  • targeted mRNA sequencing from different species and tissues

Small genomes – next generation sequencing (de novo or resequencing)

  • bacteria
  • yeast
  • viruses
  • phages
  • mitochondria

Microbial diversity – NGS 16s metagenomics

Identification of bacterial species from different sources

  • food
  • waste
  • human and animal excrections and secretions

Capillary electrophoresis

Amplicon / DNA and RNA fragment separation with resolution up to 2 nucleotides (quantitative and qualitative analysis)

DNA methylation analysis

Selected CpG islands methylation analysis by pyrosequencing (PyroMark Q24)

20130710044023pic1

Robot for DNA/RNA/protein isolation QIASymphony (Qiagen)

homogenisers

Homogenisers: TissueLyzerII (Qiagen) and CryoMill (Retsch)

nanodrops

Spectrophoto- and spectrofluorimeter NanoDrop 8000 and 3300 (ThermoFisher Scientific)

Labcycler_00012_engThermal Cycler LabCycler (SensoQuest)

rotor_gene_Q

Real-time PCR thermal cycler (CEIVD) Rotor Gene Q (Qiagen)

3.8.1.5.1.1-large

Real-time PCR HT thermal cycler Light Cycler 480 (Roche)

Laboratory pipetting robot

Pipetting robot Piro (DORNIER-LTF)

Instrument_fragment_analyzer

Capillary electrophoresis Fragment Analyzer (Advanced Analytical)

miseq-fgx-hero

Next generation sequencer MiSeq (Illumina)

pyromarkQ24

Pyrosequencer PyroMark Q24 (Qiagen)

Building 4, 3rd floor, room 4.36 – office, 4.29, 4.31, 4.32 – laboratory
Laboratory Manager
dr Anna Tracewska (Siemiątkowska)
e-mail: anna.tracewska@eitplus.pl

tel. (71) 734 72 53; 727 66 55 61

OLYMPUS DIGITAL CAMERA

Lab Manager substitute (01.12.2016-30.06.2017)
dr Andrzej Kowalczyk

Grants

SONATA 10 (National Centre of Science)

Grant no.: 2015/19/D/NZ2/031

Title: Towards the light: unraveling genetic basis of hereditary retinal diseases in Poland

Duration: 01.12.2016 – 31.05.2019

 

Laboratory Manager Publications

  • van Huet RA, Siemiatkowska AM, Ozgül RK, Yücel D, Hoyng CB, Banin E, Blumenfeld A, Rotenstreich Y, Riemslag FC, den Hollander AI, Theelen T, Collin RW, van den Born LI, Klevering BJ. Retinitis pigmentosa caused by mutations in the ciliary MAK gene is relatively mild and is not associated with apparent extra-ocular features. Acta Ophthalmol. 2015;93:83-94.
  • Bujakowska KM, Zhang Q, Siemiatkowska AM, Liu Q, Place E, Falk MJ, Consugar M, Lancelot ME, Antonio A, Lonjou C, Carpentier W, Mohand-Saïd S, den Hollander AI, Cremers FP, Leroy BP, Gai X, Sahel JA, van den Born LI, Collin RW, Zeitz C, Audo I, Pierce EA. Mutations in IFT172 cause isolated retinal degeneration and Bardet-Biedl syndrome. Hum Mol Genet. 2015;24:230-42
  • Siemiatkowska AM, Schuurs-Hoeijmakers JH, Bosch DG, Boonstra FN, Riemslag FC, Ruiter M, de Vries BB, den Hollander AI, Collin RW, Cremers FP. Nonpenetrance of the most frequent autosomal recessive Leber congenital amaurosis mutation in NMNAT1. JAMA Ophthalmol. 2014;132:1002-4.
  • Siemiatkowska AM, Collin RW, den Hollander AI, Cremers FP. Genomic approaches for the discovery of genes mutated in inherited retinal degeneration. Cold Spring Harb Perspect Med. 2014;17;4. pii: a017137. (review)
  • van Huet RA, Collin RW, Siemiatkowska AM, Klaver CC, Hoyng CB, Simonelli F, Khan MI, Qamar R, Banin E, Cremers FP, Theelen T, den Hollander AI, van den Born LI, Klevering BJ. IMPG2-associated retinitis pigmentosa displays relatively early macular involvement. Invest Ophthalmol Vis Sci. 2014;55:3939-53.
  • Roosing S, Collin RW, den Hollander AI, Cremers FP#, Siemiatkowska AM#. Prenylation defects in inherited retinal diseases. J Med Genet. 2014;51:143-51. (review)
  • Siemiatkowska AM, van den Born LI, van Genderen MM, Bertelsen M, Zobor D, Rohrschneider K, van Huet RA, Nurohmah S, Klevering BJ, Kohl S, Faradz SM, Rosenberg T, den Hollander AI, Collin RW, Cremers FP. Novel compound heterozygous NMNAT1 variants associated with Leber congenital amaurosis. Mol Vis. 2014;20:753-9.
  • Siemiatkowska AM, van den Born LI, van Hagen PM, Stoffels M, Neveling K, Henkes A, Kipping-Geertsema M, Hoefsloot LH, Hoyng CB, Simon A, den Hollander AI,Cremers FP, Collin RW. Mutations in the mevalonate kinase (MVK) gene cause nonsyndromic retinitis pigmentosa. Ophthalmology. 2013;120:2697-705.
  • Neveling K, Collin RW, Gilissen C, van Huet RA, Visser L, Kwint MP, Gijsen SJ, Zonneveld MN, Wieskamp N, de Ligt J, Siemiatkowska AM, Hoefsloot LH, Buckley MF, Kellner U, Branham KE, den Hollander AI, Hoischen A, Hoyng C, Klevering BJ, van den Born LI, Veltman JA, Cremers FP, Scheffer H. Next-generation genetic testing for retinitis pigmentosa. Hum Mutat. 2012;33:963-72.
  • Paun CC, Pijl BJ, Siemiatkowska AM, Collin RW, Cremers FP, Hoyng CB, den Hollander AI. A novel crumbs homolog 1 mutation in a family with retinitis pigmentosa, nanophthalmos, and optic disc drusen. Mol Vis. 2012;18:2447-53.
  • Siemiatkowska AM*, Astuti GD*, Arimadyo K, den Hollander AI, Faradz SM, Cremers FP, Collin RW. Identification of a novel nonsense mutation in RP1 that causes autosomal recessive retinitis pigmentosa in an Indonesian family. Mol Vis. 2012;18:2411-9.
  • Ozgül RK*, Siemiatkowska AM*, Yücel D*, Myers CA, Collin RW, Zonneveld MN, Beryozkin A, Banin E, Hoyng CB, van den Born LI; European Retinal Disease Consortium, Bose R, Shen W, Sharon D, Cremers FP, Klevering BJ, den Hollander AI, Corbo JC. Exome sequencing and cis-regulatory mapping identify mutations in MAK, a gene encoding a regulator of ciliary length, as a cause of retinitis pigmentosa. Am J Hum Genet. 2011;89:253-64.
  • Collin RW, van den Born LI, Klevering BJ, de Castro-Miró M, Littink KW, Arimadyo K, Azam M, Yazar V, Zonneveld MN, Paun CC, Siemiatkowska AM, Strom TM, Hehir-Kwa JY, Kroes HY, de Faber JT, van Schooneveld MJ, Heckenlively JR, Hoyng CB, den Hollander AI, Cremers FP. High-resolution homozygosity mapping is a powerful tool to detect novel mutations causative of autosomal recessive RP in the Dutch population. Invest Ophthalmol Vis Sci. 2011;52:2227-39.
  • Siemiatkowska AM*, Arimadyo K*, Moruz LM, Astuti GD, de Castro-Miro M, Zonneveld MN, Strom TM, de Wijs IJ, Hoefsloot LH, Faradz SM, Cremers FP, den Hollander AI, Collin RW. Molecular genetic analysis of retinitis pigmentosa in Indonesia using genome-wide homozygosity mapping. Mol Vis. 2011;17:3013-24.
  • Bandah-Rozenfeld D, Collin RW, Banin E, van den Born LI, Coene KL, Siemiatkowska AM, Zelinger L, Khan MI, Lefeber DJ, Erdinest I, Testa F, Simonelli F, Voesenek K, Blokland EA, Strom TM, Klaver CC, Qamar R, Banfi S, Cremers FP, Sharon D, den Hollander AI. Mutations in IMPG2, encoding interphotoreceptor matrix proteoglycan 2, cause autosomal-recessive retinitis pigmentosa. Am J Hum Genet. 2010;87:199-208.
  • Siemiatkowska A, Bieniaszewska M, Hellmann A, Limon J. JAK2 and MPL gene mutations in V617F-negative myeloproliferative neoplasms. Leuk Res. 2010;34:387-9.
  • Duchnowska R, Siemiatkowska A, Grala B, Smoter M. [Long-term remission after erlotinib therapy in an elderly patient with advanced non-small-cell lung cancer. Case report and conclusions for clinical practice]. Pneumonol Alergol Pol. 2008;76:451-5.
  • Kanka C, Brozek I, Skalska B, Siemiatkowska A, Limon J. Germline NBS1 mutations in families with aggregation of breast and/or ovarian cancer from north-east Poland. Anticancer Res. 2007;27:3015-8.
  • Dziadziuszko R, Siemiatkowska A, Limon J, Rzyman W, Jassem J, Bunn PA Jr, Varella-Garcia M, Hirsch FR. Unusual chemosensitivity of advanced bronchioalveolar carcinoma after gefitinib response and progression: a case report. J Thorac Oncol. 2007;2:91-2.
  • Haack B, Kupka S, Ebauer M, Siemiatkowska A, Pfister M, Kwiatkowska J, Ereciński J, Limon J, Ochman K, Blin N. Analysis of candidate genes for genotypic diagnosis in the long QT syndrome. J Appl Genet. 2004;45:375-81.31.

* – equal contribution of the first authors
# – equal contribution of the last authors

Paweł Paszkowski

Sales Manager

Paweł Paszkowski

tel: 727 663 380

pawel.paszkowski@eitplus.pl

Posted by atracewska, Posted on 10.02.2016
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