SNP genotyping using microsphere‐linked PNA and flow cytometric detection

Rockenbauer, Eszter; Petersen, Kenneth H.; Vogel, Ulla; Bolund, Lars; Kølvraa, Steen; Nielsen, Kirsten; Nexø, Bjørn A.

doi:10.1002/cyto.a.20123

Cited by 14 publications

(4 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, amplification of genomic target DNA is a prerequisite to hybridization with conjugated probes in order to score discrete genotypes [Hadd et al, 2004;Rockenbauer et al, 2005]. Because amplification is inherently logarithmic and thus difficult to control, hybridization of these target sequences to conjugated oligonucleotide probes does not reproducibly and accurately quantify genomic copy number [Earley et al, 2002;Sekar et al, 2005;Vignali, 2000].…”

Section: Introductionmentioning

confidence: 99%

Determination of genomic copy number with quantitative microsphere hybridization

et al. 2006

View full text Add to dashboard Cite

Communicated by Jing ChengWe developed a novel quantitative microsphere suspension hybridization (QMH) assay for determination of genomic copy number by flow cytometry. Single copy (sc) products ranging in length from 62 to 2,304 nucleotides [Rogan et al., 2001;Knoll and Rogan, 2004] from ABL1 (chromosome 9q34), TEKT3 (17p12), PMP22 (17p12), and HOXB1 (17q21) were conjugated to spectrally distinct polystyrene microspheres. These conjugated probes were used in multiplex hybridization to detect homologous target sequences in biotinylated genomic DNA extracted from fixed cell pellets obtained for cytogenetic studies. Hybridized targets were bound to phycoerythrin-labeled streptavidin; then the spectral emissions of both target and conjugated microsphere were codetected by flow cytometry. Prior amplification of locus-specific target DNA was not required because sc probes provide adequate specificity and sensitivity for accurate copy number determination. Copy number differences were distinguishable by comparing the mean fluorescence intensities (MFI) of test probes with a biallelic reference probe in genomic DNA of patient samples and abnormal cell lines. Concerted 5 0 ABL1 deletions in patient samples with a chromosome 9;22 translocation and chronic myelogenous leukemia were confirmed by comparison of the mean fluorescence intensities of ABL1 test probes with a HOXB1 reference probe. The relative intensities of the ABL1 probes were reduced to 0.5970.02 fold in three different deletion patients and increased 1.4270.01 fold in three trisomic 9 cell lines. TEKT3 and PMP22 probes detected proportionate copy number increases in five patients with Charcot-Marie-Tooth Type 1a disease and chromosome 17p12 duplications. Thus, the assay is capable of distinguishing one allele and three alleles from a biallelic reference sequence, regardless of chromosomal context. Hum Mutat 27(4), [376][377][378][379][380][381][382][383][384][385][386] 2006.

show abstract

Section: Introductionmentioning

confidence: 99%

Determination of genomic copy number with quantitative microsphere hybridization

et al. 2006

View full text Add to dashboard Cite

show abstract

“…Immunophenotyping is an important diagnostic tool for decision making in various diseases [1][2][3][4][5][6]. Typical examples are: -diagnosis of immunodeficiencies [7], -monitoring of systemic and chronic diseases [8][9][10][11][12], -cellular diagnostics of allergy [13,14], -functional characterization of cells [15,16], -genetic investigation [17,18], -differential diagnosis of lymphocytosis [19], -diagnosis of leukemia [5,[20][21][22][23][24], -investigation of T-cell receptor expression in Kawasaki syndrome [25][26], -investigation of pregnancy disturbances [27], -quality control in hemotherapy [28][29][30]. Furthermore, there are several indications that are presently not so important for routine diagnostics [31].…”

Section: Introductionmentioning

confidence: 99%

Age-Related Lymphocyte Subset Changes in the Peripheral Blood of Healthy Children – a Meta-Study

Sack¹,

Gerling²,

Tárnok³

2007

Transfus Med Hemother

View full text Add to dashboard Cite

Immunophenotyping is an important diagnostic tool for decision making in various diseases. Although clinical interpretation relies on measurable aberrations of the patient’s values from normal ranges, agedependent are rarely available. Materials and Methods: The present study is aimed to combine published data about normal values of peripheral blood lymphocyte subpopulations to describe age-dependent changes from the neonate to the adult. These values could serve in a better way as normal values in comparison to present ones. Furthermore, this investigation allows us to define valid data even for short periods in children’s life and to investigate the influence of technical approaches, sample preparation, antibody selection, measurement equipment, and data analysis. Results: Development-related alterations of lymphocyte subset counts in children could be extracted from the pre-existing papers for diagnostic use. These results were mostly independent from gender, ethnic factors, procedure of sample collection, anticoagulation, pre-analytical procedures, time to workbench, applied method for immunophenotyping, staining procedure, selected monoclonal antibodies, technical devices, and software products. Conclusion: Our data indicate that previous normal values are not sufficiently precise for the interpretation of lymphocyte subsets in children. Mainly during the 1st year of life, count and subset distribution of lymphocytes is different from that of adults. Therefore, a close meshed data set of normal values is required to guarantee adequate diagnostic interpretation.

show abstract

“…Rockenbauer et al reported a new method that combined allele-specific hybridization, PNA technology, and detection while using flow cytometry. These authors described a fully functional two-bead genotyping system based on PNA capture and flow cytometric detection that they used for the accurate and fast re-genotyping of a Danish basal cell carcinoma cohort [15]. Bethge et al synthesized forced intercalation probes (FIT-probes) that contained an intercalating cyanine dye, such as oxazole yellow (YO), which serves as a replacement for a canonical nucleobase.…”

Section: Introductionmentioning

confidence: 99%

SNP Discrimination by Tolane-Modified Peptide Nucleic Acids: Application for the Detection of Drug Resistance in Pathogens

et al. 2020

View full text Add to dashboard Cite

During the treatment of viral or bacterial infections, it is important to evaluate any resistance to the therapeutic agents used. An amino acid substitution arising from a single base mutation in a particular gene often causes drug resistance in pathogens. Therefore, molecular tools that discriminate a single base mismatch in the target sequence are required for achieving therapeutic success. Here, we synthesized peptide nucleic acids (PNAs) derivatized with tolane via an amide linkage at the N-terminus and succeeded in improving the sequence specificity, even with a mismatched base pair located near the terminal region of the duplex. We assessed the sequence specificities of the tolane-PNAs for single-strand DNA and RNA by UV-melting temperature analysis, thermodynamic analysis, an in silico conformational search, and a gel mobility shift assay. As a result, all of the PNA-tolane derivatives stabilized duplex formation to the matched target sequence without inducing mismatch target binding. Among the different PNA-tolane derivatives, PNA that was modified with a naphthyl-type tolane could efficiently discriminate a mismatched base pair and be utilized for the detection of resistance to neuraminidase inhibitors of the influenza A/H1N1 virus. Therefore, our molecular tool can be used to discriminate single nucleotide polymorphisms that are related to drug resistance in pathogens.

show abstract

SNP genotyping using microsphere‐linked PNA and flow cytometric detection

Cited by 14 publications

References 25 publications

Determination of genomic copy number with quantitative microsphere hybridization

Determination of genomic copy number with quantitative microsphere hybridization

Age-Related Lymphocyte Subset Changes in the Peripheral Blood of Healthy Children – a Meta-Study

SNP Discrimination by Tolane-Modified Peptide Nucleic Acids: Application for the Detection of Drug Resistance in Pathogens

Contact Info

Product

Resources

About