A Multiplex PCR for Detection of Genetic Aberrations in Non-Hodkin’s Lymphoma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4554-4554
Author(s):  
Charlotte Guldborg Nyvold ◽  
Knud Bendix ◽  
Stephen J. Hamilton-Dutoit ◽  
Dorte Melsvik ◽  
Hanne Tønder ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Long Range ◽  
Cell Lines ◽  
Clinical Setting ◽  
Residual Disease ◽  
Simultaneous Detection ◽  
Control Gene ◽  
Genetic Aberrations ◽  
Dna And Rna ◽  
Long Range Pcr

Abstract Aim: To develop a multiplex PCR method applicable in a clinical setting for the simultaneous detection of the chromosomal lesions t(11;14)(q13q32), t(14;18)(q32;q21), t(2;5)(p23;q35), t(11;18)(q21;q21), t(3q27;var), and t(8;14)(q24;q32) frequently found in non-Hodgkin lymphoma (NHL). Methods: DNA and RNA were prepared from 50μm lymph node (LN) sections by homogenization on a FastPrep instrument (Qbiogene, Cedex, France) followed by automated nucleic acid purification on a MagNa-Pure LC robot (Roche Diagnostics, Basel, Switzerland). The multiplex PCR was condensed in four PCR tubes. The first covered the MTC and mTCp94 region of BCL1/IGH fusion DNA, the MBR and MCR breakpoint regions of BCL2/IGH fusion DNA together with the control gene TCF20. The second included the API2/MLT and ALK/NPM breakpoints on cDNA along with β-ACTIN as a control gene. The third contained primers amplifying eight different fusions partners of BCL6 (IGH (14q32), IGL (22q11), HSP89α (14q32), HSP90β (6p12), PIM1 (6p21), TFR (3q26), TTF (4p13), and H4 (6p21)) on cDNA together with β-ACTIN as the control gene. The fourth tube harbored a long range PCR with primers detecting the CMYC/IGH breakpoints on genomic DNA (Cμ, Cγ, Cα, and joining region of the IGH (Basso et al., 1999, Am J Pathology)) together with ABL as a control gene. Patient samples and cell lines: One-hundred-and-twelve LN biopsies frozen in Tissue-Tek OCT Compound (Sakura, Vaerloese, Denmark) were randomly selected from consecutive patients referred with suspected hematological malignancy. The following cell lines were used as positive controls: B-CLL line JVM-2 (t(11;14)+), NHL lines DOHH-2 (t(14;18)+, t(8;14)+) and WSU-NHL (t(14;18)+), Burkitt’s lymphoma lines BL-41, BL-70 (t(8;14)+), and MD901 (t(3;22)+), T-NHL line Karpas 299 (t(2;5)+), and ALL line MD903 (t(3;14)+). Results: In pilot experiments employing cell lines and fresh LN material, this optimized multiplex PCR reaction proved to be simple and fast with a short turnover time, considering the large number of genetic aberrations detected. In a retrospective LN material encompassing 112 blinded samples, BCL1/IGH fusion DNA with breakpoint in the MTC region was detected once while BCL2/IGH was found in 20 samples (19 in MBR and one in MCR). BCL6/IGH fusion cDNA was found in three samples while the TTF gene was utilized twice as translocation partner to BCL6. Finally, CMYC/IGH fusion DNA was detected three times (1 IGHCα, 2 IGHCγ). All PCR products apart from CMYC/IGH were sequenced and verified the specific chromosomal lesions. Nineteen were excluded due to weak control bands in the first three PCR tubes, while 38 were excluded in the long range PCR detecting CMYC/IGH. Conclusion: We conclude that the NHL multiplex PCR described is an easy and timesaving method for identifying heterogeneous molecular disease markers in NHL. The standardized DNA- and RNA preparation together with the condensation into four PCR tubes, moreover, makes it convenient to the clinical setting. Application of this assay and identification of positive cases has the added advantage that quantitative real-time PCR monitoring residual disease can be applied.

scholarly journals Simultaneous Detection of CDC Category "A" DNA and RNA Bioterrorism Agents by Use of Multiplex PCR & RT-PCR Enzyme Hybridization Assays

Viruses ◽  
2009 ◽  
Vol 1 (3) ◽  
pp. 441-459 ◽  
Author(s):  
Jie He ◽  
Andrea Kraft ◽  
Jiang Fan ◽  
Meredith Van Dyke ◽  
Lihua Wang ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Simultaneous Detection ◽  
Rt Pcr ◽  
Dna And Rna ◽  
Hybridization Assays

scholarly journals Phylogenetic analysis of higher-level relationships within Hydroidolina (Cnidaria: Hydrozoa) using mitochondrial genome data and insight into their mitochondrial transcription

PeerJ ◽  
2015 ◽  
Vol 3 ◽  
pp. e1403 ◽  
Author(s):  
Ehsan Kayal ◽  
Bastian Bentlage ◽  
Paulyn Cartwright ◽  
Angel A. Yanagihara ◽  
Dhugal J. Lindsay ◽  
...  
Keyword(s):  
Long Range ◽  
High Throughput Sequencing ◽  
Phylogenetic Analyses ◽  
Mitochondrial Gene ◽  
Morphological Diversity ◽  
Sister Group ◽  
Rna Seq ◽  
Dna And Rna ◽  
Long Range Pcr ◽  
Group Relationships

Hydrozoans display the most morphological diversity within the phylum Cnidaria. While recent molecular studies have provided some insights into their evolutionary history, sister group relationships remain mostly unresolved, particularly at mid-taxonomic levels. Specifically, within Hydroidolina, the most speciose hydrozoan subclass, the relationships and sometimes integrity of orders are highly unsettled. Here we obtained the near complete mitochondrial sequence of twenty-six hydroidolinan hydrozoan species from a range of sources (DNA and RNA-seq data, long-range PCR). Our analyses confirm previous inference of the evolution of mtDNA in Hydrozoa while introducing a novel genome organization. Using RNA-seq data, we propose a mechanism for the expression of mitochondrial mRNA in Hydroidolina that can be extrapolated to the other medusozoan taxa. Phylogenetic analyses using the full set of mitochondrial gene sequences provide some insights into the order-level relationships within Hydroidolina, including siphonophores as the first diverging clade, a well-supported clade comprised of Leptothecata-Filifera III–IV, and a second clade comprised of Aplanulata-Capitatas.s.-Filifera I–II. Finally, we describe our relatively inexpensive and accessible multiplexing strategy to sequence long-range PCR amplicons that can be adapted to most high-throughput sequencing platforms.

Identification of Novel Molecular Markers for the Follow-up of Minimal Residual Disease in Hematooncological Disorders

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4905-4905
Author(s):  
David Warren Hardekopf ◽  
Tereza Jancuskova ◽  
Marie Jarosova ◽  
Lucie Krutilkova ◽  
Radek Plachy ◽  
...  
Keyword(s):  
Minimal Residual Disease ◽  
Long Range ◽  
Residual Disease ◽  
Molecular Techniques ◽  
Molecular Diagnostic ◽  
Multicolor Fish ◽  
Bac Clones ◽  
Long Range Pcr ◽  
Minimal Residual

Abstract Abstract 4905 Modern molecular diagnostic techniques allow the detection of minimal residual disease (MRD) in hematological malignancies with high sensitivity, allowing patient-specific assays of MRD levels over the course of treatment. One challenge for these techniques is finding appropriate MRD marker targets. For instance, PCR-based MRD analysis requires unique sequence-specific information for the malignant clone of interest. In many acute leukemias, such detailed information is lacking, either because no abnormality is detected (e.g. in 40–50% of adult Acute myeloid leukemia-AML), or the resolution of cytogenetic methods is too low to precisely define newly-found abnormalities. We have used multicolor-FISH (mFISH) to perform karyotyping of 141 patients. Using this approach we have identified chromosomal abnormalities in about 35% of analyzed cases. The aberrations found involved the recurrent aberrations t(15;17), t(8;21), inv(16), t(9;22),−5q, −7, +8 but also an array of unique abnormalities, such as der(15)t(5;15)(q15;q24), t(6;12)(p22;q13), and der(17)t(12;17)(q14;pter), which may serve as possible targets for molecular MRD follow-up. To further characterize such targets, we seek to bridge the gap from cytogenetic resolution down to higher-resolution molecular techniques. For this, we first employ the mBAND technique, followed by multicolor FISH using DNA probes with known cytogenetic location. These probes are drawn from the Human Minimal Genomic Clone Set (Version 1.0, Source Bioscience LifeSciences) of over 25,000 bacterial artificial chromosome (BAC) clones with human DNA inserts. These are tiling clones covering almost 100% of the genome, and therefore allow us to develop FISH probes to regions containing almost any conceivable target breakpoint. Starting with breakpoint information from mFISH/mBAND, we perform consecutive multi-color/multi-BAC hybridizations around the region of interest, looking for breakpoint-spanning probes. Using this system, we are able to move from a resolution of around 2 Mbp (the limit of mBAND) to the resolution of individual BAC clones (average 150 kbp) in three hybridizations. To achieve further resolution, long-range PCR products are designed within the region defined by breakpoint-spanning BACs, with labeling and FISH-mapping of these probes. At present, we are starting to perform these hybridizations on stretched DNA (fiber-FISH or combed DNA), where one micrometer of a stretched DNA molecule represents about 2000 base pairs, allowing visualization of probe distance and spacing, and even higher-resolution breakpoint analysis. Chromosome microdissection followed by high-throughput sequencing (Roche GS Junior) is also being pursued to identify novel breakpoints by rapidly sequencing large chromosomal regions on both sides of a translocation. The final goal is to map genomic abnormalities to a resolution amenable to long-range PCR, yielding specific targets for MRD detection, and allowing clone-specific Real-Time PCR assays for sensitive and specific monitoring of MRD in hematooncological patients. Disclosures: Smolej: GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Travel Grants; Roche: Honoraria, Travel Grants; Genzyme: Honoraria, Travel Grants.

Development of multiplex PCR for simultaneous detection of six swine DNA and RNA viruses

2012 ◽  
Vol 183 (1) ◽  
pp. 69-74 ◽  
Author(s):  
Xin-Gang Xu ◽  
Guang-Da Chen ◽  
Yong Huang ◽  
Li Ding ◽  
Zhao-Cai Li ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Rna Viruses ◽  
Simultaneous Detection ◽  
Dna And Rna

scholarly journals Long-Range PCR-Based NGS Applications to Diagnose Mendelian Retinal Diseases

2021 ◽  
Vol 22 (4) ◽  
pp. 1508
Author(s):  
Jordi Maggi ◽  
Samuel Koller ◽  
Luzy Bähr ◽  
Silke Feil ◽  
Fatma Kivrak Pfiffner ◽  
...  
Keyword(s):  
Genetic Testing ◽  
Long Range ◽  
Copy Number Variants ◽  
Retinal Disease ◽  
Promoter Regions ◽  
Long Range Pcr ◽  
Cost Efficient ◽  
Nucleotide Resolution ◽  
Genomic Regions ◽  
Next Generation Sequencing Ngs

The purpose of this study was to develop a flexible, cost-efficient, next-generation sequencing (NGS) protocol for genetic testing. Long-range polymerase chain reaction (PCR) amplicons of up to 20 kb in size were designed to amplify entire genomic regions for a panel (n = 35) of inherited retinal disease (IRD)-associated loci. Amplicons were pooled and sequenced by NGS. The analysis was applied to 227 probands diagnosed with IRD: (A) 108 previously molecularly diagnosed, (B) 94 without previous genetic testing, and (C) 25 undiagnosed after whole-exome sequencing (WES). The method was validated with 100% sensitivity on cohort A. Long-range PCR-based sequencing revealed likely causative variant(s) in 51% and 24% of proband from cohorts B and C, respectively. Breakpoints of 3 copy number variants (CNVs) could be characterized. Long-range PCR libraries spike-in extended coverage of WES. Read phasing confirmed compound heterozygosity in 5 probands. The proposed sequencing protocol provided deep coverage of the entire gene, including intronic and promoter regions. Our method can be used (i) as a first-tier assay to reduce genetic testing costs, (ii) to elucidate missing heritability cases, (iii) to characterize breakpoints of CNVs at nucleotide resolution, (iv) to extend WES data to non-coding regions by spiking-in long-range PCR libraries, and (v) to help with phasing of candidate variants.

scholarly journals BTK, NUTM2A, and PRPF19 Are Novel KMT2A Partner Genes in Childhood Acute Leukemia

Biomedicines ◽  
2021 ◽  
Vol 9 (8) ◽  
pp. 924
Author(s):  
Elena Zerkalenkova ◽  
Svetlana Lebedeva ◽  
Aleksandra Borkovskaia ◽  
Olga Soldatkina ◽  
Olga Plekhanova ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Residual Disease ◽  
Chromosomal Rearrangements ◽  
Strong Impact ◽  
Acute Leukemias ◽  
Dna And Rna ◽  
Targeted Ngs ◽  
Childhood Acute Leukemia ◽  
Next Generation Sequencing Ngs ◽  
The Given

Chromosomal rearrangements of the human KMT2A/MLL gene are associated with acute leukemias, especially in infants. KMT2A is rearranged with a big variety of partner genes and in multiple breakpoint locations. Detection of all types of KMT2A rearrangements is an essential part of acute leukemia initial diagnostics and follow-up, as it has a strong impact on the patients’ outcome. Due to their high heterogeneity, KMT2A rearrangements are most effectively uncovered by next-generation sequencing (NGS), which, however, requires a thorough prescreening by cytogenetics. Here, we aimed to characterize uncommon KMT2A rearrangements in childhood acute leukemia by conventional karyotyping, FISH, and targeted NGS on both DNA and RNA level with subsequent validation. As a result of this comprehensive approach, three novel KMT2A rearrangements were discovered: ins(X;11)(q26;q13q25)/KMT2A-BTK, t(10;11)(q22;q23.3)/KMT2A-NUTM2A, and inv(11)(q12.2q23.3)/KMT2A-PRPF19. These novel KMT2A-chimeric genes expand our knowledge of the mechanisms of KMT2A-associated leukemogenesis and allow tracing the dynamics of minimal residual disease in the given patients.

scholarly journals Poly-L-ornithine-mediated transformation of mammalian cells.

1987 ◽  
Vol 7 (6) ◽  
pp. 2286-2293 ◽  
Author(s):  
V C Bond ◽  
B Wold
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
High Efficiency ◽  
Protein Product ◽  
Marker Genes ◽  
Recipient Mouse ◽  
L Cells ◽  
Dna And Rna ◽  
Selectable Marker Genes

Poly-L-ornithine has been used to introduce DNA and RNA into mammalian cells in culture. Ornithine-mediated DNA transfer has several interesting and potentially useful properties. The procedure is technically straightforward and is easily applied to either small or large numbers of recipient cells. The efficiency of transformation is high. Under optimal conditions, 1 to 2% of recipient mouse L cells take up and continue to express selectable marker genes. DNA content of transformants can be varied reproducibly, yielding cells with just one or two copies of the new gene under one set of conditions, while under a different set of conditions 25 to 50 copies are acquired. Cotransformation and expression of physically unlinked genes occur at high efficiency under conditions favoring multiple-copy transfer. Polyornithine promotes gene transfer into cell lines other than L cells. These include Friend erythroleukemia cells and NIH 3T3 cells. Both are transformed about 1 order of magnitude more efficiently by this procedure than by standard calcium phosphate products. However, the method does not abolish the large transformation efficiency differences between these cell lines that have been observed previously by other techniques. (vi) mRNA synthesized in vitro was also introduced into cells by this method. The RNA was translated resulting in a transient accumulation of the protein product.

A novel common primer multiplex PCR (CP-M-PCR) method for the simultaneous detection of meat species

Food Control ◽  
2009 ◽  
Vol 20 (4) ◽  
pp. 366-370 ◽  
Author(s):  
Weibin Bai ◽  
Wentao Xu ◽  
Kunlun Huang ◽  
Yanfang Yuan ◽  
Sishuo Cao ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Simultaneous Detection ◽  
Meat Species ◽  
Pcr Method
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