A novel RT-PCR approach to detecting EML4-ALK fusion genes in archival NSCLC tissue.
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10535 Background: The echinoderm microtubule-associated protein-like 4–anaplastic lymphoma kinase (EML4-ALK) fusion gene can be detected in subsets of NSCLC, especially never-smoking patients with adenocarcinoma, and appears to be mutually exclusive of EGFR mutation. PF-1066, a potent and selective c-MET/ALK inhibitor, yields high response rates in ALK-positive patients, as detected by labor-intensive fluorescent in situ hybridization (FISH) methodology. Existing RT-PCR assays for these gene variants are designed to amplify large cDNA fragments (> 450 bases), while formalin-fixed paraffin-embedded (FFPE) specimens yield mostly RNA fragments of < 150 bases. Thus, our goal was to design a robust RT-PCR assay for EML4-ALK fusion gene transcripts suitable for use with commonly available FFPE tissues of limited size. Methods: Synthetic fragments representing the nine EML4-ALK fusion genes variants 1, 2, 3a, 3b, 4, 5a, 5b, 6 and 7 were generated by recursive PCR technology. The approach consisted of amplifying over-lapping fragments containing the fusion variant sequences with outside 5′ and 3′ primers. Primer probes were designed to detect specific EML4-ALK fusion gene fragments with a maximum amplicon of 170 bases by RT-PCR. Results: Functional RT-PCR assay were developed for each specific EML4-ALK variant, meeting criteria specified above for optimal clinical testing. Assays detecting multiple fusion variants without distinguishing specific variants were also designed. All nine known EML4-ALK variants can be detected using 6 RT-PCR assays. Screening of NSCLC cDNAs from the Response Genetics database is underway (molecular and clinical correlations to be presented). Conclusions: We developed RT-PCR assays capable of detecting EML4-ALK fusion gene variants from FFPE tissues. Two assays were successfully established: one that can detect and identify each individual variant, and one capable of detecting the presence of any variant as a single assay. We expect this methodology to provide a useful tool for large-scale screening of NSCLC or other FFPE tissues for the EML4-ALK fusion gene. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Response Genetics Amgen, AstraZeneca, Biodesix, Boehringer Ingelheim, Bristol-Myers Squibb, Genentech, GlaxoSmithKline, Lilly, Merck, Novartis, Pfizer, sanofi-aventis Response Genetics DxS Abbott Laboratories, Bristol-Myers Squibb, Genentech, Lilly, Merck, Novartis, PfizerKeywords:
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We applied an algorithm targeting length polymorphisms of intergenic sequences between gene-flanking regions for constructing PCR primer pairs to distinguish among serogroups of Salmonella, a major pathogen of humans and animals. From 43 constructed primer pairs, a pair capable in a single-step conven-tional PCR to categorize five serogroups of Salmonella enterica subsp enterica into three classes according to amplicon lengths (400, 800, and 900 bp, respectively). Nucleotide sequences of the amplicons were those of flanking regions rfbH and rfbJ. No amplicon was generated in other bacterial genera examined, indicative of the high specificity of this PCR primer pair. As more genetic information becomes available, the smaller number of primer pairs will be required in multiplex-PCR for differentiating Salmonella microorganisms using the novel primer design method.
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Four new primers and one published primer were used to PCR amplify hypervariable regions within the protozoal 18S rRNA gene to determine which primer pair provided the best identification and statistical analysis. PCR amplicons of 394 to 498 bases were generated from three primer sets, sequenced using Roche 454 pyrosequencing with Titanium, and analyzed using the BLAST database (NCBI) and MOTHUR version 1.29. The protozoal diversity of rumen contents from moose in Alaska was assessed. In the present study, primer set 1, P-SSU-316F and GIC758R (amplicon of 482 bases), gave the best representation of diversity using BLAST classification, and the set amplified Entodinium simplex and Ostracodinium spp., which were not amplified by the other two primer sets. Primer set 2, GIC1080F and GIC1578R (amplicon of 498 bases), had similar BLAST results and a slightly higher percentage of sequences that were identified with a higher sequence identity. Primer sets 1 and 2 are recommended for use in ruminants. However, primer set 1 may be inadequate to determine protozoal diversity in nonruminants. The amplicons created by primer set 1 were indistinguishable for certain species within the genera Bandia, Blepharocorys, Polycosta, and Tetratoxum and between Hemiprorodon gymnoprosthium and Prorodonopsis coli, none of which are normally found in the rumen.
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In forensic DNA analysis, the samples recovered from the crime scene are often highly degraded leading to poor PCR amplification of the larger sized STR loci. To avoid this problem, we have developed STR markers with redesigned primer sequences called "Miniplexes" to produce smaller amplicons. To assess the effectiveness of these kits, we have tested these primer sets with enzymatically degraded DNA and compared the amplifications to a commercial kit. We also conducted sensitivity and peak balance studies of three Miniplex sets. Lastly, we report a case study on two human skeletal remain samples collected from different environmental conditions. In both types of degraded DNA, the Miniplex primer sets were capable of producing more complete profiles when compared to the larger sized amplicons from the commercial kit. Correct genotypes were obtained at template concentrations as low as 31 pg/25 microL. Overall, our data confirm that our redesigned primers can increase the probability of obtaining a usable profile in situations where standard kits fail.
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Inflammatory myofibroblastic tumors (IMTs) are rare tumors characterized as low-to-intermediate grade sarcomas. Rearrangements of the anaplastic lymphoma kinase (ALK) gene have been reported in IMT. Here, we describe a novel fusion gene in an IMT tumor specimen. A 12-year-old male was admitted to our hospital with a bladder tumor. We identified the fibronectin 1 gene (FN1) as a fusion partner of ALK using 5'RACE. This novel fusion, FN1-ALK, resulted in ALK overexpression in the IMT. This finding should clarify the causes of IMT and facilitate development of novel therapeutics.
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Abstract Since December 2019, the coronavirus disease 2019 (COVID-19) caused by a novel coronavirus SARS-CoV-2 has rapidly spread to almost every nation in the world. Soon after the pandemic was recognized by epidemiologists, a group of biologists comprising the ARTIC Network, has devised a multiplexed polymerase chain reaction (PCR) protocol and primer set for targeted whole-genome amplification of SARS-CoV-2. The ARTIC primer set amplifies 98 amplicons, which are separated only in two PCRs, across a nearly entire viral genome. The original primer set and protocol showed a fairly small amplification bias when clinical samples with relatively high viral loads were used. However, when sample’s viral load was low, several amplicons, especially amplicons 18 and 76, exhibited low coverage or complete dropout. We have determined that these dropouts were due to a dimer formation between the forward primer for amplicon 18, 18_LEFT, and the reverse primer for amplicon 76, 76_RIGHT. Replacement of 76_RIGHT with an alternatively designed primer was sufficient to produce a drastic improvement in coverage of both amplicons. Based on this result, we replaced 12 primers in total in the ARTIC primer set that were predicted to be involved in 14 primer interactions. The resulting primer set, version N1 (NIID-1), exhibits improved overall coverage compared to the ARTIC Network’s original (V1) and modified (V3) primer set.
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The study was undertaken with an objective to evaluate a rapid PCR based method for detection of adulteration of buffalo milk in cow milk at minimum level of detection. This method utilizes primers targeting the mitochondrial encoded 12S rRNA gene as the target for species identification. PCR assay involve use of three different primers. Reverse primers specific for cow and buffalo complementary to the gene fragment of 12S rRNA along with the common forward primer. The cow specific primer, along with the common forward primer, yields a cow specific amplicon of 346 bp in the 12S rRNA gene. On the other hand, a buffalo specific primer along with the same common forward primer yields a buffalo specific amplicon of 220 bp fragment in the same gene. The method evaluated was able to detect presence of buffalo milk in cow at 0.5% level of adulteration.
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Abstract Since December 2019, the coronavirus disease 2019 (COVID-19) caused by a novel coronavirus SARS-CoV-2 has rapidly spread to almost every nation in the world. Soon after the pandemic was recognized by epidemiologists, a group of biologists comprising the ARTIC Network, has devised a multiplexed polymerase chain reaction (PCR) protocol and primer set for targeted whole-genome amplification of SARS-CoV-2. The ARTIC primer set amplifies 98 amplicons, which are separated only in two PCRs, across a nearly entire viral genome. The original primer set and protocol showed a fairly small amplification bias when clinical samples with relatively high viral loads were used. However, when sample’s viral load was low, several amplicons, especially amplicons 18 and 76, exhibited low coverage or complete dropout. We have determined that these dropouts were due to a dimer formation between the forward primer for amplicon 18, 18_LEFT, and the reverse primer for amplicon 76, 76_RIGHT. Replacement of 76_RIGHT with an alternatively designed primer was sufficient to produce a drastic improvement in coverage of both amplicons. Based on this result, we replaced 12 primers in total in the ARTIC primer set that were predicted to be involved in 14 primer interactions. The resulting primer set, version N1 (NIID-1), exhibits improved overall coverage compared to the ARTIC Network’s original (V1) and modified (V3) primer set.
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