Low-cost PCR Research Articles

Direct comparison of an ultrasensitive real-time PCR assay with droplet digital PCR for the detection of PIK3CA hotspot mutations in primary tumors, plasma cell-free DNA and paired CTC-derived gDNAs

IntroductionDetection of PIK3CA mutations in primary tumors and liquid biopsy samples is of increasing importance for treatment decisions and therapy resistance in many types of cancer. The aim of the present study was to directly compare the efficacy of a relatively inexpensive ultrasensitive real-time PCR with the well-established and highly sensitive technology of ddPCR for the detection of the three most common hotspot mutations of PIK3CA, in exons 9 and 20, that are all of clinical importance in various types of cancer.Patients and methodsWe analyzed 42 gDNAs from primary tumors (FFPEs), 29 plasma-cfDNA samples, and 29 paired CTC-derived gDNAs, all from patients with ER+ metastatic breast cancer, and plasma from 10 healthy donors. The same blood draws were used for CTC isolation using EpCAM beads for positive immunomagnetic enrichment. All FFPEs and plasma-cfDNA samples were analyzed in parallel for PIK3CA mutations by ultrasensitive real-time PCR assay and droplet digital PCR.ResultsIn gDNAs from FFPEs, using ultrasensitive real-time PCR, the p.E545K mutation was detected in 22/42(52.4%), and the p.E542K and p.H1047R mutations were detected in 14/42(33.3%) and 16/42(38.1%), respectively. Using ddPCR, the p.E545K mutation was detected in 22/42(52.4%), p.E542K in 17/42(40.5%), and p.H1047R in 19/42(45.2%) samples, revealing a concordance between the two methodologies of 81%, 78.6% and 78.6% for each mutation respectively. In plasma-cfDNA, using ultrasensitive real-time PCR, the p.E545K mutation was detected in 11/29(38%) and both p.E542K and p.H1047R mutations in 2/29(6.9%).In the same plasma-cfDNA samples using ddPCR, p.E545K was detected in 1/29(3.5%), p.E542K in 2/29(6.9%), and p.H1047R in 3/29(10.5%) samples, revealing a concordance of 65.5%,100% and 93.1% for each mutation respectively. In paired CTC-derived gDNAs p.E545K was detected in 11/29(38%), p.E542K in 3/29(10.3%), and p.H1047R in 7/29(24.1%) samples.ConclusionsThis low-cost, high-throughput and ultrasensitive real-time PCR assay provides accurate and specific detection of PIK3CA hotspot mutations in liquid biopsy samples and gives similar results to ddPCR. This assay can be performed in labs where digital PCR instrumentation is not available. In CTC-derived gDNA and paired plasma-cfDNA, PIK3CA mutations detected were not identical, revealing that CTC and plasma-cfDNA give complementary information.

Low-cost and simple PCR process for access to molecular diagnosis of HTLV-1/2 in low-resource countries

BackgroundHTLV-1/2 exhibit a widespread distribution globally and are associated with severe clinical manifestations, necessitating precise viral identification for diagnosis. Currently, there are no official diagnostic guidelines, and a variety of published protocols exists. We introduce an enhanced nested real-time PCR technique followed by high-resolution melting (rtPCR-HRM), designed to offer a cost-effective and straightforward tool for the simultaneous identification of both viruses. MethodsThe technique was tested in a retrospective, blinded study, involving a total panel of 110 samples, of which 47 were positive for HTLV-1, 12 for HTLV-2, and 51 tested negatives. Additionally, we compared the performance of this technique with a line immunoassay (LIA). ResultsThe results demonstrate 100 % sensitivity, specificity, and diagnostic accuracy for both viruses. Sensitivity analysis indicated that at least 1 viral copy of HTLV-1 and 14.4 viral copies of HTLV-2 could be reliably detected. ConclusionsOur results indicate that rtPCR-HRM is effective in confirming HTLV-1 and HTLV-2 infection, important in Latin American countries where both viruses circulate. Furthermore, the proposed strategy provides a new tool that can be used to resolve indeterminate cases identified by Western blot, with the added advantage of being faster and simpler than n-PCR and more cost-effective than other probe-based RT-PCRs.

Microsatellite Instability Detection in Cancer: A Multiplex qPCR Approach that Obviates the Need for Matching Normal Samples.

Microsatellite instability (MSI) indicates DNA mismatch repair deficiency in certain types of cancer, such as colorectal cancer. The current gold standard technique, PCR-capillary electrophoresis (CE), requires matching normal samples and specialized instrumentation. We developed VarTrace, a rapid and low-cost quantitative PCR (qPCR) assay, to evaluate MSI using solely the tumor sample DNA, obviating the requirement for matching normal samples. One hundred and one formalin-fixed paraffin-embedded (FFPE) tumor samples were tested using VarTrace and compared with the Promega OncoMate assay utilizing PCR-CE. Tumor percentage limit of detection was evaluated on contrived samples derived from clinical high MSI (MSI-H) samples. Analytical sensitivity, specificity, limit of detection, and input requirements were assessed using synthetic commercial reference standards. VarTrace successfully analyzed all 101 clinical FFPE samples, demonstrating 100% sensitivity and 98% specificity compared to OncoMate. It detected MSI-H with 97% accuracy down to 10% tumor. Analytical studies using synthetic samples showed a limit of detection of 5% variant allele frequency and a limit of input of 0.5 ng. This study validates VarTrace as a swift, accurate, and economical assay for MSI detection in samples with low tumor percentages without the need for matching normal DNA. VarTrace's capacity for highly sensitive MSI analysis holds potential for enhancing the efficiency of clinical work flows and broadening the availability of this test.

Genótipos de Circovírus Suíno Tipo 2 e PCV3 em Amostras Clínicas de Suínos no Brasil

Abstract Porcine circovirus type 2 (PCV2), an important pathogen in swine, causes PCV disease (PCVD). Although PCVD is effectively controlled using commercial vaccines, its clinical presentation is changing. Moreover, PCV2 is genetically evolving, with new genotypes emerging in vaccinated or unvaccinated pigs. In this study, we aimed to verify the presence of the PCV2a, PCV2b, and PCV2d genotypes in PCV-positive porcine samples. Furthermore, to identify coinfections between the PCV2 genotypes and/or PCV3, which can also induce disease in pigs, we employed a quick, effective, and low-cost PCR diagnostic test. In this study, 333 PCV2 PCR and clinically positive samples from various production stages and herds across Brazil were analyzed. Among these, 266 samples were genotyped, with PCV2b emerging as the most predominant genotype (56.77% of the positive samples), mainly observed in nursery pigs. PCV2d was also identified in 33.10% of the samples, primarily from finishing pigs and breeding sows. The employed PCR test was compared with a commercial kit, proving effective in PCV2 genotyping. This study demonstrates the significance of PCV2 genotyping, showing PCV2b as the most predominant genotype responsible for disease in pig farms in Brazil. PCV2a, the prevalent genotype used in commercial vaccines, was not detected in any of the analyzed samples. While pigs infected with other PCV2 genotypes may receive some heterologous protection from PCV2a vaccines, adequate diagnosis and vaccine monitoring for updates must be considered.

Development of a disposable and easy-to-fabricate microfluidic PCR device for DNA amplification

The production of microfluidic PCR devices within lab settings in a straightforward process and large numbers is limited due to complex methods of fabrication and high cost of development. In this study, we developed a unique disposable, low-cost, and easy-to-fabricate microfluidic PCR chip, comprised of a passive micromixer, reaction well, microheater, and a dynamic temperature control system. In addition to being compact, this device has the advantages of microfluidic PCR systems, such as low power and sample consumption, as well as being disposable and easy to fabricate. The disposable part of the chip is easy to fabricate and eliminates the run-to-run carryover contamination while its integration with a reusable heating system reduces the cost of producing a micro PCR device. The geometry of the micromixer and the pattern of microheater electrodes are analyzed through experiments to achieve comparable amplification to a conventional thermocycler. The temperature control system is designed to monitor the temperature within the reaction well with ±1 °C precision. Finally, the reliability and repeatability of the microfluidic device were verified by amplifying lambda (λ) DNA. The proposed device demonstrated high-quality amplification comparable to the conventional PCR and is an adaptable and low-cost on-chip PCR device for reliable molecular diagnostics.

Rapid PCR-lateral flow assay for the onsite detection of Atlantic white shrimp

The Atlantic white shrimp (Litopenaeus setiferus) is of great economic importance to the United States and risk being substituted with imported species due to a shortage in domestic production. To improve the current methods used for the identification of the Atlantic white shrimp species, we designed and validated a robust multiplex PCR-lateral flow assay for the onsite identification of L. setiferus. The standardized assay was validated using a miniaturized, low-cost PCR instrument with 68 shrimp, prawn, and fish samples, spread over fourteen seafood species. L. setiferus was simultaneously amplified by the multiplex assay to give three visual bands, which distinguished it from other species having either one or two bands on the dipstick. The standardized assay showed 100% inclusivity for target L. setiferus samples, 100% exclusivity for non-target samples and can be completed in less than two hours. The assay standardized in this study can be used for onsite testing of L. setiferus samples at processing facilities, restaurants, and wholesalers' facilities.

A Disposable Electromagnetic Bi-Directional Micropump Utilizing a Rotating Multi-Pole Ring Magnetic Coupling.

Electromagnetic bi-directional micropumps (EMBM) are indispensable for the development of portable devices which enable fluid transportation in forward and reverse directions. However, the high disposal cost of rare-earth magnets attached to the fluidic part and the low pump density due to multiple motors limit their practical application in disposable multi-channel microfluidic applications such as droplet-based oscillatory-flow (DBOF) rapid PCR amplification. Therefore, this paper presented a low-cost, disposable, high-pump-density EMBM. To reduce the disposal cost, we separated the magnets from the disposable fluidic part and used cylindrical holes to store and guide the magnet, which resulted in the ability to reuse all the magnets. To increase the pump density, we used the combination of one motor and one multi-pole ring magnet to drive several channels simultaneously. A proof-of-concept prototype with a pump density of 0.28 cm−2 was fabricated and experimentally evaluated. The fabricated micropump exhibited a maximum flow rate of 0.86 mL/min and a maximum backpressure of 0.5 kPa at a resonant frequency around 50 Hz. The developed multi-channel micropump with reusable magnets is highly beneficial to the development of low-cost and high-throughput rapid PCR amplification microchips and therefore can have a significant impact on timely infectious disease recognition and intervention.

Development of a low-cost multi-channel nucleic acid detection PCR instrument and clinical detection application of COVID-19

Since the COVID-19 outbreak at the end of December 2019, a variety of novel Coronavirus nucleic acid detection methods have been proposed at home and abroad. Because of the disadvantages of most existing PCR instruments on the market such as long reaction time and high cost, this study developed a more timesaving and cheaper two-channel real-time quantitative PCR instrument. In this instrument, a PCR system combining a thermal cycle system and real-time fluorescence quantitative technology was designed. The software system and data processing, optical system, thermal cycle module, and hardware module of the PCR instrument were studied. The low-cost, portable real-time quantitative PCR system has been validated with consistent results compared to Bio-rad CFX Connect. At the same time, the same samples were used for the contract experiment with the hospital instrument, and the amplification result was better than the existing instrument in the hospital.

An integrated digital PCR system with high universality and low cost for nucleic acid detection.

Digital PCR is the most advanced PCR technology. However, due to the high price of the digital PCR analysis instrument, this powerful nucleic acid detection technology is still difficult to be popularized in the general biochemistry laboratory. Moreover, one of the biggest disadvantages of commercial digital PCR systems is the poor versatility of reagents: each instrument can only be used for a few customized kits. Herein, we built a low-cost digital PCR system. The system only relies on low-cost traditional flat-panel PCR equipment to provide temperature conditions for commercial dPCR chips, and the self-made fluorescence detection system is designed and optically optimized to meet a wide range of reagent requirements. More importantly, our system not only has a low cost (<8000 US dollars) but also has a much higher universality for nucleic acid detection reagents than the traditional commercial digital PCR system. In this study, several samples were tested. The genes used in the experiment were plasmids containing UPE-1a fragment, TP53 reference DNA, hepatitis B virus DNA, leukemia sample, SARS-COV-2 DNA, and SARS-COV-2 RNA. Under the condition that DNA can be amplified normally, the function of the dPCR system can be realized with simpler and low-price equipment. Some DNA cannot be detected by using the commercial dPCR system because of the special formula when it is configured as the reaction solution, but these DNA fluorescence signals can be clearly detected by our system, and the concentration can be calculated. Our system is more applicable than the commercial dPCR system to form a new dPCR system that is smaller and more widely applicable than commercially available machinery.

An in house qPCR System to Evaluate Transcripts Stimulated by an Immune Innate Agonist (CIGB2020) during the Covid19 Pandemic Scenario

Background: A low cost and simple real-time PCR method to quantify different transcripts in human samples is a need in most developing countries. A quantitative real-time PCR assay to evaluate the relative RNA expression stimulation of specific transcripts during the Pandemic COVID19 scenario was developed at the Center for Genetic Engineering and Biotechnology in Havana, using a commercial amplification system with standards and specific primers that are produced in house. Methods: Oro-pharyngeal scrape samples from patients that were treated previously with an innate immune agonist, the CIGB2020, were employed to RNA purification, cDNA synthesis, and evaluation of different gene transcripts by the in house qPCR assay using different standard curves constructed with specific primers that amplify a pool of the own evaluated transcripts. Results: Preliminary results showed a typical standard curve from the in house qPCR system developed. A typical standard curve during the evaluation of the TLR3 gene transcript was described as example, and was calculated automatically by plotting the Ct values against each standard of known concentration, and the linear regression of this curve was also calculated as: M=-3.31641, B=42.73084, R=0.9516. It was also observed the influence of the CIGB2020 on the stimulation of the three toll like receptors from the innate immune system. Conclusions: This study demonstrated that the in house qPCR system is useful to quantify the relative induction of the expression of several genes transcripts considered as immunological markers of the immune innate system

Statistical meta-analysis to investigate the association between the Interleukin-6 (IL-6) gene polymorphisms and cancer risk.

A good number of genome-wide association studies (GWAS), including meta-analyses, reported that single nucleotide polymorphisms (SNPs) of the IL-6 gene are significantly associated with various types of cancer risks, though some other studies reported insignificant association with cancers, in the literature. These contradictory results may be due to variations in sample sizes and/or deficiency of statistical modeling. Therefore, an attempt is made to provide a more comprehensive understanding of the association between the IL-6 gene SNPs (rs1800795, rs1800796, rs1800797) and different cancer risks, giving the weight on a large sample size, including different cancer types and appropriate statistical modeling with the meta-dataset. In order to attain a more reliable consensus decision about the association between the IL-6 gene polymorphisms and different cancer risks, in this study, we performed a multi-case statistical meta-analysis based on the collected information of 118 GWAS studies comprising of 50053 cases and 65204 control samples. Results from this Meta-analysis indicated a significant association (p-value < 0.05) of the IL-6 gene rs1800796 polymorphism with an overall increased cancer risk. The subgroup analysis data based on cancer types exhibited significant association (p-value < 0.05) of the rs1800795 polymorphism with an overall increased risk of cervical, liver and prostate cancers; the rs1800796 polymorphism with lung, prostate and stomach cancers; and the rs1800797 polymorphism with cervical cancer. The subgroup analysis of ethnicity data showed a significant association (p-value < 0.05) of an overall cancer risk with the rs1800795 polymorphism for the African and Asian populations, the rs1800796 polymorphism for the Asian only and the rs1800797 polymorphism in the African population. Comparative discussion showed that our multi-case meta-analyses received more support than any previously reported individual meta-analysis about the association between the IL-6 gene polymorphisms and cancer risks. Results from this study, more confidently showed that the IL-6 gene SNPs (rs1800795, rs1800796 and rs1800797) in humans are associated with increased cancer risks. Therefore, these three polymorphisms of the IL-6 gene have the potential to be evaluated as a population based rapid, low-cost PCR prognostic biomarkers for different types of cancers diagnosis and research.

A multi-volume microfluidic device with no reagent loss for low-cost digital PCR application

A multi-volume multi-level vertical branching microchannel microfludic chip is presented in this study for low-cost and easy-operation digital PCR (dPCR). The chip with multi-volume design can achieve a dynamic range of over 104 for nucleic acid quantification with only 1792 chambers, which consist of two types of chambers with different volumes and quantities. Meanwhile, the dPCR chip realizes 100 % sample compartmentalization without any loss. In addition, the chip can partition samples autonomously in a simple way through negative pressure provided by a degassed PDMS layer. Furthermore, a glass-PDMS-glass “sandwich” structure was employed in the chip to form a robust support. The quantitative capability of the digital PCR chip is evaluated by measuring a 10-fold serial dilution of the KRAS plasmid template. Owing to its characteristics of easy operation process, low cost, relative wide dynamic range, and no sample loss, the proposed dPCR chip is expected to further promote the extensive application of digital PCR, especially in the POCT field.

Fast and robust sample self-digitization for digital PCR

We present a facile sample partitioning method to enable rapid and low-cost digital PCR (dPCR) assays. By subdividing a high percentage of the sample volume into a large number of equal volume compartments with a self-digitization (SD) chip, this method can achieve a low-waste and high-order sample discretization in a matter of minutes. The SD chip contains a set of parallel microfluidic channels used for sample delivery, and each channel is connected with two rows of cylindrical wells to hold the discretized sample. By utilizing a degassed PDMS sealing slab as a detachable vacuum pumping source, the SD chip automatically generate large arrays of small sample volumes without requirement of external pumping and valving components. Unlike most microfluidic chamber-based methods for sample discretization, our detachable SD chip allows for discretizing sample with air flushing, then peeling off the cover PDMS slab and sealing the digitized samples with oil layer. Due to obviation of time-consuming oil flushing, such microfluidic device can achieve much faster digitization of sample volumes. Furthermore, this digitization chip can partition more than 90% of a sample volume, which is important for the applications where the amount of material available is small. We also demonstrated the utility of the proposed SD chip by applying it to a dPCR assay.

Lack of detection of Candida nivariensis and Candida bracarensis among 440 clinical Candida glabrata sensu lato isolates in Kuwait.

Occurrence of Candida nivariensis and Candida bracarensis, two species phenotypically similar to Candida glabrata sensu stricto, in human clinical samples from different geographical settings remains unknown. This study developed a low-cost multiplex PCR (mPCR) and three species-specific singleplex PCR assays. Reference strains of common Candida species were used during development and the performance of mPCR and singleplex PCR assays was evaluated with 440 clinical C. glabrata sensu lato isolates. The internal transcribed spacer (ITS) region of rDNA was also sequenced from 85 selected isolates and rDNA sequence variations were used for determining genetic relatedness among the isolates by using MEGA X software. Species-specific amplicons for C. glabrata (~360 bp), C. nivariensis (~250 bp) and C. bracarensis (~180 bp) were obtained in mPCR while no amplicon was obtained from other Candida species. The three singleplex PCR assays also yielded expected results with reference strains of Candida species. The mPCR amplified ~360 bp amplicon from all 440 C. glabrata sensu lato isolates thus identifying all clinical isolates in Kuwait as C. glabrata sensu stricto. The results of mPCR were confirmed for all 440 isolates as they yielded an amplicon only in C. glabrata sensu stricto-specific singleplex PCR assay. The rDNA sequence data identified 28 ITS haplotypes among 85 isolates with 18 isolates belonging to unique haplotypes and 67 isolates belonging to 10 cluster haplotypes. In conclusion, we have developed a simple, low-cost mPCR assay for rapid differentiation of C. glabrata sensu stricto from C. nivariensis and C. bracarensis. Our data obtained from a large collection of clinical C. glabrata sensu lato isolates show that C. nivariensis and C. bracarensis are rare pathogens in Kuwait. Considerable genetic diversity among C. glabrata sensu stricto isolates was also indicated by rDNA sequence analyses.

A self-digitization chip integrated with hydration layer for low-cost and robust digital PCR

We present a self-discretization and zero-water-loss microfluidic digital PCR (dPCR) device to enable low-cost and robust quantitative nucleic acid assays. In this device, a thin void is integrated beneath the reaction chamber array. By utilizing the permeability of polydimethylsiloxane (PDMS) film, the integrated void serves a dual function: vacuum “accumulator” and hydration “reservoir”. The combination of pre-stored pumping energy and water compensation for evaporation loss enables simple, robust and reliable single-DNA-molecule amplification and detection in 10,000 reactors of picoliter volume. Compared to the conventional degassing PDMS pumps, the vacuum accumulator exhibits superior performance due to more vacuum storage and shorter diffusion distance. We also evaluated the performance of the embedded hydration layer in suppressing evaporation loss at elevated temperatures, and verified that zero-water-loss could be achieved for all reaction chambers in our dPCR chip during thermal cycling. By performing quantitative detection of T790M DNA from 10 to 104 copies/μL, the proposed dPCR chip demonstrated high accuracy and excellent performance for the absolute quantification of the target gene with a dynamic range of 104. The simplicity and robustness of our dPCR chip make it well suited for low-cost molecular diagnostic assays under resource-limited settings.

Digital polymerase chain reaction technology - recent advances and future perspectives.

Digital polymerase chain reaction (dPCR) technology has remained a "hot topic" in the last two decades due to its potential applications in cell biology, genetic engineering, and medical diagnostics. Various advanced techniques have been reported on sample dispersion, thermal cycling and output monitoring of digital PCR. However, a fully automated, low-cost and handheld digital PCR platform has not been reported in the literature. This paper attempts to critically evaluate the recent developments in techniques for sample dispersion, thermal cycling and output evaluation for dPCR. The techniques are discussed in terms of hardware simplicity, portability, cost-effectiveness and suitability for automation. The present paper also discusses the research gaps observed in each step of dPCR and concludes with possible improvements toward portable, low-cost and automatic digital PCR systems.

Reduced-cost Chlamydia trachomatis-specific multiplex real-time PCR diagnostic assay evaluated for ocular swabs and use by trachoma research programmes

IntroductionTrachoma, caused by the intracellular bacterium Chlamydia trachomatis (Ct), is the leading infectious cause of preventable blindness. Many commercial platforms are available that provide highly sensitive and specific detection of Ct DNA. However, the majority of these commercial platforms are inaccessible for population-level surveys in resource-limited settings typical to trachoma control programmes. We developed two low-cost quantitative PCR (qPCR) tests for Ct using readily available reagents on standard real-time thermocyclers. MethodsEach multiplex qPCR test targets one genomic and one plasmid Ct target in addition to an endogenous positive control for Homo sapiens DNA. The quantitative performance of the qPCR assays in clinical samples was determined by comparison to a previously evaluated droplet digital PCR (ddPCR) test. The diagnostic performance of the qPCR assays were evaluated against a commercial assay (artus C. trachomatis Plus RG PCR, Qiagen) using molecular diagnostics quality control standards and clinical samples. We examined the yield of Ct DNA prepared from five different DNA extraction kits and a cold chain-free dry-sample preservation method using swabs spiked with fixed concentrations of human and Ct DNA. ResultsThe qPCR assay was highly reproducible (Ct plasmid and genomic targets mean total coefficients of variance 41.5% and 48.3%, respectively). The assay detected 8/8 core specimens upon testing of a quality control panel and performed well in comparison to commercially marketed comparator test (sensitivity and specificity>90%). Optimal extraction and sample preservation methods for research applications were identified. ConclusionWe describe a pipeline from collection to diagnosis providing the most efficient sample preservation and extraction with significant per test cost savings over a commercial qPCR diagnostic assay. The assay and its evaluation should allow control programs wishing to conduct independent research within the context of trachoma control, access to an affordable test with defined performance characteristics.

A microfluidic chip based on surfactant-doped polydimethylsiloxane (PDMS) in a sandwich configuration for low-cost and robust digital PCR

Digital PCR (dPCR) is a powerful method for the absolute quantification and rare mutation detection of nucleic acids. However, the available dPCR platforms require either expensive instruments or complicated operation, which limit their extensive application. This paper describes a robust self-partition chip to perform dPCR in a very simple and inexpensive format. The chip is constructed by sandwiching a micromolded surfactant-doped PDMS layer between two glass slides, containing 10,000 reaction compartments (0.785nL each). The two simple strategies, “glass-PDMS-glass” sandwich configuration and surfactant-doped surface modification, are used to enhance PCR compatibility and enable efficient amplification in the chip by minimizing water loss and nonspecific adsorption of protein. Furthermore, powered by pre-degassing of its PDMS substrate and an attached PDMS slab, the chip autonomously loads and partitions sample into a high-density array of microwells for performing dPCR analysis. We evaluated the quantitative capabilities of the dPCR chip by measuring a ten-fold serial dilution of genomic DNA from lung cancer cell line H1975. Finally, we also demonstrate its feasibility in the accurate measurement of small copy number variations, which exhibited better accuracy than real-time quantitative PCR (qPCR). Such simple and low-cost platform promises to accelerate the widespread use of dPCR.

PELMO, an optimised in-house cloning vector

DNA cloning is an essential tool regarding DNA recombinant technology as it allows the replication of foreign DNA fragments within a cell. pELMO was here constructed as an in-house cloning vector for rapid and low-cost PCR product propagation; it is an optimally designed vector containing the ccdB killer gene from the pDONR 221 plasmid, cloned into the pUC18 vector’s multiple cloning site (Thermo Scientific). The ccdB killer gene has a cleavage site (CCC/GGG) for the SmaI restriction enzyme which is used for vector linearisation and cloning blunt-ended products. pELMO transformation efficiency was evaluated with different sized inserts and its cloning efficiency was compared to that of the pGEM-T Easy commercial vector. The highest pELMO transformation efficiency was observed for ~500 bp DNA fragments; pELMO vector had higher cloning efficiency for all insert sizes tested. In-house and commercial vector cloned insert reads after sequencing were similar thus highlighting that sequencing primers were designed and localised appropriately. pELMO is thus proposed as a practical alternative for in-house cloning of PCR products in molecular biology laboratories.

CNV-RF Is a Random Forest–Based Copy Number Variation Detection Method Using Next-Generation Sequencing

Simultaneous detection of small copy number variations (CNVs) (<0.5 kb) and single-nucleotide variants in clinically significant genes is of great interest for clinical laboratories. The analytical variability in next-generation sequencing (NGS) and artifacts in coverage data because of issues with mappability along with lack of robust bioinformatics tools for CNV detection have limited the utility of targeted NGS data to identify CNVs. We describe the development and implementation of a bioinformatics algorithm, copy number variation-random forest (CNV-RF), that incorporates a machine learning component to identify CNVs from targeted NGS data. Using CNV-RF, we identified 12 of 13 deletions in samples with known CNVs, two cases with duplications, and identified novel deletions in 22 additional cases. Furthermore, no CNVs were identified among 60 genes in 14 cases with normal copy number and no CNVs were identified in another 104 patients with clinical suspicion of CNVs. All positive deletions and duplications were confirmed using a quantitative PCR method. CNV-RF also detected heterozygous deletions and duplications with a specificity of 50% across 4813 genes. The ability of CNV-RF to detect clinically relevant CNVs with a high degree of sensitivity along with confirmation using a low-cost quantitative PCR method provides a framework for providing comprehensive NGS-based CNV/single-nucleotide variant detection in a clinical molecular diagnostics laboratory.