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Rapid and zero-cost DNA extraction from soft-bodied insects for routine PCR-based applications.

ABSTRACT: Nucleic acid extraction is the first and foremost step in molecular biology studies. Extraction of DNA from small, soft-bodied insects is often time-consuming and costly. A fast, easy, and cost-effective DNA extraction method with greater yield and purity of DNA would aid in the rapid diagnostics, screening of large populations, and other routine PCR-based applications. The present study evaluated and standardized a rapid and zero-cost DNA extraction from soft-bodied small insects for routine molecular studies. Five rapid DNA extraction methods viz. extraction in sterile distilled water (SDW), 1X phosphate-buffered saline (PBS, pH 7.4), 1.4 M sodium chloride (NaCl), 20 mM ethylenediaminetetraacetic acid (EDTA, pH 8.0), and elution from blotted nitrocellulose membrane (NCM) were compared with standard CTAB extraction buffer and DNeasy® Blood and Tissue Kit methods. The average yield, purity, storage stability, time, and cost of extraction were assessed for all the methods and compared. A method of DNA extraction by simply crushing the soft-bodied insects in SDW was ideal in terms of yield, purity, storability, and performing routine PCR-based applications including detection of pathogens from vector species. The extraction could be accomplished in 2.5 min only with zero-reagent cost. The method would be useful in rapid molecular diagnostics and screening large populations of soft-bodied insects.

SUBMITTER: Jangra S

PROVIDER: S-EPMC9286237 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Rapid and zero-cost DNA extraction from soft-bodied insects for routine PCR-based applications.

Jangra Sumit S Ghosh Amalendu A

PloS one 20220715 7

Nucleic acid extraction is the first and foremost step in molecular biology studies. Extraction of DNA from small, soft-bodied insects is often time-consuming and costly. A fast, easy, and cost-effective DNA extraction method with greater yield and purity of DNA would aid in the rapid diagnostics, screening of large populations, and other routine PCR-based applications. The present study evaluated and standardized a rapid and zero-cost DNA extraction from soft-bodied small insects for routine mo ...[more]

PMID: 35839213

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Project description:Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (Ct) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98-100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score genotypes. ASQ is cost-effective because universal fluorescent probes negate the necessity of designing expensive probes for each locus.

Project description:BackgroundMany modern molecular diagnostic assays targeting nucleic acids are typically confined to developed countries or to the national reference laboratories of developing-world countries. The ability to make technologies for the rapid diagnosis of infectious diseases broadly available in a portable, low-cost format would mark a revolutionary step forward in global health. Many molecular assays are also developed based on polymerase chain reactions (PCR), which require thermal cyclers that are relatively heavy (>20 pounds) and need continuous electrical power. The temperature ramping speed of most economical thermal cyclers are relatively slow (2 to 3 °C/s) so a polymerase chain reaction can take 1 to 2 hours. Most of all, these thermal cyclers are still too expensive ($2k to $4k) for low-resource setting uses.Methodology/principal findingsIn this article, we demonstrate the development of a low-cost and rapid water bath based thermal cycler that does not require active temperature control or continuous power supply during PCR. This unit costs $130 to build using commercial off-the-shelf items. The use of two or three vacuum-insulated stainless-steel Thermos food jars containing heated water (for denaturation and annealing/extension steps) and a layer of oil on top of the water allow for significantly stabilized temperatures for PCR to take place. Using an Arduino-based microcontroller, we automate the "archaic" method of hand-transferring PCR tubes between water baths.Conclusions/significanceWe demonstrate that this innovative unit can deliver high speed PCR (17 s per PCR cycle) with the potential to go beyond the 1,522 bp long amplicons tested in this study and can amplify from templates down to at least 20 copies per reaction. The unit also accepts regular PCR tubes and glass capillary tubes. The PCR efficiency of our thermal cycler is not different from other commercial thermal cyclers. When combined with a rapid nucleic acid detection approach, the thermos thermal cycler (TTC) can enable on-site molecular diagnostics in low-resource settings.

Dataset Information

Rapid and zero-cost DNA extraction from soft-bodied insects for routine PCR-based applications.

Publications

Rapid and zero-cost DNA extraction from soft-bodied insects for routine PCR-based applications.

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