Template Sequence Research Articles

PyPCRtool: A python package for in silico PCR and primer verification

In Silico PCR is a computational technique used to predict PCR outcomes, improve primer specificity, and optimize experimental conditions prior to conducting laboratory work. Numerous web-based tools with pre-loaded genome templates have been developed for conducting In Silico PCR simulations. However, there is an increasing demand for a flexible, user-friendly package that allows users to upload or define their own custom template sequences and operates offline, ensuring data privacy and security during In Silico PCR simulations and primer verification. This paper introduces PyPCRtool, a Python package designed to perform In Silico PCR simulations and verify primer specificity. The tool aims to offer a flexible, user-friendly solution that handles data locally, facilitating the prediction of DNA fragment amplification and the visualization of PCR product bands through gel electrophoresis simulations. PyPCRtool allows users to input and specify template DNA sequence files, forward and reverse primer sequences and customize mismatch tolerances. An example scenario demonstrates PyPCRtool's functionality, showcasing its ability to predict PCR product formation and visualize gel electrophoresis results. The tool provides detailed outputs, including PCR product sequences, sizes, and binding site information, assisting in experimental planning and analysis. PyPCRtool offers a robust and versatile solution for In Silico PCR and primer verification. By integrating flexible Python-based operations with local data handling for privacy, it serves as an invaluable resource for students and researchers in molecular biology and biotechnology, enhancing the accuracy and efficiency of PCR experimental planning and result interpretation.

Effect of poly(dA) stretch in a template sequence on the rate and yield of polymerase chain reaction with electroactive modified 2′-deoxyuridine-5′-triphosphates

The effective incorporation of electroactive labeled nucleotides into DNA during its enzymatic amplification provides the ground for developing assays based on direct electrochemical detection of amplification products. Yet, whether the template sequence can affect the incorporation efficiency of modified nucleotides is still poorly understood. Here we examined the effects of a poly(dA) stretch in template sequence on the rate and yield of polymerase chain reaction (PCR) in the presence of modified 2′-deoxyuridine-5′-triphosphates (dUTP) in the reaction mixture. Four dUTP derivatives carrying 4-hydroxyphenyl (tyrosine; dUTP-Y1 and dUTP-Y2), 4-nitrophenyl (dUTP-N1), or indolyl (tryptophan; dUTP-W1) aromatic groups were tested as substrates for Taq and KTN (lacking 3′-5′ exonuclease activity) polymerases at 100 % substitution of 2′-deoxythymidine-5′-triphosphate (dTTP) in the PCR mixture. The 120 base pair long synthetic DNA templates contained poly(dA)·poly(dT) tracts of various length (dAn·dTn, where n = 2–10). In all cases, full-size amplicons have been obtained. Overall, in the presence of modified dUTP, the PCR yield was higher with KTN polymerase than that with Taq polymerase. The PCR yield slightly (for dUTP-Y1 and dUTP-Y2) or significantly (for dUTP-N1 and dUTP-W1) decreased with an increase of n in the dAn·dTn tract. In most cases, in the presence of modified dUTP, the PCR rate substantially decreased with n ≥ 4 in dAn·dTn. No changes in amplicon sequences due to potential incorrect nucleotide pairing during amplification with the complete substitution of dTTP with dUTP-Y2 were revealed. Electrochemical activity of tyrosine labeled PCR products dsDNA-Y2 differing in the length of stretches of modified nucleotides was similar. The findings demonstrate that the incorporation efficiency for base-modified nucleotides can in general depend on template sequence, in particular on the presence of long poly(dA) stretches when the modified dUTP are used. That has to be taken into account when developing electrochemical or other DNA assays with a specific modified nucleotide.

Blocking uncertain mispriming errors of PCR

The polymerase chain reaction (PCR) plays a central role in genetic engineering and is routinely used in various applications, from biological and medical research to the diagnosis of viral infections. PCR is an extremely sensitive method for detecting target DNA sequences, but it is substantially error prone. In particular, the mishybridization of primers to contaminating sequences can result in false positives for virus tests. The blocker method, also called the clamping method, has been developed to suppress mishybridization errors. However, its application is limited by the requirement that the contaminating template sequence be known in advance. Here, we demonstrate that a mixture of multiple blocker sequences effectively suppresses the amplification of contaminating sequences even in the presence of uncertainty. The blocking effect was characterized by a simple model validated by experiments. Furthermore, the modeling allowed us to minimize the errors by optimizing the blocker concentrations. The results highlighted an inherent robustness of the blocker method in that fine-tuning the blocker concentrations is not necessary. Our method extends the applicability of PCR and other hybridization-based techniques, including genome editing, RNA interference, and DNA nanotechnology, by improving their fidelity.

Quantitative Characterization of RCA-Based DNA Hydrogels - Towards Rational Materials Design.

DNA hydrogels hold significant promise for biomedical applications and can be synthesized through enzymatic Rolling Circle Amplification (RCA). Due to the exploratory nature of this emerging field, standardized RCA protocols specifying the impact of reaction parameters are currently lacking. This study varied template sequences and reagent concentrations, evaluating RCA synthesis efficiency and hydrogel mechanical properties through quantitative PCR (qPCR) and indentation measurements, respectively. Primer concentration and stabilizing additives showed minimal impact on RCA efficiency, while changes in polymerase and nucleotide concentrations had a stronger effect. Concentration of the circular template exerted the greatest influence on RCA productivity. An exponential correlation between hydrogel viscosity and DNA amplicon concentration was observed, with nucleobase sequence significantly affecting both amplification efficiency and material properties, particularly through secondary structures. This study suggests that combining high-throughput experimental methods with structural folding prediction offers a viable approach for systematically establishing structure-property relationships, aiding the rational design of DNA hydrogel material systems.

Leishmania major telomerase RNA knockout: From altered cell proliferation to decreased parasite infectivity

This study focuses on the biological impacts of deleting the telomerase RNA from Leishmania major (LeishTER), a parasite responsible for causing leishmaniases, for which no effective treatment or prevention is available. TER is a critical player in the telomerase ribonucleoprotein complex, containing the template sequence copied by the reverse transcriptase component during telomere elongation. The success of knocking out both LeishTER alleles was confirmed, and no off-targets were detected. LmTER−/− cells share similar characteristics with other TER-depleted eukaryotes, such as altered growth patterns and partial G0/G1 cell cycle arrest in early passages, telomere shortening, and elevated TERRA expression. They also exhibit increased γH2A phosphorylation, suggesting that the loss of LeishTER induces DNA damage signaling. Moreover, pro-survival autophagic signals and mitochondrion alterations were shown without any detectable plasma membrane modifications. LmTER−/− retained the ability to transform into metacyclics, but their infectivity capacity was compromised. Furthermore, the overexpression of LeishTER was also deleterious, inducing a dominant negative effect that led to telomere shortening and growth impairments. These findings highlight TER's vital role in parasite homeostasis, opening discussions about its potential as a drug target candidate against Leishmania.

Robust Sequence Design Space for the Isothermal Exponential Amplification of Short Oligonucleotides.

Advances in isothermal amplification techniques have accelerated development in biosensing applications and the design of complex molecular devices. The exponential amplification reaction technique, or EXPAR, is uniquely positioned to process molecular information from short oligonucleotide strands (≈10nucleotides length) typically encountered in molecular computing or microRNA detection. Despite its conceptual simplicity (requiring only a template strand and two enzymes), the issue of nonspecific background amplification has hindered broader adoption. In this work, a new system configuration is established at 37 °C to achieve significantly improved performance. Critical sequence motifs responsible for the excellent signal-to-background profile are identified and generalized as a universal adapter design framework. Orthogonal template sequences generated from the framework are implemented for a triplex reaction and successfully evaluated mixtures of multiple-target inputs in a single-step, one-pot format without the need for exogenous agents.

Ribosome-Free Translation up to Pentapeptides via Template Walk on RNA Sequences.

The origin of translation is one of the most difficult problems of molecular evolution. Identifying molecular systems that translate an RNA sequence into a peptide sequence in the absence of ribosomes and enzymes is a challenge. Recently, single-nucleotide translation via coupling of 5' phosphoramidate-linked amino acids to 2'/3'-aminoacyl transfer-NMPs, as directed by the sequence of an RNA template, was demonstrated for three of the four canonical nucleotides. How single-nucleotide translation could be expanded to include all four bases and to produce longer peptides without translocation along the template strand remained unclear. Using transfer strands of increasing length containing any of the four bases that interrogate adjacent positions along the template, we now show that pentapeptides can be produced in coupling reactions and subsequent hydrolytic release in situ. With 2'/3'-aminoacylated mono-, di-, tri- and tetranucleotides we thus show how efficient translation can be without biomacromolecules.

Ribosome‐Free Translation up to Pentapeptides via Template Walk on RNA Sequences

AbstractThe origin of translation is one of the most difficult problems of molecular evolution. Identifying molecular systems that translate an RNA sequence into a peptide sequence in the absence of ribosomes and enzymes is a challenge. Recently, single‐nucleotide translation via coupling of 5′ phosphoramidate‐linked amino acids to 2′/3′‐aminoacyl transfer‐NMPs, as directed by the sequence of an RNA template, was demonstrated for three of the four canonical nucleotides. How single‐nucleotide translation could be expanded to include all four bases and to produce longer peptides without translocation along the template strand remained unclear. Using transfer strands of increasing length containing any of the four bases that interrogate adjacent positions along the template, we now show that pentapeptides can be produced in coupling reactions and subsequent hydrolytic release in situ. With 2′/3′‐aminoacylated mono‐, di‐, tri‐ and tetranucleotides we thus show how efficient translation can be without biomacromolecules.

A bivariate fluorescence biosensor based on Janus DNA nanoarchitecture-loaded dual-emissive Ag nanoclusters as bi-responsive signaling reporters

Constructing label-free bivariate fluorescence biosensor would be intriguing and desired for the recognizable and accurate detection of two specific DNA segments, yet the design of functional DNA structures with low overlapped interference might be challenging. Herein in this work, a double-faced Janus DNA nanoarchitecture (JDNA) with bi-responsive recognition regions on opposite sides was assembled, which consisted of two substrate strands and two template strands for loading green-/red-emissive Ag nanoclusters (gAgNC and rAgNC) as bivariate signaling reporters. Of note, the hybridized double helix in the middle rationally oriented two flank faces and stabilized the rigid conformation of JDNA, while the template sequences of bicolor clusters were blocked to minimize non-specific background leakage. Upon inputting two targets, the discernible hairpins lost their hairpin structures due to forming two dsDNA complexes. They were executed to simultaneously invade JDNA for activating two individual target-recycled strand displacement (TRSD) events, guiding signal transduction and efficient amplification. Consequently, the clustering templates were unlocked via the tailored conformation switch of JDNA, in which gAgNC and rAgNC were in situ synthesized in two diagonal positions, thereby significantly emitting bi-responsive signal without cross interference. Benefited from the logic integration of double-faced JDNA and TRSD, a label-free, sensitive and specific bivariate fluorescence approach was developed, which would open a new avenue for the potential application in biosensing and bioanalysis.

Association of a rapidly selected 4.3kb transposon-containing structural variation with a P450-based resistance to pyrethroids in the African malaria vector Anopheles funestus.

Deciphering the evolutionary forces controlling insecticide resistance in malaria vectors remains a prerequisite to designing molecular tools to detect and assess resistance impact on control tools. Here, we demonstrate that a 4.3kb transposon-containing structural variation is associated with pyrethroid resistance in central/eastern African populations of the malaria vector Anopheles funestus. In this study, we analysed Pooled template sequencing data and direct sequencing to identify an insertion of 4.3kb containing a putative retro-transposon in the intergenic region of two P450s CYP6P5-CYP6P9b in mosquitoes of the malaria vector Anopheles funestus from Uganda. We then designed a PCR assay to track its spread temporally and regionally and decipher its role in insecticide resistance. The insertion originates in or near Uganda in East Africa, where it is fixed and has spread to high frequencies in the Central African nation of Cameroon but is still at low frequency in West Africa and absent in Southern Africa. A marked and rapid selection was observed with the 4.3kb-SV frequency increasing from 3% in 2014 to 98% in 2021 in Cameroon. A strong association was established between this SV and pyrethroid resistance in field populations and is reducing pyrethroid-only nets' efficacy. Genetic crosses and qRT-PCR revealed that this SV enhances the expression of CYP6P9a/b but not CYP6P5. Within this structural variant (SV), we identified putative binding sites for transcription factors associated with the regulation of detoxification genes. An inverse correlation was observed between the 4.3kb SV and malaria parasite infection, indicating that mosquitoes lacking the 4.3kb SV were more frequently infected compared to those possessing it. Our findings highlight the underexplored role and rapid spread of SVs in the evolution of insecticide resistance and provide additional tools for molecular surveillance of insecticide resistance.

Search for genes gained by horizontal gene transfer in an entomopoxvirus, with special reference to the analysis of the transfer of an ABC transporter gene

Although it is generally believed that large DNA viruses capture genes by horizontal gene transfer (HGT), the detailed manner of such transfer has not been fully elucidated. Here, we searched for genes in the coleopteran entomopoxvirus (EV) Anomala cuprea entomopoxvirus (ACEV) that might have been gained by ACEV by HGT. We classified the potential source organisms for HGT into three categories: the host A. cuprea; other organisms, including viruses unrelated to EVs; and organisms with uncertain host attribution. Of the open reading frames (ORFs) of the ACEV genome, 2.1 % were suggested to have been gained from the host by ACEV or its recent ancestor via HGT; 8.7 % were possibly from organisms other than the host, and 3.7 % were possibly from the third category of organisms via HGT. The analysis showed that ACEV contains some interesting ORFs obtained by HGT, including a large ATP-binding cassette protein (ABC transporter) ORF and a tenascin ORF (IDs ACV025 and ACV123, respectively). We then performed a detailed analysis of the HGT of the ACEV large ABC transporter ORF—the largest of the ACEV ORFs. mRNA sequences obtained by RNA-seq from fat bodies—sites of ACEV replication—and midgut tissues—sites of initial infection—of the virus's host A. cuprea larvae were subjected to BLAST analysis. One type of ABC transporter ORF from the fat bodies and two types from the midgut tissues, one of which was identical to that in the fat bodies, had the greatest identity to the ABC transporter ORF of ACEV. The two types from the host had high levels of identity to each other (approximately 95 % nucleotide sequence identity), strongly suggesting that the host ABC transporter group consisting of the two types was the origin of ACV025. We then determined the sequence (12,381 bp) containing a full-length gene of the A. cuprea ABC transporter. It turned out to be a transcription template for the abovementioned mRNA found in both tissues. In addition, we determined a large part (ca. 6.9 kb) of the template sequence for the mRNA found only in the midgut tissues. The results showed that the ACEV ABC transporter ORF is missing parts corresponding to introns of the host ABC transporter genes, indicating that the ORF was likely acquired by HGT in the form of mRNA. The presence of definite duplicated sequences adjacent to the ACEV ABC transporter genes—a sign of LINE-1 retrotransposon-mediated HGT—was not observed. An approximately 2-month ACV025 transcription experiment suggested that the transporter sequence is presumed to be continuously functional. The amino acid sequence of ACV025 suggests that its product might function in the regulation of phosphatide in the host-cell membranes.

Global kinetic mechanism describing single nucleotide incorporation for RNA polymerase I reveals fast UMP incorporation

RNA polymerase I (Pol I) is responsible for synthesizing ribosomal RNA, which is the rate limiting step in ribosome biogenesis. We have reported wide variability in the magnitude of the rate constants defining the rate limiting step in sequential nucleotide additions catalyzed by Pol I. in this study we sought to determine if base identity impacts the rate limiting step of nucleotide addition catalyzed by Pol I. To this end, we report a transient state kinetic interrogation of AMP, CMP, GMP, and UMP incorporations catalyzed by Pol I. We found that Pol I uses one kinetic mechanism to incorporate all nucleotides. However, we found that UMP incorporation is faster than AMP, CMP, and GMP additions. Further, we found that endonucleolytic removal of a dimer from the 3′ end was fastest when the 3′ terminal base is a UMP. It has been previously shown that both downstream and upstream template sequence identity impacts the kinetics of nucleotide addition. The results reported here show that the incoming base identity also impacts the magnitude of the observed rate limiting step.

A persistent variant telomere sequence in a human pedigree

The telomere sequence, TTAGGG, is conserved across all vertebrates and plays an essential role in suppressing the DNA damage response by binding a set of proteins termed shelterin. Changes in the telomere sequence impair shelterin binding, initiate a DNA damage response, and are toxic to cells. Here we identify a family with a variant in the telomere template sequence of telomerase, the enzyme responsible for telomere elongation, that led to a non-canonical telomere sequence. The variant is inherited across at least one generation and one family member reports no significant medical concerns despite ~9% of their telomeres converting to the novel sequence. The variant template disrupts telomerase repeat addition processivity and decreased the binding of the telomere-binding protein POT1. Despite these disruptions, the sequence is readily incorporated into cellular chromosomes. Incorporation of a variant sequence prevents POT1-mediated inhibition of telomerase suggesting that incorporation of a variant sequence may influence telomere addition. These findings demonstrate that telomeres can tolerate substantial degeneracy while remaining functional and provide insights as to how incorporation of a non-canonical telomere sequence might alter telomere length dynamics.

CD59 gene: 143 haplotypes of 22,718 nucleotides length by computational phasing in 113 individuals from different ethnicities.

CD59 deficiency due to rare germline variants in the CD59 gene causes disabilities, ischemic strokes, neuropathy, and hemolysis. CD59 deficiency due to common somatic variants in the PIG-A gene in hematopoietic stem cells causes paroxysmal nocturnal hemoglobinuria. The ISBT database lists one nonsense and three missense germline variants that are associated with the CD59-null phenotype. To analyze the genetic diversity of the CD59 gene, we determined long-range CD59 haplotypes among individuals from different ethnicities. We determined a 22.7 kb genomic fragment of the CD59 gene in 113 individuals using next-generation sequencing (NGS), which covered the whole NM_203330.2 mRNA transcript of 7796 base pairs. Samples came from an FDA reference repository and our Ethiopia study cohorts. The raw genotype data were computationally phased into individual haplotype sequences. Nucleotide sequencing of the CD59 gene of 226 chromosomes identified 216 positions with single nucleotide variants. Only three haplotypes were observed in homozygous form, which allowed us to assign them unambiguously as experimentally verified CD59 haplotypes. They were also the most frequent haplotypes among both cohorts. An additional 140 haplotypes were imputed computationally. We provided a large set of haplotypes and proposed three verified long-range CD59 reference sequences, based on a population approach, using a generalizable rationale for our choice. Correct long-range haplotypes are useful as template sequences for allele calling in high-throughput NGS and precision medicine approaches, thus enhancing the reliability of clinical diagnostics. Long-range haplotypes can also be used to evaluate the influence of genetic variation on the risk of transfusion reactions or diseases.

Benchmarking AlphaFold-Generated Structures of Chemokine-Chemokine Receptor Complexes.

AlphaFold and AlphaFold-Multimer have become two essential tools for the modeling of unknown structures of proteins and protein complexes. In this work, we extensively benchmarked the quality of chemokine-chemokine receptor structures generated by AlphaFold-Multimer against experimentally determined structures. Our analysis considered both the global quality of the model, as well as key structural features for chemokine recognition. To study the effects of template and multiple sequence alignment parameters on the results, a new prediction pipeline called LIT-AlphaFold (https://github.com/LIT-CCM-lab/LIT-AlphaFold) was developed, allowing extensive input customization. AlphaFold-Multimer correctly predicted differences in chemokine binding orientation and accurately reproduced the unique binding orientation of the CXCL12-ACKR3 complex. Further, the predictions of the full receptor N-terminus provided insights into a putative chemokine recognition site 0.5. The accuracy of chemokine N-terminus binding mode prediction varied between complexes, but the confidence score permitted the distinguishing of residues that were very likely well positioned. Finally, we generated a high-confidence model of the unsolved CXCL12-CXCR4 complex, which agreed with experimental mutagenesis and cross-linking data.

Characterisation of the Arabidopsis thaliana telomerase TERT-TR complex

Most eukaryotic organisms employ a telomerase complex for the maintenance of chromosome ends. The core of this complex is composed of telomerase reverse transcriptase (TERT) and telomerase RNA (TR) subunits. The TERT reverse transcriptase (RT) domain synthesises telomeric DNA using the TR template sequence. The other TERT domains contribute to this process in different ways. In particular, the TERT RNA-binding domain (TRBD) interacts with specific TR motif(s). Using a yeast 3-hybrid system, we show the critical role of Arabidopsis thaliana (At) TRBD and embryophyta-conserved KRxR motif in the unstructured linker preceding the TRBD domain for binding to the recently identified AtTR subunit. We also show the essential role of the predicted P4 stem and pseudoknot AtTR structures and provide evidence for the binding of AtTRBD to pseudoknot and KRxR motif stabilising interaction with the P4 stem structure. Our results thus provide the first insight into the core part of the plant telomerase complex.

A nascent riboswitch helix orchestrates robust transcriptional regulation through signal integration

Widespread manganese-sensing transcriptional riboswitches effect the dependable gene regulation needed for bacterial manganese homeostasis in changing environments. Riboswitches – like most structured RNAs – are believed to fold co-transcriptionally, subject to both ligand binding and transcription events; yet how these processes are orchestrated for robust regulation is poorly understood. Through a combination of single-molecule and bulk approaches, we discover how a single Mn2+ ion and the transcribing RNA polymerase (RNAP), paused immediately downstream by a DNA template sequence, are coordinated by the bridging switch helix P1.1 in the representative Lactococcus lactis riboswitch. This coordination achieves a heretofore-overlooked semi-docked global conformation of the nascent RNA, P1.1 base pair stabilization, transcription factor NusA ejection, and RNAP pause extension, thereby enforcing transcription readthrough. Our work demonstrates how a central, adaptable RNA helix functions analogous to a molecular fulcrum of a first-class lever system to integrate disparate signals for finely balanced gene expression control.

Design and application of universal primers for mitochondrial genome sequencing of synanthropic mite species (Astigmatina: Acaridae)</p >

This study aims to design and develop a set of universal primers for amplification of the entire mitochondrial genome of Acaridae mites based on polymerase chain reaction (PCR). The conserved regions were screened by comparing the mitochondrial genomes of known Acaridae mites in GenBank. To ensure that the primers cover the entire mitochondrial sequence, the mitochondrial genome was divided into six overlapping fragments based on the conserved region. After synthesizing primers, we extracted the total DNA of Acaridae mites collected from grain stores. Then the total DNA of individual Acaridae mites was used as the template for PCR, Sanger sequencing, and sequence splicing. Agarose gel electrophoresis revealed that the lengths of the six amplification products were between 1.2 kb and 3.8 kb, consistent with design expectations. The resulting sequences could be successfully spliced to form a loop structure, confirming that the primers cover the entire mitochondrial genome. The splicing results were consistent with the full sequence released in GenBank. Developed universal primers can achieve rapid amplification and help to obtain mitochondrial genome from a single acarid mite with high efficiency.

Micromechanical Indentation Platform for Rapid Analysis of Viscoelastic Biomolecular Hydrogels.

The advent of biomedical applications of soft bioinspired materials has entailed an increasing demand for streamlined and expedient characterization methods meant for both research and quality control objectives. Here, a novel measurement system for the characterization of biological hydrogels with volumes as low as 75µL was developed. The system is based on an indentation platform equipped with micrometer drive actuators that allow the determination of both the fracture points and Young's moduli of relatively stiff polymers, including agarose, as well as the measurements of viscosity for exceptionally soft and viscous hydrogels, such as DNA hydrogels. The sensitivity of the method allows differentiation between DNA hydrogels produced by rolling circle amplification based on different template sequences and synthesis protocols. In addition, the polymerization kinetics of the hydrogels can be determined by time-resolved measurements, and the apparent viscosities of even more complex DNA-based nanocomposites can be measured. The platform presented here thus offers the possibility to characterize a broad variety of soft biomaterials in a targeted, fast, and cost-effective manner, holding promises for applications in fundamental materials science and ensuring reproducibility in the handling of complex materials.

A Comparison of Different Protocols for the Extraction of Microbial DNA Inhabiting Synthetic Mars Simulant Soil.

Compared with typical Earth soil, Martian soil and Mars simulant soils have distinct properties, including pH > 8.0 and high contents of silicates, iron-rich minerals, sulfates, and metal oxides. This unique soil matrix poses a major challenge for extracting microbial DNA. In particular, mineral adsorption and the generation of destructive hydroxyl radicals through cationic redox cycling may interfere with DNA extraction. This study evaluated different protocols for extracting microbial DNA from Mars Global Simulant (MGS-1), a Mars simulant soil. Two commercial kits were tested: the FastDNA SPIN Kit for soil ("MP kit") and the DNeasy PowerSoil Pro Kit ("PowerSoil kit"). MGS-1 was incubated with living soil for five weeks, and DNA was extracted from aliquots using the kits. After extraction, the DNA was quantified with a NanoDrop spectrophotometer and used as the template for 16S rRNA gene amplicon sequencing and qPCR. The MP kit was the most efficient, yielding approximately four times more DNA than the PowerSoil kit. DNA extracted using the MP kit with 0.5 g soil resulted in 28,642-37,805 16S rRNA gene sequence reads and 30,380-42,070 16S rRNA gene copies, whereas the 16S rRNA gene could not be amplified from DNA extracted using the PowerSoil kit. We suggest that the FastDNA SPIN Kit is the best option for studying microbial communities in Mars simulant soils.