Artificial DNA Templates Research Articles

Selective measurement of \u03b1 smooth muscle actin: why \u03b2-actin can not be used as a housekeeping gene when tissue fibrosis occurs

BackgroundPrevalence of fibroproliferative diseases, including chronic kidney disease is rapidly increasing and has become a major public health problem worldwide. Fibroproliferative diseases are characterized by increased expression of α smooth muscle actin (α-SMA) that belongs to the family of the six conserved actin isoforms showing high degree homology. The aim of the present study was to develop real-time PCRs that clearly discriminate α-SMA and ß-actin from other actin isoforms.ResultsReal-time PCRs using self-designed mouse, human and rat specific α-SMA or ß-actin primer pairs resulted in the specific amplification of the artificial DNA templates corresponding to mouse, human or rat α-SMA or ß-actin, however ß-actin showed cross-reaction with the housekeeping γ-cyto-actin. We have shown that the use of improperly designed literary primer pairs significantly affects the results of PCRs measuring mRNA expression of α-SMA or ß-actin in the kidney of mice underwent UUO.ConclusionWe developed a set of carefully designed primer pairs and PCR conditions to selectively determine the expression of mouse, human or rat α-SMA and ß-actin isoforms. We demonstrated the importance of primer specificity in experiments where the results are normalized to the expression of ß-actin especially when fibrosis and thus increased expression of α-SMA is occur.

Artificial DNA Templates for the Correction of PCR Bias in Next Generation Sequencing Based MRD Analysis for Childhood Acute Lymphoblastic Leukaemia: The Influence of Secondary DNA Structure

PCR bias is a potential confounder for PCR-based NGS-MRD quantitation. A method was published using artificial DNA constructs (gBlocks, IDT Technologies, Coralville, IO) to assess PCR primer bias and correct for it(1). We sought to confirm those findings by assessing PCR bias of the Biomed-2 TRG primer set.36 synthetic DNA templates of 495bp length gBlocks combining each TRG V (V 2,3,4,5,7,8,9,10,11) & J gene (JG1-01, JG1-02, JP1, JP2) amplified by the Biomed-2 primer set were synthesised containing external MiSeq flow cell adaptor sequence (universal primer (UP)) binding sites, VG and JG sequence and junctional regions. Barcodes inserted internal to the universal primer sites and centrally enabled accurate template identification (fig 1).Individual gBlocks supplied at a nominal concentration of 10ng/uL (30nM) were quantitated by TapeStation (Agilent, Santa Clara, CA) and KAPA (Kapa Biosystems, Wilmington, MA) methodologies and pooled (unadjusted). gBlockswithin the pool were further quantitated by both 6 and 8 cycles of PCR with universal MiSeq adaptor primers.Next the gBlock pool was amplified in triplicate in a 2-stage PCR process: 1) Using Biomed-2 TRG primers that contained partial MiSeq adaptor sequences. The resulting products were purified with Agencourt AMPure XP beads (Beckman Coulter, Jersey City, NJ). 2) These amplicons were further amplified by primers containing indices and full MiSeq adaptor sequences. After purification, amplicons were Qubit (InvitroGen, Carlsbad, CA) and TapeStation analysed for normalisation and to construct the sequencing library, which was KAPA quantitated to ensure ideal cluster density. Bi-directional sequencing was performed using an Illumina MiSeq 500 cycle cartridge and Nano Reagent Kit (Illumina, San Diego, CA).Bioinformatics analysis was performed using the Vidjil platform (Bonsai team, CRIStAL, Lille, France).TapeStation quantitation showed a mean concentration of 3.3ng/uL (10nM) (range 1.1-6.3ug/uL). KAPA quantitation by contrast showed low quantities of all gBlocks (2nM, 0.67ug/uL) with the exception of all VG9 and most VG10 constructs (16nM, 5ug/uL)(fig 2a).Comparison of 6 vs 8 cycles of amplification using universal primers showed good correlation (R2=0.991) confirming that PCR cycle length does not affect the UP amplification (fig 2b), but there was an 8.5-fold variation in gBlock quantitation by UP PCR. There was no correlation between NGS quantitation and with either TapeStation or KAPA quantitation.Family specific V & J gene amplification efficiency for the Biomed-2 primers is shown in fig 3. Amplification efficiencies of V gene primers were <2-fold different with the exception of VY9 and VY10 which were 5-fold more efficient. Amplification efficiency for J gene primers showed <2-fold differences.This study has shown that there are clear PCR efficiency differences between the V primers in the Biomed-2 primer set, with VG9 and 10 being 5x more efficient. This could be adjusted for by reducing the concentrations of the VG9/10 primers. However, the variable results of the 3 methods used to quantitate the artificial templates raises significant questions about the rationale. TapeStation quantitation is not PCR based and produced broadly equivalent (but lower than anticipated) gBlock concentrations for the individual constructs. PCR based KAPA showed very low concentrations in all but the VG9/VG10 templates. It is notable that this exactly parallels the results for the locus specific PCR. The most plausible explanation is that unexpected secondary structure reduces the amplifiability of the templates in PCR reactions. The near 10-fold variation in template concentration as assessed by UP NGS-PCR is concerning, suggesting significant secondary structure effects, though not dissimilar to the 5-fold difference seen in the published method(1). It is impossible to know if the postulated secondary structure effect that we see is replicated in vivo. In that regard it is reassuring that published results of NGS-MRD appear to correlate well with RQ-PCR results.In conclusion, secondary structure issues potentially affect the in vitro method for eliminating PCR bias. Within the EuroClonality consortium, studies at other loci are underway to confirm these preliminary findings, and to design primer sets with minimal risk of PCR bias.1. Carlson CS, Emerson RO, Sherwood AM et al. Nat Commun. 2013;4:2680. [Display omitted] [Display omitted] [Display omitted] DisclosuresMoppett:Jazz Pharmaceuticals: Honoraria.

Use of artificial DNA with multiple probe sites as reference DNA templates for quantitative real-time PCR to examine methanogen communities

Isolation of reference DNA templates for quantitative real-time PCR assays is an expensive, labor-intensive and time-consuming process if they are not readily available. Two artificial DNA templates with multiple probe sites were designed for quantifying methanogens and their 10 subgroups, based on the methyl coenzyme M reductase gene (mcrA). Their standards were comparable to each other. PCR amplification efficiencies (cycle vs. cumulative fluorescence) of the artificial DNAs were also comparable to those of the observed methanogen groups from anaerobic digesters. The artificial templates can be alternatives to the actual references.

A new DNA diagnostic system for the detection of human CYP21 gene mutations associated with adrenal cortex hyperplasia

Congenital Adrenal Hyperplasia (CAH) is one of the most widespread severe autosomal recessive hereditary diseases. CAH is caused by the impaired biosynthesis of the key human hormones cortisol and aldosterone and is accompanied by the excess synthesis of androgens. Over 90% of CAH cases are caused by a deficiency of the steroid 21-hydrohylase (P450c21). The degree of damage in this enzyme is responsible for the severity of the clinical manifestation of CAH from potentially lethal to mild symptoms. Various mutations of the gene encoding this enzyme are the main source of the reduced activity of the 21-hydrolase. The location of the highly homological pseudogene CYP21P in close proximity to the functional gene impedes the DNA diagnostics of CAH. To detect the eight most frequent CYP21 gene mutations associated with CAH, we developed a new real-time PCR-based system of DNA diagnostics using new allele-specific primers and TaqMan probes for the analyzed mutations. The method was primarily tested on artificial DNA templates, where the analyzed mutations were introduced by site-directed mutagenesis. Then, it was tested on DNA samples from 43 patients with clinical and biochemical manifestations of CAH; seven patients were used as a control. Two mutant alleles were detected in two different individuals: the nonsense Q318X and the missense V281L mutations.

Chain-like assembly of gold nanoparticles on artificial DNA templates via ‘click chemistry’

We present a new type of azide-functionalized gold nanoparticle and their coupling to an alkyne-modified DNA duplex using the copper(I)-catalyzed Huisgen cycloaddition ('click chemistry'), resulting in a chain-like assembly of nanoparticles on the DNA template.

Formation of Bimetallic Ag–Au Nanowires by Metallization of Artificial DNA Duplexes

Uniform bimetallic nanowires, tunable in size, have been grown on artificial DNA templates via a two-step metallization process. Alkyne-modified cytosines were incorporated into 900-base-pair polymerase-chain-reaction fragments. The alkyne modifications serve as addressable metal-binding sites after conversion to a sugar triazole derivative via click chemistry. Reaction of the Tollens reagent with these sugar-coated DNA duplexes generates Ag0 metallization centers around the sugar modification sites of the DNA. After a subsequent enhancement step using gold, nanowires < or = 10 nm in diameter with a homogeneous surface profile were obtained. Furthermore, the advantage of this two-step procedure lies in the high selectivity of the process, due to the exact spatial control of modified DNA base incorporation and hence the confinement of metallization centers at addressable sites. Besides experiments on a membrane as a proof for the selectivity of the method, atomic force microscopy (AFM) studies of the wires produced on Si-SiO2 surfaces are discussed. Furthermore, we demonstrate time-dependent metallization experiments, monitored by AFM.

Altered DNA polymerase iota expression in breast cancer cells leads to a reduction in DNA replication fidelity and a higher rate of mutagenesis.

The recently discovered human enzyme DNA polymerase iota (pol iota) has been shown to have an exceptionally high error rate on artificial DNA templates. Although there is a considerable body of in vitro evidence for a role for pol iota in DNA lesion bypass, there is no in vivo evidence to confirm this action. We report here that pol iota expression is elevated in breast cancer cells and correlates with a significant decrease in DNA replication fidelity. We also demonstrate that UV treatment of breast cancer cells additionally increases pol iota expression with a peak occurring between 30 min and 2 h after cellular insult. This implies that the change in pol iota expression is an early event after UV-mediated DNA damage. That pol iota may play a role in the higher mutation frequencies observed in breast cancer cells was suggested when a reduction in mutation frequency was found after pol iota was immunodepleted from nuclear extracts of the cells. Analysis of the UV-induced mutation spectra revealed that > 90% were point mutations. The analysis also demonstrated a decreased C --> T nucleotide transition and an increased C --> A transversion rate. Overall, our data strongly suggest that pol iota may be involved in the generation of both increased spontaneous and translesion mutations during DNA replication in breast cancer cells, thereby contributing to the accumulation of genetic damage.

Reconstitution of complete SV40 DNA replication with purified replication factors.

The identification and purification of human cell proteins required for the production of form I DNA following DNA replication from the simian virus 40 (SV40) origin is described. Using these proteins, complete SV40 DNA replication was reconstituted with only purified DNA replication factors: SV40 large tumor antigen (TAg), replication protein A (RPA), DNA topoisomerases I and II, DNA polymerase alpha-primase, replication factor C (RFC), the proliferating cell nuclear antigen (PCNA), DNA polymerase delta, maturation factor 1 (MF1), and DNA ligase I. MF1, a 5' to 3' exonuclease and DNA ligase I were both identified as essential components for production of covalently closed circular relaxed (form I) DNA. MF1 is probably the same exonuclease previously shown by others to function during DNA synthesis on artificial DNA templates or in conjunction with DNA polymerase alpha from the SV40 origin. Combined with these previous studies, our results suggest that MF1 functions to remove an RNA primer attached to every Okazaki fragment during lagging strand DNA synthesis. Interestingly, whereas mammalian DNA ligase I functioned in the reconstituted replication system, mammalian DNA ligase III did not substitute and the phage T4 DNA ligase functioned inefficiently, suggesting that DNA ligase I has a specific role as a replicative DNA ligase in eukaryotic cells.

In vitro cell cycle arrest induced by using artificial DNA templates.

In cell extracts of Xenopus eggs which oscillate between S and M phases of the cell cycle, the onset of mitosis is blocked by the presence of incompletely replicated DNA. In this report, we show that several artificial DNA templates (M13 single-stranded DNA and double-stranded plasmid DNA) can trigger this feedback pathway, which inhibits mitosis. Single-stranded M13 DNA is much more effective than double-stranded plasmid DNA at inhibiting the onset of mitosis. Furthermore, we have shown that low levels of M13 single-stranded DNA and high levels of double-stranded plasmid DNA can elevate the tyrosine kinase activity responsible for phosphorylating p34cdc2, thereby inactivating maturation-promoting factor and inhibiting entry into mitosis. This constitutes a simplified system with which to study the signal transduction pathway from the DNA template to the tyrosine kinase responsible for inhibiting p34cdc2 activity.

In vitro cell cycle arrest induced by using artificial DNA templates.

In cell extracts of Xenopus eggs which oscillate between S and M phases of the cell cycle, the onset of mitosis is blocked by the presence of incompletely replicated DNA. In this report, we show that several artificial DNA templates (M13 single-stranded DNA and double-stranded plasmid DNA) can trigger this feedback pathway, which inhibits mitosis. Single-stranded M13 DNA is much more effective than double-stranded plasmid DNA at inhibiting the onset of mitosis. Furthermore, we have shown that low levels of M13 single-stranded DNA and high levels of double-stranded plasmid DNA can elevate the tyrosine kinase activity responsible for phosphorylating p34cdc2, thereby inactivating maturation-promoting factor and inhibiting entry into mitosis. This constitutes a simplified system with which to study the signal transduction pathway from the DNA template to the tyrosine kinase responsible for inhibiting p34cdc2 activity.