Comparison of real time PCR and conventional PCR by identifying genomic DNA of bovine and porcine

Bovine and porcine are poultry meat that consumed worldwide particularly in Southeast Asia.Both of them are prone to food counterfeit owing to several factors such as price, appetite and Halal status. Sensitive and selective analytical methods are required to control meat products that distributed to markets. This paper studied the sensitivity between real – time and conventional PCR or known as qPCR and cPCR, respectively. Bovine and porcine were samples used to verify the sensitivity of them. Nevertheless, those instruments did not show a specific difference during DNA analysis of bovine and porcine. In conventional PCR, two pairs of DNA primers targeted cytochrome b (Cyt b) was analyzed, resulting of 120 and 131 amplicons, respectively. While qPCR applied to analyze porcine and bovine DNA. The detection limit of qPCR after porcine and bovine analysis were at 0.004 and 0.007 µg/µL, respectively. Results demonstrated the qPCR was reliable for verifying porcine and bovine DNA compared to conventional PCR. Furthermore, the study concluded that the developed assay can be easily employed for the identification of porcine and bovine tissue in food products in low resource areas.

Arabidopsis thaliana PrimPol is a primase and lesion bypass DNA polymerase with the biochemical characteristics to cope with DNA damage in the nucleus, mitochondria, and chloroplast

PrimPol is a novel Primase–Polymerase that synthesizes RNA and DNA primers de novo and extents from these primers as a DNA polymerase. Animal PrimPol is involved in nuclear and mitochondrial DNA replication by virtue of its translesion DNA synthesis (TLS) and repriming activities. Here we report that the plant model Arabidopsis thaliana encodes a functional PrimPol (AtPrimPol). AtPrimPol is a low fidelity and a TLS polymerase capable to bypass DNA lesions, like thymine glycol and abasic sites, by incorporating directly across these lesions or by skipping them. AtPrimPol is also an efficient primase that preferentially recognizes the single-stranded 3′-GTCG-5′ DNA sequence, where the 3′-G is cryptic. AtPrimPol is the first DNA polymerase that localizes in three cellular compartments: nucleus, mitochondria, and chloroplast. In vitro, AtPrimPol synthesizes primers that are extended by the plant organellar DNA polymerases and this reaction is regulated by organellar single-stranded binding proteins. Given the constant exposure of plants to endogenous and exogenous DNA-damaging agents and the enzymatic capabilities of lesion bypass and re-priming of AtPrimPol, we postulate a predominant role of this enzyme in avoiding replication fork collapse in all three plant genomes, both as a primase and as a TLS polymerase.

Human PrimPol Discrimination against Dideoxynucleotides during Primer Synthesis

PrimPol is required to re-prime DNA replication at both nucleus and mitochondria, thus facilitating fork progression during replicative stress. ddC is a chain-terminating nucleotide that has been widely used to block mitochondrial DNA replication because it is efficiently incorporated by the replicative polymerase Polγ. Here, we show that human PrimPol discriminates against dideoxynucleotides (ddNTP) when elongating a primer across 8oxoG lesions in the template, but also when starting de novo synthesis of DNA primers, and especially when selecting the 3′nucleotide of the initial dimer. PrimPol incorporates ddNTPs with a very low efficiency compared to dNTPs even in the presence of activating manganese ions, and only a 40-fold excess of ddNTP would significantly disturb PrimPol primase activity. This discrimination against ddNTPs prevents premature termination of the primers, warranting their use for elongation. The crystal structure of human PrimPol highlights Arg291 residue as responsible for the strong dNTP/ddNTP selectivity, since it interacts with the 3′-OH group of the incoming deoxynucleotide, absent in ddNTPs. Arg291, shown here to be critical for both primase and polymerase activities of human PrimPol, would contribute to the preferred binding of dNTPs versus ddNTPs at the 3′elongation site, thus avoiding synthesis of abortive primers.

Assessment of Genetic Stability on In Vitro And Ex Vitro Plants of Ficus Carica Var. Black Jack Using Issr and Damd Markers

In vitro propagation has been significant in producing a large number of genetically stable regenerated plants. Regenerated Ficus carica var. Black Jack plantlets were established using woody plant medium (WPM) supplemented with 20 µM 6-Benzylaminopurine (BAP) and 8 µM Indole-3-acetic acid (IAA) under different light treatments such as normal fluorescent white light (60 µmol.m− 2.s− 1), and four different LED spectra, white (400– 700nm), blue (440nm), red (660nm) and blue + red (440nm + 660nm). Genetic stability analysis was performed on the in vitro and ex vitro plants of Ficus carica var. Black Jack. Ten (10) primers of each ISSR and DAMD molecular marker were used to assess the genetic stability of the eight (8) samples of Ficus carica var. Black Jack, acquired over two years. The findings of this study revealed that inter simple sequence repeats (ISSR) and directed amplification of minisatellite DNA (DAMD) markers (DNA primers) are efficient in determining the polymorphism and monomorphism percentage among the in vitro and ex vitro samples of Ficus carica var. Black Jack. ISSR markers showed 97.87% of monomorphism whereas DAMD markers showed 100% monomorphism. Polymorphism of 2.13% was observed for the UBC840 ISSR – DNA primer which was negated under the genetic similarity index analysis for the eight samples. It is recommended that genetic stability analysis should be performed for long-term maintenance of micropropagated plants.

Assessment of Genetic Stability of Micropropagated Bambusa balcooa Roxb. using RAPD Marker

Bambusa balcooa Roxb. was in vitro propagated by optimizing protocol using nodal segment from secondary branches with 100% success in MS liquid media containing 100 mg/l Myo-inositol, 3% sucrose supplement with 4.4 - 26.64 μM BAP for shoot multiplication, and 2.69 - 32.26 μM NAA for root induction. The highest shoot multiplication (14.53 ± 0.33 folds), shoot length (5.9 ± 0.6 cm), shoot number per explants (4.0 ± 0.24), and rooting (89.3 ± 0.33%) was obtained in MS liquid media supplement with 13.32 μM BAP (shooting) and 26.88 μM NAA (rooting) and 1% aqueous leaf extract of Artemisia vulgaris L. (EAV). Twenty RAPD (Random amplified Polymorphic DNA) primers were used individually to amplify DNA of tissue culture-raised plants and the mother plant where 8 primers yielded monomorphic banding patterns with reproducible, clear, scorable bands (2.8 per primer) ranging from 250 to 1800 bp respectively which revealed the micropropagated plants of B. balcooa retained their genetic stability. Plant Tissue Cult. & Biotech. 31(1): 81-95, 2021 (June)

Resistance to a Nucleoside Analog Antiviral Drug from More Rapid Extension of Drug-Containing Primers

ABSTRACT Nucleoside analogs are mainstays of antiviral therapy. Although resistance to these drugs hinders their use, understanding resistance can illuminate mechanisms of the drugs and their targets. Certain nucleoside analogs, such as ganciclovir (GCV), a leading therapy for human cytomegalovirus (HCMV), contain the equivalent of a 3′-hydoxyl moiety, yet their triphosphates can terminate genome synthesis (nonobligate chain termination). For ganciclovir, chain termination is delayed until incorporation of the subsequent nucleotide, after which viral polymerase idling (repeated addition and removal of incorporated nucleotides) prevents extension. Here, we investigated how an alanine-to-glycine substitution at residue 987 (A987G), in conserved motif V in the thumb subdomain of the catalytic subunit (Pol) of HCMV DNA polymerase, affects polymerase function to overcome delayed chain termination and confer ganciclovir resistance. Steady-state enzyme kinetic studies revealed no effects of this substitution on incorporation of ganciclovir-triphosphate into DNA that could explain resistance. We also found no effects of the substitution on Pol’s exonuclease activity, and the mutant enzyme still exhibited idling after incorporation of GCV and the subsequent nucleotide. However, despite extending normal DNA primers similarly to wild-type enzyme, A987G Pol more rapidly extended ganciclovir-containing DNA primers, thereby overcoming chain termination. The mutant Pol also more rapidly extended RNA primers, a previously unreported activity for HCMV Pol. Structural analysis of related Pols bound to primer-templates provides a rationale for these results. These studies uncover a new drug resistance mechanism, potentially applicable to other nonobligate chain-terminating nucleoside analogs, and shed light on polymerase functions. IMPORTANCE While resistance to antiviral drugs can hinder their clinical use, understanding resistance mechanisms can illuminate how these drugs and their targets act. We studied a substitution in the human cytomegalovirus (HCMV) DNA polymerase that confers resistance to a leading anti-HCMV drug, ganciclovir. Ganciclovir is a nucleoside analog that terminates DNA replication after its triphosphate and the subsequent nucleotide are incorporated. We found that the substitution studied here results in an increased rate of extension of drug-containing DNA primers, thereby overcoming termination, which is a new mechanism of drug resistance. The substitution also induces more rapid extension of RNA primers, a function that had not previously been reported for HCMV polymerase. Thus, these results provide a novel resistance mechanism with potential implications for related nucleoside analogs that act against established and emerging viruses, and shed light on DNA polymerase functions.

Gene flow and genetic structure between populations of Hesperis L. (Brassicaceae) species using molecular markers

Genetic variability and populations, structure were studied in seven geographical populations of Hesperis L. Genetic diversity parameters were determined in these populations. 5 of 10 random amplified polymorphic DNA primers produced 62 reproducible bands with average of 7.1 bands per primer and 55% of polymorphism. Hesperis hyrcana showed the highest number of effective allele (Ne), Shannon index (I) and genetic diversity (H). The highest values of genetic diversity were obtained in Hesperis hyrcana. NJ trees grouped the populations in two different clusters/groups, indicating their genetic difference which is discussed in details. The results of this study showed that the level of genetic variation in Hesperis is relatively high. NJ-based dendrogram showed a close relationship between members of Hesperis straussii and Hesperis hyrcana while the Hesperis luristanica protected population differ the most from the other populations. Principal component analysis, however, showed some minor differences with NJ-based dendrograms.

Comparative DNA Analysis of Coconut (Cocos nucifera L.) palms with polyembryonic and monoembryonic origins

Makapuno is a rare, high-value coconut in the Philippines known for its extraordinary thick gelatinous meat with various uses in the food industry. Homozygous makapuno embryos do not germinate in vivo so plantlets are produced in vitro. where one plantlet grows from an embryo. Rare cases of polyembryony were observed in makapuno hybrids developed bythe Visayas State University, Knowledge of the genetic control of polyembryony may be used to increase the production of planting materials of these rare coconut types. DNA analysis of two sets of twins (polyembryonic), three monoembryonic hybrid palms, and their monoembryonic parental cultivars Coconiño and tall makapuno was done using seven DNA primers to determine differences which may be associated with polyembryony in the hybrids. Polyacrylamide Gel Electrophoresis of PCR products showed DNA fragments amplified by primers CAC2 and CAC56 which are unique to the twins suggesting that polyembryony might have a genetic origin.

New Real-Time PCRs to Differentiate Rickettsia spp. and Rickettsia conorii

Rickettsia species are an important cause of emerging infectious diseases in people and animals, and rickettsiosis is one of the oldest known vector-borne diseases. Laboratory diagnosis of Rickettsia is complex and time-consuming. This study was aimed at developing two quantitative real-time PCRs targeting ompB and ompA genes for the detection, respectively, of Rickettsia spp. and R. conorii DNA. Primers were designed following an analysis of Rickettsia gene sequences. The assays were optimized using SYBR Green and TaqMan methods and tested for sensitivity and specificity. This study allowed the development of powerful diagnostic methods, able to detect and quantify Rickettsia spp. DNA and differentiate R. conorii species.

A novel DNA primase-helicase pair encoded by SCCmec elements

Mobile genetic elements (MGEs) are a rich source of new enzymes, and conversely, understanding the activities of MGE-encoded proteins can elucidate MGE function. Here, we biochemically characterize three proteins encoded by a conserved operon carried by the Staphylococcal Cassette Chromosome (SCCmec), an MGE that confers methicillin resistance to Staphylococcus aureus, creating MRSA strains. The first of these proteins, CCPol, is an active A-family DNA polymerase. The middle protein, MP, binds tightly to CCPol and confers upon it the ability to synthesize DNA primers de novo. The CCPol-MP complex is therefore a unique primase-polymerase enzyme unrelated to either known primase family. The third protein, Cch2, is a 3’-to-5’ helicase. Cch2 additionally binds specifically to a dsDNA sequence downstream of its gene that is also a preferred initiation site for priming by CCPol-MP. Taken together, our results suggest that this is a functional replication module for SCCmec.