REDigest: a Python GUI for In-Silico Restriction Digestion Analysis of Genes or Complete Genome Sequences

Restriction fragment length polymorphism (RFLP) is a technology for the molecular characterization of DNA and widely used genome mapping, medical genetics, molecular microbiology and forensics etc. Terminal restriction fragment length polymorphism (T-RFLP), a variant of RFLP is extensively used in environmental microbiology for the microbial community profiling based on the restriction digestion profile of marker gene (16S rRNA, FTHFS etc.) amplicons. At present, there is a lack of a tool which can perform in-silico restriction digestion of a large number of sequences at a time, in an interactive way and as an output produce sequences of the restriction fragments and visualization plot. I have developed a graphical user interface based software REDigest for the in-silico restriction digestion analysis for gene or genome sequences. The REDigest software program with a graphical user interface is freely available at https://github.com/abhijeetsingh1704/REDigest.

Cloning, expression, purification and amyloid degradation study of recombinant serrapeptase from Serratia marcescens

Background Serrapeptase (SP) is a proteolytic enzyme which plays a prominent role in the degradation of dead tissue. It can also use as a therapeutic enzyme for the treatment of Insulin amyloids. It has reported that the SP protein has the fibrinolytic activity, but the activities towards insulin amyloid degradation not reported. Our study has focused the cloning and expressing SP gene and studying its cytotoxic reactivity studies and amyloids degradation. Results SP gene was cloned in pET-28 a (+), and the enzyme was expressed using E.coli BL 21 (DE3) strain. The clone was confirmed using colony PCR, sequencing and restriction digestion. Overexpression of enzyme and purification performed using IPTG and Ni-NTA column. The molecular weight of serrapeptase protein was determined using SDS-PAGE, which found to be 52 kDa. The purified enzyme showed a zone of clearance on casein plate, slight cytotoxic reactivity to PC-12 cells after 24 h contact and 25% insulin amyloid degradation confirmed after 24 h of incubation. Conclusion This study shows that cloning and the expression of SP will reduce the time and cost of production of SP from wild type strain which can be used for the treatment of amyloid-related diseases.

EzRAD detail protocol v1

This protocol describes the methodologies used for DNA extraction, quantification, restriction digestion, and make libraries from non-model organisms following Toonen el al. (2013). Toonen, R. J., Puritz, J. B., Forsman, Z. H., Whitney, J. L., Fernandez-Silva, I., Andrews, K. R., et al. (2013). ezRAD: a simplified method for genomic genotyping in non-model organisms. PeerJ 1, e203. doi:10.7717/peerj.203.

First report on the presence of natural Wolbachia population from major malarial vector mosquitoes Anopheles culicifacies s.l., and Anopheles stephensi from Tamil Nadu, India

Wolbachia is an alpha-proteobacteria present in several arthropods. The present study focussed on the identification of Wolbachia in wild malarial vector mosquitoes. This was achieved by molecular identification of Wolbachia from collected mosquitoes. A total of four hundred and eight seven mosquito samples were collected. Morphometric and molecular analysis revealed that they belong to Anopheles culicifacies s.l., (48.25%) and Anopheles stephensi (51.75%). The presence of Wolbachia was identified using 16S rRNA, wsp and FtsZ genes, where nested PCR of 16S rRNA alone was successful and then sequenced. Only seven mosquitoes (1.4%) were positive for Wolbachia. In silico and restriction digestion of 16S rRNA gene product using RsaI enzyme showed that the identified Wolbachia belongs to supergroup B. The prevalence rate of natural Wolbachia was lesser in native malarial vector An. culicifacies s.l. and An. stephensi was about 1.7% and 1.2%, respectively. This is the first report on the presence of Wolbachia in Anopheles culicifacies s.l. and Anopheles stephensi.

Molecular Analysis of Pathogenicity Differences of Avian Paramyxovirus 1 Genotypes VI and VII in Chickens

Background: Genotypes VI and VII of (APMV-1) have different host range and pathogenicity in pigeons and chickens. However, the molecular determinants of these differences are still unclear. Methods: Here, we aligned the DNA sequences of 56 genotype VI and 33 genotype VII APMV-1 strains. Sequence alignment results revealed that there are 17 amino acids sites differed between APMV-1 strains of these two genotypes. We then constructed a plasmid based on the full-length genome of rSG10 APMV-1 strain, which belongs to genotype VII but was mutated with these 17 VI-genotype-specific amino acids, and rescued as rSG10-17 strain. The restriction digestion and ligation and overlapping PCR methods were used in the construction of plasmids with amino acids mutation. This virus was evaluated for its virulence and growth characteristics. Results and conclusion: The results indicated that the virulence and the growth characteristics have no obvious difference between the rSG10-17 virus and its parental strain rSG10. The simultaneous mutation of 17 genotype-specific amino acids did not affect the virulence of APMV-1 in chickens. Further analysis of these amino acids is required by taking into consideration of the functions of encoded proteins.

Analysis and Comparative Genomics of the SXT/R391 Family ICE, pMERPH

The aim of this study was to analyse pMERPH, the first integrative and conjugative element (ICE) of the SXT/R391 family isolated in the United Kingdom and to determine its relationship to other members of the SXT/R391 family of ICEs. Whole Genome Sequencing of Escherichia coli isolate KH802 (which contains the ICE pMERPH) was performed using Illumina sequencing technology. pMERPH was evaluated by de novo assembly of the sequenced genome, via gene prediction and annotation tools, and phenotypic analysis via comparative genomics to other members of the SXT/R391 ICEs. pMERPH has a size of 110 Kb and has 112 predicted ORFs making it one of the bigger SXT/R391 ICE’s thus far characterised. The “Hotspot regions” of the element were found to contain putative restriction digestion systems, insertion sequences and heavy metal resistance genes that give resistance to mercury and arsenate. pMERPH is closely related to the SXT-like elements from widely dispersed geographic areas. The sequencing of pMERPH increases the knowledge of the earliest isolated SXT/R391family members and may provide insight on the emergence of such elements.

Genetic polymorphism of β-Lactoglobulin gene in indigenous Nigerian goat breeds

Polymorphism at the ?-Lactoglobulin (?-LG) gene of three Nigerian goat breeds, namely: the West African Dwarf, Sahel and Red Sokoto goats, was investigated using the Polymerase Chain Reaction-Random Fragment Length Polymorphism (PCR-RFLP) method. The restriction endonucleases used in the study were Rsal and Mspl, respectively. The results revealed the existence of only one polymorphic variant (allele A) with a gene frequency of 1.0 in all the three goat breeds studied. The amplified products were observed at 120 bp and the restriction digestion with Rsal revealed just one genotype at the ?-LG locus. It was concluded that there was the absence of polymorphism at the ?-LG locus of the goats investigated.