Novel primer designing and PCR-AFLP approach for an expeditious detection of coliforms in potable waters

Molecular techniques involving 16S rRNA gene have long been proved to be a mainstay of sequence-based bacterial analysis and enhance the competence of bacterial removal in drinking water and food. The main goal of this analysis was to reduce the time of detection of total coliforms by developing 16S rRNA based DNA markers by targeting variable region in the 16S rRNA gene position of V2 and V9. Coliform specific primers (189F and 1447R) were designed to amplify total coliform with an amplicon size of 1300 bp. The PCR product was later digested with Hind III and BseRI (restriction enzymes) to differentiate the type of contamination caused by fecal and non-fecal coliforms respectively. The digested amplicons were run on agarose gel electrophoresis and contamination levels were estimated based on the respective band pattern. This method can be applicable to know the coliform contamination levels of potable waters, in food and beverage industries within a short period of time. To our knowledge, this is the first report on newly designed primers which not only amplify coliform bacteria, followed by various restriction digestions of these amplicons but also provides unique band patterns to identify coliforms at genus level.

Identification, Isolation and Amplification of BRCA1 Gene Involved on Breast Cancer.

Cancer is a disease that begins in the cells of the body which is characterized by uncontrolled, uncoordinated and undesirable cell division. If a cell accumulates critical mutations in five or six of the protooncogenes, tumour suppressor genes and DNA repair genes are likely to result in a fully malignant cell, capable of forming a tumour. In this work we described the isolation and amplification of the BRCA1 gene. Primers were designed and synthesised later used to amplify the BRCA1 gene. The total new workflow includes all steps from purified DNA to data analysis, and includes PCR for all amplicons covering the gene, PCR cleanup, cycle sequencing, electrophoresis, and data analysis. To simplify workflows and decrease the time-to-result, we focused on the method “one sample, one assay” approach. The success of this workflow was the 24-well plate design, which contained prespotted PCR primers covering the gene and also included multiplex nontemplate controls. The workflow was developed using a Genetic Analyzer and bands were observed.

Characterization and Primer Design for Simple Sequence Repeat Loci in Salvia miltorrhiza Genome / 世界科学技术-中医药现代化

Salvia miltorrhiza Bge. is a perennial deciduous flowering plant. Its medicinal root and rhizomes part is widely used in the treatment of various diseases. In this study, bioinformatics analysis was performed to identify 4832 genome SSR loci with length longer than or equal to 40 bp from the draft genome assembly of S. miltorrhiza. The re-sults showed that the dinucleotide repeat motifs and trinucleotide repeat motifs constitute the main types of genome SSR loci, accounting for 37.3% and 61.3% respectively. SSR types enriched with A/T bases showed significantly higher abundance than other types, including AT/TA AAT/ATT, ATA/TAT, TAA/TTA, accounting for 30.5%, 21.6%, 17.1%, 20.4% of the total number of SSR loci, respectively. 1079 primer pairs were designed for these genome SSR loci. These primers can be used for genomic diversity analysis, genetic map construction, genetic marker screening. These data could lay the foundation for population genetics and genomics research of S. miltorrhiza.

Selection of reference genes for normalization of quantitative real-time PCR in cell cultures of Cyclamen persicum

As a prerequisite for gene expression analyses in cell cultures of the ornamental crop Cyclamen persicum basic parameters for quantitative real-time polymerase chain reaction (qRT-PCR) have been established including the selection of reference genes using the software tools ‘geNorm’ and ‘NormFinder’. Five potential reference genes have been tested (elongation factor tu (Ef-Tu), putative ABC transporter ATPase, putative conserved oligomeric Golgi (COG) complex component, V-ATPase G subunit 1 and Histone H3-K9 methyltransferase 4 (H3-K9-HMTase 4)). ‘NormFinder’ as well as ‘geNorm’ identified Ef-Tu to be the least stable reference gene while the ranking of the most stable genes differed depending on the algorithm. According to ‘NormFinder’ COG complex component displayed the most stable expression whereas ‘geNorm’ indicated V-ATPase G subunit 1 and a putative ABC transporter ATPase to be the most reliable reference genes. Hence, we concluded to use a normalization factor calculated from the four reference genes V-ATPase G subunit 1, ABC transporter ATPase, Histone H3-K9 methyltransferase 4 (H3-K9-HMTase 4) and COG complex component for normalization of qRT-PCR in cell cultures of Cyclamen persicum.

Distinctively variable sequence-based nuclear DNA markers for multilocus phylogeography of the soybean- and rice-infecting fungal pathogen Rhizoctonia solani AG-1 IA

A series of multilocus sequence-based nuclear DNA markers was developed to infer the phylogeographical history of the Basidiomycetous fungal pathogen Rhizoctonia solani AG-1 IA infecting rice and soybean worldwide. The strategy was based on sequencing of cloned genomic DNA fragments (previously used as RFLP probes) and subsequent screening of fungal isolates to detect single nucleotide polymorphisms (SNPs). Ten primer pairs were designed based on these sequences, which resulted in PCR amplification of 200-320 bp size products and polymorphic sequences in all markers analyzed. By direct sequencing we identified both homokaryon and heterokaryon (i.e. dikaryon) isolates at each marker. Cloning the PCR products effectively estimated the allelic phase from heterokaryotic isolates. Information content varied among markers from 0.5 to 5.9 mutations per 100 bp. Thus, the former RFLP codominant probes were successfully converted into six distinctively variable sequence-based nuclear DNA markers. Rather than discarding low polymorphism loci, the combination of these distinctively variable anonymous nuclear markers would constitute an asset for the unbiased estimate of the phylogeographical parameters such as population sizes and divergent times, providing a more reliable species history that shaped the current population structure of R. solani AG-1 IA.

COCAW: A Genome-wide Pattern Search System for Designing Microbial Probes

A few bioinformatics tools have been used to find out conserved regions as probes. We have developed a system based on a heuristic method with web interfaces to find out conserved regions against microbial genomes. The system runs in real time by using relative entropy in limited narrow regions and detecting similar regions between pair regions with local alignment. The system could be useful to find out conserved regions as genome-wide scale.

Genetic polymorphisms of the pentanucleotide repeat loci Penta D and Penta E in Wuhan Han population by re-designed primers / 中国法医学杂志

Objective To study the primer design for Penta D and Penta E genotyping and investigate the genetic polymorphisms of these two loci in Chinese Han population in Wuhan.Methods 281 unrelated Chinese individuals living in Wuhan were typed by hot-start PCR with re-designed Penta D and Penta E genotyping primers and PAGE technique,and the results were compared with those by the PowerPlex TM 16 system of Promega.Results The amplified fragment size of Penta D and Penta E loci with re-designed primers ranged between 153～198bp and 107～212bp respectively,which were consistent with the results obtained by the re-designed primers and PowerPlex TM 16 system,and the re-designed primers had a higher sensitivity than PowerPlex TM 16 system (0.2ng vs 0.5ng) by silver staining.10 and 21 alleles were observed for Penta D and Penta E in Chinese Han population in Wuhan,and the genotype distributions of the two loci were in accordance with Hardy-Weinberg equilibrium.Family studies confirmed Mendelian inheritance of alleles.The power of discrimination (DP) for Penta D and Penta E were 0.926 2 and 0.986 0,and the power of exclusion (PE) were 0.665 1 and 0.832 5 respectively.Conclusion The re-designed primers for Penta D and Penta E genotyping are reliable.These two loci are highly polymorphic in Chinese Han population and can be used in forensic identification and paternity test.