Phenol Chloroform Isoamyl Alcohol Method Research Articles

Complete mitochondrial genome of critically endangered catfish Hemibagrus punctatus (Jerdon, 1849) and comparative analysis for insights into the phylogeny of hemibagrids through mitogenomic approach.

Hemibagrus punctatus (Jerdon, 1849) is a critically endangered bagrid catfish endemic to the Western Ghats of India, whose population is declining due to anthropogenic activities. The current study aims to compare the mitogenome of H. punctatus with that of other Bagrid catfishes and provide insights into their evolutionary relationships. Samples were collected from Hemmige Karnataka, India. In the present study, the mitogenome of H. punctatus was successfully assembled, and its phylogenetic relationships with other Bagridae species were studied. The total genomic DNA of samples was extracted following the phenol-chloroform isoamyl alcohol method. Samples were sequenced, and the Illumina paired-end reads were assembled to a contig length of 16,517bp. The mitochondrial genome was annotated using MitoFish and MitoAnnotator (Iwasaki et al., 2013). A robust phylogenetic analysis employing NJ (Maximum composite likelihood) and ASAP methods supports the classification of H. punctatus within the Bagridae family, which validates the taxonomic status of this species. In conclusion, this research enriches our understanding of H. punctatus mitogenome, shedding light on its evolutionary dynamics within the Bagridae family and contributing to the broader knowledge of mitochondrial genes in the context of evolutionary biology. The study's findings contribute to a better understanding of the mitogenome of H. punctatus and provide insights into the evolutionary relationships within other Hemibagrids.

Detection of Tannerella forsythia from saliva samples in different ethnic majority groups in Sarawak

Nowadays racial and ethnic differences in health care has become a growing concern. It is one of the critical determinant in influencing the genotype of the host in which may results in some diseases such as periodontal disease. Tannerella forsythia can be found in oral cavity and have the strongest relation in resulting on the destruction of connective tissue in periodontal disease. This research is aim to investigate the prevalence of periodontal pathogens, particularly T. forsythia in four major ethnic groups in Sarawak which may result in periodontal disease in Sarawak. This disease may due to the results from the infection of the tissue supporting the teeth. A total of (n = 40) saliva samples consist of 10 samples for each ethnic groups such as Iban, Malay, Chinese and Bidayuh were collected in Kuching and Kota Samarahan using culture-independent method. The DNA was extracted from saliva based on Phenol Chloroform Isoamyl Alcohol method. After that, 16S rRNA gene was then amplified via PCR for bacterial detection using 27 F and 1492 R primers, followed by PG-F and PG-R primers set in identifying T. forsythia. The PCR product was observed on 1.5% gel electrophoresis. As a result, the presence of bacteria T. forsythia was found more frequently from saliva samples of ethnic in Iban (70%), followed by Malay (60%), Bidayuh (60%) and lastly Chinese (50%). The differences of demographic, certain cultural beliefs and practices might affect the oral health status. This finding show that it may help to identify the risk groups and has contributed an additional evidence for the association between ethnicity and periodontal disease.

A pilot study of DNA yield from bloodstains on various surfaces using Phenol chloroform isoamyl alcohol (PCIA) and Chelex DNA extraction methods

Presently, Short Tandem Repeats (STRs) based forensic DNA typing technology is being globally used in solving a diverse range of forensic cases such as paternity, identification of unknown dead bodies/skeletal remains, or suspect in a case of rape or mass rape. The technology has invaded its tentacles in almost all areas of criminal investigation in the last few decades. The present forensic DNA technology is based on capillary electrophoresis and utilizes short tandem repeats(STRs).On one hand, the technology is extensively used in the investigation of crime in highly sensitive cases, but on the another hand, obtaining DNA profile from forensic samples are highly challenging many times. Advent of PCR has been a boon for handling the challenging samples in forensic DNA analysis. The quality DNA profiles from challenging samples rely on the yield and quality of DNA, which is mainly dependent upon the method used for DNA extraction. Any specific method can never be thought of to be useful for all variety of samples. Still, Phenol Chloroform Isoamyl Alcohol (PCIA) organic extraction method has been proven to be useful for a wide variety of samples from the simplest saliva/blood to complex teeth and bone samples. In the present study, we compared the yield of DNA from blood stains recovered from various surfaces using the PCIA extraction method and Chelex DNA extraction methods and their compatibility with present-day STR based capillary electrophoresis typing. The mean value of DNA yield was found 50.5 ng/ µl and 32.25 ng/ µl by PCIA and Chelex DNA extraction methods, respectively. Overall, the highest yield was observed from all the tested samples from the PCIA method.

EFFICIENCY OF TRADITIONAL DNA EXTRACTION METHOD IN PCR DETECTION OF PORCINE DNA IN MEAT MIXTURES

Through the advancement of biotechnology, DNA-based methods are the most effective techniques in species identification, as they are rapid and have higher stability in harsh conditions compared to protein-based methods. This study was conducted to determine the efficiency of the traditional DNA extraction method, phenol/chloroform/isoamyl alcohol (PCIA), and comparing it with the commercially available kit by evaluating the purity, concentration, and suitability for amplification of porcine DNA in raw chicken and beef mixtures. The quantity and quality of the DNA extracts were assessed using a UV-Vis spectrophotometer. Polymerase chain reaction (PCR) was performed using species-specific primers targeting mitochondrial DNA cytochrome b (cyt b) gene of chicken (227-bp), beef (274-bp), and pork (398-bp), to confirm the template usability and quality of the DNA extracts. High DNA concentrations and purity were obtained from meat samples extracted using the PCIA method. The visualization of pork DNA on 2% agarose gel was able to detect pork contamination in raw meat mixtures up to minute proportion (1%). The existence of pork in chicken and beef was indicated with the presence of a specific 398-bp DNA band. Thus, the PCIA method can be recommended as a cost-effective and an excellent alternative to more expensive extraction kits in detecting pork DNA in raw meat mixtures.

Comparative Analysis of the Genomic DNA Isolation Methods on <i>Inula</i> sp. (Asteraceae)

Simple, fast, low-cost and high throughput protocols are required for DNA isolation of plant species. In this study, phenol chloroform isoamyl alcohol and commercial (Sigma) DNA isolation kit methods were applied on some Inula species that belong to Asteraceae family. Genomic DNA amounts, A260, A280, A260/A230 and purity degrees (A260/A280) that were obtained through both methods were measured through electrophoresis and spectrophotometer. Additionally, PCR amplification was realized by primer pairs specific to nrDNA ITS, cpDNA ndhF (972F-1603R) and trnL-F regions. Results showed that maximum genomic DNA in nanograms obtained by phenol chloroform isoamyl alcohol method. The study also revealed that I. macrocephala had the maximum DNA and I. heterolepis had the minimum DNA amount. A260/A280 purity degrees showed that the highest and lowest purity in gDNAs obtained through phenol-choloform isoamyl alcohol method were in I.aucheriana and I. salicina, respectively. The highest and lowest purity degrees of gDNAs obtained through commercial kit was observed in I. fragilis and I. macrocephala samples, respectively. PCR amplification results showed that while band profiles of each three regions (ITS, trnL-F and ndhF) did not yield positive results in PCR amplifications using phenol-choloform isoamyl alcohol method; PCR band profiles obtained through commercial kit yielded positive results. As a result, it is fair to say that the relation of genomic DNA with PCR was found to be more efficient although the maximum amount of genomic DNA was obtained through phenol chloroform isoamyl alcohol method.

Sequence Analysis of the Internal Transcribed Spacer (ITS) Region of the Nuclear Ribosomal DNA (nrDNA) and Chloroplast trnL-F Region (cpDNA) of Some <i>Lactuca</i> L. (Asteraceae) Species in Turkey

In the current study, sequence analysis of some Turkish Lactuca L. species using nrITS DNA and trnL-F cpDNA sequences were performed to elucidate phylogenetic relationships among the taxa under study. Hieracium umbellatum was used as an outgroup. Different plant materials of Lactuca were collected from different parts of Turkey during excursions of summer 2013. Plant materials were either kept in silica gel or kept fresh for immediate DNA isolation. Both phenol chloroform-isoamyl alcohol method and commercial kits were used to extract genomic DNA for PCR reactions. ITS4 and ITS5A primers were utilized for ITS region, while trnLe and trnLf primers were used to amplify the trnL-F region. Obtained DNA sequences were edited both manually and by using BioEdit 7.0.4.1. Sequencing data were aligned via ClustalW program and analyzed using PAUP 4.01b10 software. nrITS sequences varied from 639 nucleotides to 735 nucleotides. Average nucleotide composition for nrITS was 22.1% (T), 27.9% (C), 23.2% (A) and 26.8% (G). It was also found that divergence values differed between 0.0000 and 0.10290. The trnL-F sequences varied from 296 nucleotides to 385 nucleotides. Average nucleotide composition of trnL-F sequences was 34.1% (T), 18.4% (C), 31.6% (A) and 16.0% (G). It was also found that divergence values differed between 0.0000 and 0.09674. Neighbour Joining (NJ) trees were constructed in order to identify the relationships among Lactuca species. Phylogenetic trees based on ITS region were found to be more useful than phylogenetic trees based on trnL-F region. After analysis of the results obtained, the data suggest that Lactuca contains 2 clades, with clade 1 having 2 subclades. These results support the prior phylogenetic studies on Lactuca and hence provide an up to date review of Turkish Lactuca species.

Assessing DNA recovery from chewing gum.

The purpose of this study was to evaluate which DNA extraction method yields the highest quantity of DNA from chewing gum. In this study, several popular extraction methods were tested, including Chelex-100, phenol-chloroform-isoamyl alcohol (PCIA), DNA IQ, PrepFiler, and QIAamp Investigator, and the quantity of DNA recovered from chewing gum was determined using real-time polymerase chain reaction with Quantifiler. Chewed gum control samples were submitted by anonymous healthy adult donors, and discarded environmental chewing gum samples simulating forensic evidence were collected from outside public areas (e.g., campus bus stops, streets, and sidewalks). As expected, results indicate that all methods tested yielded sufficient amplifiable human DNA from chewing gum using the wet-swab method. The QIAamp performed best when DNA was extracted from whole pieces of control gum (142.7 ng on average), and the DNA IQ method performed best on the environmental whole gum samples (29.0 ng on average). On average, the QIAamp kit also recovered the most DNA from saliva swabs. The PCIA method demonstrated the highest yield with wet swabs of the environmental gum (26.4 ng of DNA on average). However, this method should be avoided with whole gum samples (no DNA yield) due to the action of the organic reagents in dissolving and softening the gum and inhibiting DNA recovery during the extraction.

Comparative evaluation of polymerase chain reaction assay with microscopy for detection of asymptomatic carrier state of theileriosis in a herd of crossbred cattle.

Aim:This study aims to develop and to standardize a polymerase chain reaction (PCR) assay that will diagnose clinical as well as carrier state of the disease and to compare the results with conventional microscopy technique.Materials and Methods:A herd of crossbred cattle with the previous history of theileriosis in village Lahli, district Rohtak, Haryana, was selected for this study. A total of 29 blood samples were collected randomly from cows including five clinically ill cattle. Blood smears from all animals and lymph node biopsy smears from animal with swollen lymph nodes were examined microscopically after conventional Giemsa staining. Phenol chloroform isoamyl alcohol method was used for extracting DNA from blood. Previously published primers targeting cytochrome b gene sequence of Theileria annulata were used in the PCR assay that was standardized to use in the laboratory.Results:Out of 29 samples tested,18 (62.06%) were found positive for theileriosis by PCR assay, whereas only 10 (34.48%) samples were detected positive by conventional microscopic technique using Giemsa staining method.Conclusions:On the basis results of comparative studies, it can be concluded that PCR assay is a more sensitive than microscopic examination for detection of theileriosis. This can be attributed to the ability of PCR assay to detect small amounts of genomic DNA of T. annulata or low parasitemia in cows. Therefore, PCR assay can serve as a more sensitive tool to detect Theileria for detection of theileriosis even in asymptomatic carrier cattle which is important for the implementation of successful control programs.

Análisis comparativo de diferentes métodos de extracción de DNA y su eficiencia de genotipificación en población mexicana

Los estudios de variabilidad genetica han reportado inconsistencias de resultados entre poblaciones, en gran medida, debido a que los metodos de extraccion de acido desoxirribonucleico (DNA, por sus siglas en ingles) y las tecnicas de genotipificacion son altamente variables entre ellas, lo que conduce a la asignacion erronea de genotipos. El objetivo de este estudio es comparar distintas tecnicas de extraccion de DNA y de genotipificacion de polimorfismos. Para llevar a cabo el estudio, se analizo el DNA de 10 muestras sanguineas correspondientes a individuos mestizos mexicanos, y se purifico por los metodos de fenol-cloroformo-alcohol isoamilico (FCI), gradiente de sales (GS), gradiente de sacarosa (GSC), DNAzol® y DNeasy Blood & Tissue Kit ®. Se genotipificaron los polimorfismos GSTT1 *0 y GSTM1*0 por reaccion en cadena de la polimerasa (PCR, por sus siglas en ingles) multiple, CYP1A1*2C por RFLP’s y AhR Arg554Lys por PCR tiempo real. Los resultados mostraron diferencias significativas (p < 0.001) en cantidad, pureza e integridad entre los distintos metodos. En los analisis moleculares se observo que el metodo de FCI presenta inhibidores de la reaccion de PCR, ya que no fue posible amplificar los fragmentos por PCR multiple y PCR punto final, aunque si hubo amplificacion en el PCR tiempo real. Los metodos GS y GSC amplificaron todas las muestras en las tres modalidades de PCR y mostraron resultados concordantes, mientras que solo el 80% de las muestras extraidas mediante DNAzol ® y DNeasy ® amplificaron, y los resultados no fueron concordantes para DNAzol ® (50%) y DNeasy ® (80%) en el analisis de PCR - RFLP’s. Los metodos de extraccion GS y GSC mostraron mayor recuperacion de DNA, con parametros de calidad e integridad optimos para los analisis moleculares por PCR

Microsatellite based genetic diversity study in indigenous chicken ecotypes of Karnataka.

Aim:The current study was the first of its kind taken upon indigenous ecotypes of the Karnataka in order to unravel the diversity details at 20 chicken microsatellite regions.Materials and Methods:210 indigenous chicken belonging to six districts of Bangalore and Mysore division formed the target sample for the present study. The genomic deoxyribonucleic acid was isolated by phenol chloroform isoamyl alcohol method. A panel of 20 microsatellite regions, including 14 recommended by FAO and six identified from published scientific literature became the targeted chicken genomic region. 27-33 samples were successfully genotyped in each of the six ecotypes through simplex or multiplex polymerase chain reactions, polyacrylamide gel electrophoresis and silver staining for the selected microsatellite panel.Results:The chickens of Ramanagara and Chamrajnagara were most distant with a Nei’s genetic distance value of 0.22. The chickens of Bangalore rural and Mysore were least distant with a value of 0.056. The Ramanagara and Chamrajnagara pair had Nei’s genetic identity value of 0.802, which is least among all pairs of ecotypes. There were five main nodes from which the six ecotypes evolved on the basis 20 microsatellite markers used in this study. This study indicates that the four ecotypes Ramnagara, Bangalore Rural, Chickaballapura and Mysore are genetically identical due to their common ancestral evolution while, Mandya and Chamrajnagara ecotypes formed a relatively different cluster due to a separate common ancestral chicken population and less number of generations since drifting from bifurcation node.Conclusion:Twenty microsatellite markers based genetic diversity study on six indigenous ecotypes indicated lower genetic distances as well as lower FST values compared to the distinguished breeds reported. There were two main clusters, which differentiated into six ecotypes. They may differentiate into more distinct varieties if bred in isolation for a longer number of generations.

New &amp;lt;i&amp;gt;Spa&amp;lt;/i&amp;gt; Types among MRSA and MSSA Isolates in North of Iran

Staphylococcus aureus typing using gene encoding protein A (spa typing) seems to have a high potential discriminatory power for typing this bacterium. This study was designed based on spa typing method to compare the S. aureus types among healthy carrier vs patients, and MRSA vs MSSA isolates. Method: This study was carried out on 182 spa typeable S. aureus isolates, including 52 MRSA. DNA was extracted by phenol chloroform-isoamyl alcohol method and it was amplified by specific primer of polymorphic X region of spa. Spa types were determined by Ridom Staph Type software. The spa types distribution among MRSA vs MSSA and healthy carrier vs patients, isolates were statistically compared by X2 method and P < 0.05 was considered as significant. Result: The most common types of spa in our region were t037 (18.3%) and t937 (13.9%) from 50 spa types which were identified in this study. spa types in this study were isolated from various age groups but t660 spa types were only isolated from children. Distribution of all spa types among MRSA and MSSA isolates was 16 and 38 types, respectively which is significant. In this study we found seven new spa types (belong to twelve isolates) which are reported for the first time. In 5 out of the above 7 new types the 24 bp repeated sequences in spa gene X region were already recognized but their 24 bp arrangement is introduced in our present investigation. In the remaining 2 new types we found new 24 bp nucleotide sequences which are also introduced for the first time in our present study. Conclusion: The distribution of spa types in MSSA strains was significantly higher than MRSA isolates, but there are not any specific spa types for discrimination between MRSA and MSSA. The novelty of our study is the introducing 7 new spa types including 2 new 24 bp repeated sequences in the X region of spa gene.

Molecular Detection of Adenoviruses in Lakes and Rivers of Goiânia, Goiás, Brazil

Adenoviruses are a highly important public health issue, since they are among the most persistent and ubiquitous viruses present in water and associated with a variety of clinical manifestations. The aim of this study was to use molecular techniques for the detection of adenovirus in public and recreational water supplies in Goiânia, Brazil. From December 2007 to November 2008, 54 water samples were collected in 5 different sites in 2 lakes and 2 rivers of the city. The samples were filtered in a positively charged nylon membrane, and the DNA was extracted using the phenol–chloroform–isoamyl alcohol method. Semi-nested PCR was used to detect adenovirus DNA, and sequence analysis of the semi-nested PCR products was performed to identify the recovered viruses. Adenovirus DNA was detected in 43% (24 of 54) of samples collected. Considering all examined sites, MP1 presented the highest occurrence of adenovirus (6 positive from 10 collected samples), followed by MP2 (3 positive from 6 collected samples), JL (10 positive from 21 collected samples), VB (3 positive from 9 collected samples), and BB (2 positive from 8 collected samples), respectively. The methodology employed proved to be feasible, fast, low-cost, and suitable to be used as screening approach on adenovirus detection in water for public sanitation companies.

DYS19, DYS385, DYS389 (I and II), DYS390, DYS391, DYS392, DYS393 Haplotypes in a Population Sample from Catalonia (North-East Spain)

Abstract Blood samples (n = 224) from healthy unrelated individuals (males) were collected. DNA was extracted by using the phenolchloroform-isoamyl alcohol method (1). Hot-start PCR triplex (Triplex I: DYS19 and DYS389 I/II; Triplex II: DYS390, DYS391 and DYS393) and singleplex (DYS392) amplifications were accomplished with fluorescein labelled primers by the method published by Gené et al. (2). The primers used were those described by Kayser et al. (3) and de Knijff et al. (4). DYS385 locus was analyzed following the method and primers described by EDNAP group (5). Genotypes were analyzed in denaturing 6% polyacrilamide gel electrophoresis, using a monochrome automated laser fluorescence sequencer. The alleles from all loci reported were typed according to the published nomenclatures and the ISFG guidelines for STR analyses. Our group have received the certify of excellence in the Y-STR Haplotyping Quality Assurance Exercise 2000–2001 (http:/www.ystr.org).