Quality Deoxyribonucleic Acid Research Articles

Extraction of DNA suitable for PCR applications from mature leaves of Mangifera indica L.

Good quality deoxyribonucleic acid (DNA) is the pre-requisite for its downstream applications. The presence of high concentrations of polysaccharides, polyphenols, proteins, and other secondary metabolites in mango leaves poses problem in getting good quality DNA fit for polymerase chain reaction (PCR) applications. The problem is exacerbated when DNA is extracted from mature mango leaves. A reliable and modified protocol based on the cetyltrimethylammonium bromide (CTAB) method for DNA extraction from mature mango leaves is described here. High concentrations of inert salt were used to remove polysaccharides; Polyvinylpyrrolidone (PVP) and β-mercaptoethanol were employed to manage phenolic compounds. Extended chloroform-isoamyl alcohol treatment followed by RNase treatment yielded 950-1050 µg of good quality DNA, free of protein and RNA. The problems of DNA degradation, contamination, and low yield due to irreversible binding of phenolic compounds and coprecipitation of polysaccharides with DNA were avoided by this method. The DNA isolated by the modified method showed good PCR amplification using simple sequence repeat (SSR) primers. This modified protocol can also be used to extract DNA from other woody plants having similar problems.

Genomic Deoxyribonucleic Acid Extraction from Post Mortem Fetal Tissue

A major challenge in post-mortem (PM) genetic testing in fetuses is the difficulty in obtaining high quality deoxyribonucleic acid (DNA) due to maceration. We examined the yield and quality of DNA from various PM fetal tissues.Methods: Fresh tissue samples from the muscle, kidney, liver, heart and placenta from 14 consecutive fetuses were collected at the time of autopsy and stored at -20 °C. We extracted DNA using Promega Wizard Genomic DNA Purification Kit and assessed the DNA yield and quality using the Thermo Scientific Nano-drop and by agarose gel electrophoresis. Polymerase chain reaction (PCR) for genomic testing using 'in house' control primers was performed (Ethics approval: 10/H0713/26).Results: The median (inter quartile range) gestation of the fetuses was 22 (17, 31) weeks and the PM interval was 5.5 (3.7, 7.7) days. Muscle, kidney and placenta gave the highest average yields for non-degraded genomic DNA. Liver DNA had lowest quality;this was due to tissue degradation PM interval or gestational age did correlate with DNA yield or quality. Figure [DNA yield from various fetal tissues] Figure [DNA quality (L=liver, K=kidney, H=Heart, M=muscle)] Conclusion: High quality genomic DNA can be extracted from fetal kidney, muscle and placenta at autopsy, despite advanced maceration.

Comparison of DNA Isolation Kits to Extract DNA from Whole Blood Samples

Background and aim Genomic deoxyribonucleic acid (DNA) from white blood cells (WBCs) is widely used for polymerase chain reaction (PCR). Although kits for DNA isolation are in common use, there is scarce information about their performance and the PCR quality of the genomic DNA obtained. Hence, three different kits, QIAamp blood mini kit, High Pure PCR Template Preparation Kit and the Puregene DNA isolation kit were evaluated on these aspects. Materials and methods Genomic DNA was isolated from whole blood samples with WBC counts ranging from 0.5 to 20 x 10 WBC/l. The WBC count was used to calculate the amount of genomic DNA. The actual amount of genomic DNA isolated, was determined spectrophotometrically. The DNA extraction efficiency was obtained by dividing the actual amount of DNA by the calculated amount yielded. PCR quality was analysed by measuring Cycle threshold (Ct) values with a quantitative real-time PCR β-globin assay. Results The extraction efficiency of the three DNA isolation kits was 20% to 40%. Spectrophotometrically determined DNA concentrations correlated inversely with Ct values, regardless of the DNA isolation kit applied, whereas the strongest correlation was obtained for the Puregene DNA isolation kit. WBC counts also correlated inversely with Ct values and here the strongest correlation was found for the QIAamp blood mini kit. Conclusion The overall performance of the DNA isolation kits was quite comparable (DNA recoveries of 20 40%), albeit the QIAamp blood mini kit yielded the most reproducible extraction efficiencies and lowest Ct values within every WBC count category. Comparison of DNA isolation kits⏐59

Effects of Processing Techniques on the Forensic DNA Analysis of Human Skeletal Remains

Human remains processed by forensic anthropologists may potentially be used for genetic analysis. Therefore, the condition of the deoxyribonucleic acid (DNA) in processed remains may become an issue for future analysis. Processing techniques employed by anthropologists are highly variable and scanning electron microscopy reveals significant alterations to the bone surface depending upon the technique used. Such damage to the bone indicates differences may exist in quality and quantity of DNA extracted. This study assessed how five processing procedures used by major forensic anthropology laboratories around the country affects the amounts of DNA extracted from human rib bones and the subsequent DNA analysis. The DNA was analyzed using the short tandem repeat (STR) locus CSF1PO and amelogenin. The findings indicate processing procedures used by forensic anthropologists do not adversely affect DNA analysis but prolonged exposure to heat during processing may decrease the yield of information from the DNA.

Genomic DNA extraction from small amounts of serum to be used for α1-antitrypsin genotype analysis

If laboratory diagnosis of alpha1-antitrypsin (alpha1-AT) deficiency is usually based on its phenotype identification by isoelectric focusing, alpha1-antiprotease inhibitor (Pi)S and PiZ genotypes can also be determined by deoxyribonucleic acid (DNA)-based methods. Recently, several methods have been described for preparing genomic DNA from serum. The aim of the current study was to determine the Pi allele from serum extracted DNA by polymerase chain reaction (PCR) and to compare these results with those obtained with whole blood extracted DNA. Serum alpha1-AT concentration and phenotypic identification were systematically performed in 43 hospitalised patients. Genomic DNA was simultaneously purified from whole blood and from serum. The mutation detection was found using a PCR-mediated site-directed mutagenesis method. Concerning phenotypic identification, 29 patients were MM homozygotes, 11 were heterozygotes for S (MS = 7) or for Z (MZ = 4) and three showed a ZZ phenotype. Genotyping analyses gave identical results with serum and whole blood extracted DNA and all the results were in agreement with the phenotyping results. The authors found that the deoxyribonucleic acid-based test proved to be a reliable tool for alpha1-antitrypsin deficiency diagnosis and appears to be an alternative for the labour intensive alpha1-antitrypsin determination by isoelectric focusing. The authors also concluded that this method yields good quality deoxyribonucleic acid from serum, equal to that extracted from whole blood and is helpful in retrospective studies of multiple genetic markers.

Molecular Papanicolaou tests in the twenty-first century: Molecular analyses with fluid-based Papanicolaou technology

Objective: This study was undertaken to demonstrate the feasibility of performing molecular analyses at the deoxyribonucleic acid, ribonucleic acid and protein levels of cervical cytologic examination with a methanol fluid-based Papanicolaou specimen collection system. Study Design: Genomic deoxyribonucleic acid and total ribonucleic acid were extracted from cell pellets obtained from the residual fluid-based Papanicolaou specimen collection buffer after clinical processing. Genomic and human papillomavirus deoxyribonucleic acid polymerase chain reaction and reverse transcriptase–polymerase chain reaction were performed. Messenger ribonucleic acid transcript analysis and human papillomavirus 16 E6 mutational analysis were also performed. Methylation-specific polymerase chain reaction was used to evaluate hypermethylation status of the p16 gene and the gene for E-cadherin. Immunohistochemical staining for protein expression was performed on the processed monolayer slides. Results: Cell pellets from the residual fluid-based cytologic specimen yielded good quality deoxyribonucleic acid and ribonucleic acid. Molecular analyses of genomic deoxyribonucleic acid were successful for the identification of human papillomavirus E6 and p53 polymorphism status by means of restriction enzyme digestion and direct sequencing. Methylation status of the promotor regions of the p16 tumor suppressor gene and the gene for E-cadherin were also successfully identified. Ribonucleic acid was used as the template for transcript analysis and mutational analysis of the corresponding complementary deoxyribonucleic acid of the p53 gene. Protein expression analysis was demonstrated by immunohistochemical staining for carcinoembryonic antigen. Conclusion: It is feasible to conduct multiple molecular analyses at the deoxyribonucleic acid, ribonucleic acid, and protein levels of the cervicovaginal cell pellets from the residual fluid-based Papanicolaou cytologic specimen. This relatively simple and widely used collection system will allow significant advances in molecular epidemiology and eventual development of a molecular Papanicolaou test. (Am J Obstet Gynecol 2000;183:39-45.)

The Effects of Environment and Substrata on Deoxyribonucleic Acid (DNA): The Use of Casework Samples from New York City

This study was designed to analyze the effects of the environment and substrata on the quality of deoxyribonucleic acid (DNA) isolated from evidentiary specimens. The quality of DNA isolated from actual casework specimens was determined by measuring its size by agarose gel electrophoresis. The information obtained could be used to predict the suitability of the DNA in the samples for restriction fragment length polymorphism (RFLP) analysis. The evidentiary specimens chosen for DNA were classified according to substrate (scrapings, plastic bags, synthetics, denim, and carpet) and according to a subjective evaluation of the condition of the stain (soiled, damp, or putrefied) and to its size (small or large). The results show that DNA of sufficient quality and high molecular weight (HMW) can be reliably isolated from bloodstains deposited on evidentiary items which have an unknown environmental history and which have dried onto a variety of substrata. Subsequent RFLP analysis of a selected number of these samples verified that the DNA was suitable for this type of analysis.

Nuclear Deoxyribonucleic Acid Ploidy in Squamous Cell Bladder Cancer

Flow cytometric analysis of nuclear deoxyribonucleic acid content was performed on 76 primary squamous cell bladder carcinomas treated between January 1970 and December 1975. Patients were followed for a median of 10.1 years. Nuclei were extracted from paraffin-embedded archival material and isolated nuclei were stained with propidium iodide. Of the 76 tumors 73 were evaluable by flow cytometry providing high quality deoxyribonucleic acid histograms: 27 (37 per cent) showed a deoxyribonucleic acid diploid or normal pattern, 17 (23 per cent) exhibited a significant increase in the 4C peak (deoxyribonucleic acid tetraploid) and 29 (40 per cent) showed a distinct aneuploid peak. High grade (grades 3 and 4) and high stage (stages T2 to T4) tumors had a significantly higher incidence of abnormal (either tetraploid or aneuploid) deoxyribonucleic acid patterns than low grade (grades 1 and 2) and low stage (stages Tis/Ta/Tl) tumors (p less than 0.005). The 5 and 10-year rate free of disease for patients with deoxyribonucleic acid diploid tumors was 67 per cent compared to 22 and 18 per cent, respectively, for patients with tumors showing abnormal ploidy patterns (p less than 0.0005). At 5 and 10 years after diagnosis an estimated 18 per cent of the patients with deoxyribonucleic acid diploid tumors will die of bladder cancer. In contrast, an estimated 53 per cent of the patients with tetraploid tumors and 82 per cent and 86 per cent of those with aneuploid tumors will die of squamous cell bladder carcinoma by 5 and 10 years after diagnosis (p less than 0.0001). These results demonstrate that nuclear deoxyribonucleic ploidy measured by flow cytometry is an important objective prognostic variable for patients with squamous cell carcinoma of the bladder.