Quality Deoxyribonucleic Acid Research Articles

Effect Of Gentamicin And Neomycin+Bacitracin On Deoxyribonuclease Activity I On The Quality Of Human DNA Of Salivary Origin

Deoxyribonucleic acid (DNA) can be found in saliva and various other sources. Saliva contains epithelial cells and leukocytes that are released into the oral cavity. Cells in saliva are potential sources of DNA for diagnostic purposes. DNA in saliva can be degraded due to the activity of deoxyribonuclease (DNase), an enzyme that catalyzes the hydrolysis of DNA. DNase I is expressed in exocrine cells while DNase II is expressed in macrophages. Inhibition of DNase activity will preserve DNA. Besides being able to kill bacteria, aminoglycosides (i.e. gentamicin and neomycin) can inhibit DNase. Saliva was collected from 8 subjects. Furthermore, saliva samples from each subject were divided into 4 groups, namely negative control (K1), positive control (K2), gentamicin (K3), and neomycin + bacitracin (K4). DNA was extracted from saliva using the spin column method. DNA from groups K3 and K4 were added to 1 mg/mL gentamicin and 20 mM neomycin + bacitracin, respectively, before adding 2.5 g/mL DNase I (DNA degradation assay). The quality of human genomic DNA from saliva was determined by the presence of the NOTCH2 gene amplicons (~704 bp). DNA was separated by 1% agarose gel electrophoresis and recorded using the gel documentation system. The DNA extracted from saliva using the spin column method had an average concentration of 26.49 + 30.70 ng/mL and an average purity of 1.819 + 0.122. The administration of distilled water as a negative control in the DNA degradation assay showed that no DNA was digested.

The Effect Of Streptomycin And Clindamycin On Dnase I Activity On The Quality Of Dna Of Salivary Origin

Deoxyribonucleic acid (DNA) contains genetic material with all an individual’s information. Saliva has the potential to be a good source of human DNA. When compared to blood sampling, saliva is easier to collect and the collection process is non-invasive. However, human DNA collected from saliva has the potential to be degraded due to deoxyribonuclease (DNase) activity in saliva. DNase activity can be inhibited by antibiotics, such as aminoglycoside antibiotics and lincomycin derivatives. This study aimed to determine the effect of streptomycin and clindamycin on DNAse I activity against the quality of human genomic DNA from saliva. The sampling technique was that samples were collected from 9 subjects with each subject flowing into a saliva pot. Before saliva collection, subjects were asked to rinse their mouth with a solution of chlorhexidine gargle (Listerin™) for 30 seconds, then the samples were divided into four groups. The average concentration of DNA extracted from the spin-column method was 32.91 g/mL (7.10-99.45 g/mL) while the average purity was 1.813 g/mL (1.639-2.043 g/mL). With clindamycin treatment, PCR was able to amplify the human NOTCH2 gene (~704 bp) while various concentrations of streptomycin produced multiple bands of ~100 bp. The concentration of 3.2 mM clindamycin effectively inhibits DNAse I activity and can amplify the human NOTCH2 gene while streptomycin cannot protect.

Genetic diversity and population structure of some Nigerian accessions of Bambara groundnut (Vigna subterranea (L.) Verdc.,) using DArT SNP markers

Bambara groundnut is one of the crops with inadequate molecular research to show its full potentials. Previous studies showed morphological diversity with inadequate information to confirm genetic variations. In the quest to reveal the genetic potentials, deoxyribonucleic acid (DNA) of the selected accessions was extracted through leaf samples at 3 weeks old, using Dellaporta Miniprep for Plant DNA Isolation procedure. The high quality DNA was sequenced using Diversity Arrays Technology (DArT) markers to unlock diversity among Bambara groundnut of Nigerian origin. Cluster analysis (neighbor-joining clustering) of the single nucleotide polymorphisms (SNP’s) were used to generate sub-population to show relatedness and differences. Seven sub-populations were generated with 5927 (50.13%) high quality DArT markers out of the 11,821 SNPs generated. This revealed high genetic diversity existed among the selected Bambara groundnut accessions in Nigeria. This also revealed that DArT markers were highly efficient in classifying the accessions based on molecular expressions. This study also identified markers responsible for genetic variation that could facilitate the characterization of larger collections for further utilization of genetic resources and most importantly Bambara groundnut for the purpose of crop improvement.

Influence of preanalytical variables on the quality of cell-free DNA. Biobanking of cell-free DNA material

The search for early disease markers and the development of diagnostic systems has recently been expanding within genomics. Genomic deoxyribonucleic acid (DNA), cell-free DNA (cfDNA) and microbiome DNA obtained from different types of samples (tissues, blood and its derivatives, feces, etc.) are used as objects of genetic research. It has been shown that cfDNA that enters the bloodstream, in particular, as a result of apoptosis, necrosis, active tumor secretion and metastasis, is of great importance for studying molecular mechanisms of the pathological process and application in clinical practice. Circulating nucleic acid analysis can be used to monitor response to treatment, assess drug resistance, and quantify minimal residual disease. The review article reflects the following information about the biomaterial: source of cfDNA, methods of cfDNA isolation, storage and use for the diagnosis of certain diseases. Cell-free DNA can be present in biological fluids such as blood, urine, saliva, synovial and cerebrospinal fluid. In most cases, cfDNA is isolated from blood derivatives (serum and plasma), while it is most correct to use blood plasma for cfDNA isolation. Optimal and economically justifiable is the use of ethylenediaminetetra-acetic acid tubes for taking blood and obtaining plasma with subsequent cfDNA isolation. There is evidence that the optimal shelf life in an ethylenediaminetetra-acetic acid tube from the moment of blood sampling to subsequent isolation is a 2-hour interval. After centrifugation, cfDNA in plasma (or serum) can be stored for a long time at a temperature of -80O C. Storage at -20O C is undesirable, since DNA fragmentation increases.

A simple and cost-effective method for DNA extraction suitable for PCR in “sucupira branca”

“Sucupira branca” is a plant found in the Brazilian Cerrado and is adapted to low fertility soils, and its fruit extract has anti-inflammatory, healing, antiulcerogenic, antimicrobial, cercaricidal, leishmanicidal and antioxidant activities. Furthermore, it provides protection against oxidative stress, is a natural biocontrol agent of Aedes aegypti, has very resistant wood, is a melliferous plant and has been used in reforestation programs. The development of conservation strategies is important for maintaining diversity in natural populations of “sucupira branca” since these populations are in the process of genetic erosion. Molecular biology techniques, which are important for characterizing the genetic diversity of plants to develop conservation strategies, require sufficient high-quality genomic deoxyribonucleic acid (DNA). This study aimed to compare five methods to extract DNA from “sucupira branca”. The quality and concentration of DNA were revealed by agarose gel electrophoresis, and only the protocols of Dellaporta, Wood and Hicks et al. (1983) and Khanuja et al. (1999) did not result in satisfactory quantities of DNA. When PCR (Polymerase Chain Reaction) was performed with three inter-simple sequence repeat (ISSR) primers, DNA was successfully amplified from extractions performed with the protocols proposed by Doyle and Doyle (1987), Romano and Brasileiro (1998) and Ferreira and Grattapaglia (1995), which are less expensive than commercial purification kits. These protocols resulted in DNA of sufficient quality and quantity after the amplification reactions were performed.

Development of a High Quality, Rapid, Efficient and Economical DNA Extraction Protocol from Climate Resilient Pearl Millet Crop Without Liquid Nitrogen

Extraction of good quality genomic deoxyribonucleic acid (DNA) from plants is a major prerequisite for molecular biology experiments. An efficient genomic DNA protocol must be simple, fast and cost effective with high yield and purity. Presence of polyphenols, polysaccharides and secondary metabolites in some plants hamper with DNA extraction making it a very laborious, difficult and time consuming procedure. Here, we portrayed a modified protocol based on the cetyl trimethyl ammonium bromide (CTAB) method to isolate DNA from climate resilient pearl millet leaf tissues having higher amount of polysaccharides. It also excludes the use of expensive chemicals and equipments like proteinase K, liquid nitrogen and tissue lyser. This method includes extraction of DNA using a buffer (pH 8.0) containing 200 mM Tris-HCl, 20 mM ethylenediamine tetracetic acid (EDTA),1.4 M NaCl, 2% CTAB, 2% sodium dodecyl sulphate (SDS) and 1.0 % β-mercaptoethanol followed by purification of DNA with phenol, chloroform, isoamyl alcohol and finally precipitation of DNA by sodium acetate and isopropanol. Good quality genomic DNA with sharp and clear bands was obtained from 48 pearl millet genotypes using this protocol. The yield of DNA varied from 105.2 to 328.3 ng/μl. The purity of DNA sample ranged from 1.74 to 1.95 based on the absorbance at A260/A280 ratio indicating that it’s free from ribonucleic acid (RNA) and protein contamination. PCR analysis using simple sequence repeat (SSR) primers exhibited consistent and reliable amplification products ranging from 150 to 650 bp.This study reveals a fast, simple, efficient, specific, reproducible, reliable and cost effective method for extraction of DNA from small to large number of plant samples amenable to PCR amplification and could be stored for longer duration.

English

Girardinia diversifolia (Link) Friis is a perennial herb commonly known as Himalayan giant nettle which belongs to the family Urticaceae. The plant has cultural, medicinal and economic importance. The plant contains high concentration of polysaccharides, polyphenols and secondary metabolites which obstructs the process of isolation of the Deoxyribonucleic Acid (DNA) and inhibits downstream Polymerase Chain Reaction (PCR) amplifications. This study protocol developed a DNA extraction protocol from leaf-tissue based on the Cetyltrimethylammonium bromide, and optimized the PCR protocol for Inter Simple Sequence Repeat (ISSR) analysis. Genomic DNA extraction process was conducted using modified Doyle and Doyle method to obtain good quality DNA. The method yielded 445 ng/µL of DNA, where the purity ranged from 1.8-2.0 indicating minimum contamination of metabolites. The optimum condition for ISSR analysis was established using 4 mM MgCl2 , 0.6 mM dNTPs, 2.0 U Taq polymerase, 50 ng template DNA, and 0.7 µM primer. PCR program was optimized in the sequence of denaturation at 94°C for 3 min, subsequently followed by 45 cycles at 94°C for 30 s, annealing temperature at 45°C for 30 s, extension at 72°C for 2 min, and final extension at 72°C for 10 min. The modified technique was found to be ideal for isolation of genomic DNA and optimization of PCR process for ISSR analysis of G. diversifolia. The results of the research are beneficial for future molecular characterization and genetic diversity analysis of allied taxa. Key words: Girardinia diversifolia, Himalayan nettle, DNA isolation.

Isolation of genomic DNA from groundnut plant by modified, rapid and efficient protocol

DNA isolation is prerequisite to study the crop at molecular level. However, DNA (Deoxyribonucleic Acid) isolation is difficult in groundnut due to presence of large amount of polyphenols and polysaccharides. The age and growth stage of the plant also affect the DNA purity during isolation. In this study, newly expanded leaves and stems of five days old seedlings and matured leaves of 20 days old seedlings were used for optimization of extraction protocol. High quality DNA was obtained from all plant materials by this modified CTAB (Cetyltrimethylammonium bromide) protocol without separate purification. Optimized protocol was used to isolate DNA from newly expanded leaves of 5 days old seedlings of 100 groundnut genotypes. DNA obtained ranged from 6.3-22.1 µg/ g of sample when quantified by Nano Drop spectrophotometer and was able to amplify with Simple sequence repeats (SSR) primers during Polymerase Chain Reaction (PCR).

Comparison of Methods for Genomic Deoxyribonucleic Acid (DNA) Extraction Suitable for Whole-Genome Genotyping in Traditional Varieties of Rice

The utilization and conservation traditional rice genotypes have attracted global attention. Optimization of DNA isolation protocol for genetic characterization of plants is a necessary and primary step. Over the recent years, next generation sequencing and microarray technologies have revolutionized scientific research, with their applications to high-throughput analysis of biological systems. Isolation of high quantities of pure, intact, double stranded, highly concentrated DNA is prerequisite for successful and reliable large-scale genotyping analysis. Therefore, standardization of DNA isolation is a basic requirement. Here we employed three methods of DNA isolation namely, Dellaporta, Hi-purA and modified CTAB techniques for isolation of genomic DNA from 25 indigenous rice genotypes. From the results, it was found that genomic DNA isolated by modified CTAB method to be the most appropriate for extracting high quality and maximum quantity DNA suitable for genotyping. The quality and quantity of the differentially extracted DNA was subsequently assessed by spectrophotometric measurements and gel electrophoresis.

DNA extraction from placental, fetal and neonatal tissue at autopsy: what organ to sample for DNA in the genomic era?

Incorporation of genome and exome sequencing into fetal and neonatal autopsy investigations has been shown to improve diagnostic yield. This requires deoxyribonucleic acid (DNA) to be extracted from either the placenta or autopsy tissue for molecular testing. However, the sources and quality of DNA obtained are highly variable and there are no adequate published data on what tissue is most ideal to sample for DNA extraction in this setting. Here we compare the quality of DNA extracted from sampling the placenta and various solid organs at fetal and neonatal autopsy, thereby determining the optimal tissue from which to source DNA for ancillary testing as part of the modern perinatal autopsy. A total of 898 tissue samples were obtained at autopsy from 176 fetuses (gestational ages 17-40 weeks) and 44 neonates (age range 0-28 days) at our tertiary institution. Fetal tissue was processed using the QIAsymphony DSP DNA Mini kit and placental tissue was extracted using the New iGENatal Kit. DNA concentration was quantified using the Qubit dsDNA BR Assay Kit. DNA integrity, as stratified by gel electrophoresis was classified as high (≥5 kb) or low quality (<5 kb). Genome sequencing was performed on the extracted DNA, together with respective parental DNA from blood samples, and confirmed absence of maternal contamination in all cases. Analyses used logistic mixed models to test for associations between tissue types, intrauterine retention times, delivery to autopsy and death to autopsy intervals with DNA quality. In the fetal cohort, the placenta had the highest proportion of high quality DNA samples (93.1%), and liver had the lowest proportion (35.3%). Among the neonates, all tissue samples with the exception of liver had over 88% high DNA quality with the placenta also yielding the highest quality (100%). There was statistically significant deterioration in DNA quality with prolonged time interval between demise and autopsy (≥5 days). In the 726 fetal samples, the odds of obtaining higher quality DNA from the placenta, thymus, and spleen were 70.4 [95% confidence interval (CI) 29.2-169.6], 3.6 (95% CI 2.0-6.6) and 3.3 (95% CI 1.8-6.1) times, respectively, more likely than samples from the liver (p values <0.001). DNA yield from other fetal solid organs investigated was not significantly superior to that from the liver. This study shows that, when available, refrigerated unfixed placenta is the most suitable source of high quality DNA during perinatal investigations. Of the solid fetal organs sampled at autopsy, lymphocyte-rich, lytic enzymes-poor organs such as thymus and spleen were significantly more likely to yield good quality DNA than the liver.

English

Extraction of non-degraded and contaminant-free DNA from field specimen requires collection under liquid nitrogen which is not readily available in resource constrained laboratories in low and middle income countries (LMICs). A method of extracting DNA from silica gel-preserved common bean (Proteus vulgaris L.) leaves is presented. The method, which does not involve the use of phenol, chloroform or isoamyl alcohol also obviates the need for low temperature incubation during the DNA extraction steps and the grinding of desiccated leaf tissue in liquid nitrogen. It relies on inactivating proteins using SDS and proteinase K along with precipitation of polysaccharides using a high salt solution (0.8 M NaCl). DNA is further purified by exploiting its insolubility in aqueous media. High quality pure DNA (mean concentration 2.84 ± 0.013 µg/g of dry leaf tissue) with mean DNA purity values of 2.1 ± 0.1 was extracted. The DNA was also found to be free of protein and polysaccharide contamination. This method enables DNA amplification using molecular markers routinely used in molecular biology laboratories like random amplified polymorphic (RAPD) markers, inter simple sequence repeat (ISSR) markers, sequence-characterized amplified region (SCAR) markers and simple sequence repeat (SSR) markers. The findings of this study show that it is possible to obtain high quality DNA from leaf tissue preserved in silica gel. The method used in this research will be invaluable to resource constrained laboratories in low and middle income countries (LMICs) that cannot afford to buy or access liquid nitrogen in order to extract high quality DNA and for research groups undertaking field surveys that require several days or weeks off station without laboratory freezers to maintain the integrity of the tissues which is crucial for obtaining high quality DNA. Key words: Random amplified polymorphic (RAPD), inter simple sequence repeat (ISSR), simple sequence repeat (SSR), sequence-characterized amplified region (SCAR), deoxyribonucleic acid (DNA), low and middle income countries (LMICs).

Smith-Lemli-Opitz Mutations in Unexplained Stillbirths.

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive syndrome caused by a defect in cholesterol biosynthesis with mutations in 7-dehydrocholesterol reductase (DHCR7). A total of 3% of Caucasians carry DHCR7 mutations, theoretically resulting in a homozygote frequency of 1/4000. However, SLOS occurs in only 1/20,000 to 60,000 live births. Our objective was to assess DHCR7 mutations in unexplained stillbirths. Prospective, multicenter, population-based case-control study of all stillbirths and a representative sample of live births enrolled in five geographic areas. Cases with stillbirth due to obstetric complications, infection, or aneuploidy, and those with poor quality deoxyribonucleic acid (DNA) were excluded. DNA was extracted from placental tissue stored at -80°C, and exons 3 to 9 of the DCHR7 gene were amplified, purified, and subjected to bidirectional sequencing to identify mutations. One-hundred forty four stillbirths were unexplained and had adequate DNA for analysis. Nine stillbirths of 139 (6.5%) had a single mutation in one allele in coding exons 3 to 9 of DHCR7 (Table 1). One case (0.7%) was a compound heterozygote for mutations in exons 3 to 9 of DHCR7; this fetus had no clinical or histologic features of SLOS. We detected SLOS mutations in only 0.7% of stillbirths. This does not support a strong association between unrecognized DHCR7 mutations and stillbirth.

English

Yam (Dioscorea species) and banana (Musa species) leaf sample contain high levels of polysaccharide and polyphenolic compounds. Extraction of good quality DNA from such leaves is usually problematic. Therefore, there is a need to extract good quality DNA in order to perform downstream DNA analysis, especially for the detection of viruses due to their low titration in some infected leaves. The cetyltrimethylammonium bromide (CTAB) DNA extraction method was selected in order to optimise DNA extraction from plant material with high level of polysaccharides. Four steps of the Lodhi et al. (1994) method were modified to be user friendly in less resource laboratories of developing countries. Yield and purity of the extracted DNAs were quantified using a NanoDrop 2000 spectrophotometer and by migration on an agarose gel after polymerase chain reaction (PCR) amplification. The results of the DNA yields and purity were in the range of 287.40&plusmn;2.23 to 424.95&plusmn;1.85 ng/ul and 2.10&plusmn;0.05 to 2.19&plusmn;0.04, respectively. Modification was found to yield DNA of reasonable quantity, quality and purity. Furthermore, the method can be used in a laboratory with less sophisticated waste disposal system. Key words: Deoxyribonucleic acid (DNA), cetyltrimethyl ammonium bromide (CTAB), yam, banana, tomato.

Quality Improvement of the DNA extracted by boiling method in Gram negative bacteria

Several methods of Deoxyribonucleic acid (DNA) extraction have been applied to extract bacterial DNA. The amount and the quality of the DNA obtained for each one of those methods are variable. The study aimed to evaluate bacterial DNA extraction using conventional boiling method followed by alcohol precipitation. DNA extraction from Gram negative bacilli was extracted and precipitated using boiling method with further precipitation by ethanol. The extraction procedure performed using the boiling method resulted in high DNA yields for both E. coli and K. pneumoniae bacteria in (199.7 and 285.7μg/ml, respectively) which was close to control method (229.3 and 440.3μg/ml). It was concluded that after alcohol precipitation boiling procedure was easy, cost-effective, and applicable for high-yield quality of DNA in Gram-negative bacteria.

Sperm quality and DNA integrity of coke oven workers exposed to polycyclic aromatic hydrocarbons.

The objective of this study was to assess sperm quality and deoxyribonucleic acid (DNA) integrity of coke oven workers exposed to polycyclic aromatic hydrocarbons (PAHs) as compared to control subjects. The coke oven workers (N = 52) and administrative staff (N = 35) of a steel plant served as the exposed and control groups, respectively. Exposure to PAHs was assessed by measuring 1-hydroxypyren. Analysis of sperm quality (concentration, motility, vitality, and morphology) was performed simultaneously with sperm DNA integrity analysis, including DNA fragmentation, denaturation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo). A questionnaire was conducted to collect demographic and potential confounding data. The coke oven workers had lower percentages of sperm motility, vitality and normal morphology than the control group, but the difference was not significant. For DNA integrity, the coke oven workers had significantly higher concentrations of bulky DNA adducts and 8-oxo-dGuo than the control subjects (p = 0.009 and p = 0.048, respectively). However, DNA fragmentation percentages did not significantly increase as compared to those in the subjects from the control group (p = 0.232). There was no correlation between sperm quality parameters and DNA integrity indicators. Occupational exposure of the coke oven workers to PAHs was associated with decreased sperm DNA integrity. Int J Occup Med Environ Health 2016;29(6):915-926.

Chaos-based image encryption using a hybrid genetic algorithm and a DNA sequence

The paper studies a recently developed evolutionary-based image encryption algorithm. A novel image encryption algorithm based on a hybrid model of deoxyribonucleic acid (DNA) masking, a genetic algorithm (GA) and a logistic map is proposed. This study uses DNA and logistic map functions to create the number of initial DNA masks and applies GA to determine the best mask for encryption. The significant advantage of this approach is improving the quality of DNA masks to obtain the best mask that is compatible with plain images. The experimental results and computer simulations both confirm that the proposed scheme not only demonstrates excellent encryption but also resists various typical attacks.

A comparison of the efficiency of five different commercial DNA extraction kits for extraction of DNA from faecal samples

Differences in the composition of the gut microbiota have been associated with a range of diseases using culture-independent methods. Reliable extraction of nucleic acid is a key step in identifying the composition of the faecal microbiota. Five widely used commercial deoxyribonucleic acid (DNA) extraction kits (QIAsymphony® Virus/Bacteria Midi Kit (kit QS), ZR Fecal DNA MiniPrep™ (kit Z), QIAamp® DNA Stool Mini Kit (kit QA), Ultraclean® Fecal DNA Isolation Kit (kit U) and PowerSoil® DNA Isolation Kit (kit P)) were evaluated, using human faecal samples. Yield, purity and integrity of total genomic DNA were compared spectrophotometrically and using gel electrophoresis. Three bacteria, commonly found in human faeces were quantified using real time polymerase chain reaction (qPCR) and total bacterial diversity was studied using denaturing gradient gel electrophoresis (DGGE) as well as terminal restriction fragment length polymorphism (T-RFLP). The measurements of DNA yield and purity exhibited variations between the five kits tested in this study. Automated kit QS exhibited the best quality and highest quantity of DNA. All kits were shown to be reproducible with CV values≤0.46 for DNA extraction. qPCR results showed that all kits were uniformly efficient for extracting DNA from the selected target bacteria. DGGE and T-RFLP produced the highest diversity scores for DNA extracted using kit Z (H′=2.30 and 1.27) and kit QS (H′=2.16 and 0.94), which also extracted the highest DNA yields compared to the other kits assessed.

N-Methylimidazolium modified magnetic particles as adsorbents for solid phase extraction of genomic deoxyribonucleic acid from genetically modified soybeans

N-Methylimidazolium modified magnetic particles (MIm-MPs) were prepared and applied in the solid phase extraction of genomic deoxyribonucleic acid (DNA) from genetically modified soybeans. The adsorption of MIm-MPs for DNA mainly resulted from the strong electrostatic interaction between the positively charged MPs and the negatively charged DNA. The elution of DNA from MPs–DNA conjugates using phosphate buffer resulted from the stronger electrostatic interaction of phosphate ions with MPs than DNA. In the extraction procedure, no harmful reagents (e.g. phenol, chloroform and isopropanol, etc.) used, high yield (10.4μg DNA per 30mg sample) and high quality (A260/A280=1.82) of DNA can be realized. The as-prepared DNA was used as template for duplex-polymerase chain reaction (PCR) and the PCR products were analyzed by a sieving capillary electrophoresis method. Quick and high quality extraction of DNA template, and fast and high resolution detection of duplex PCR products can be realized using the developed method. No toxic reagents are used throughout the method.

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.