DNA Isolation Protocol Research Articles

Effect of formalin fixation on pcr amplification of DNA isolated from healthy autopsy tissues

The aim of this study is to investigate the effects of formalin fixation on the degradation of DNA molecules in five different healthy tissues exempted during the autopsy, as well as the selection of the method that is most suitable for the DNA isolation. Heart muscle, liver, brain, lung and kidney tissue obtained from the healthy people who suddenly died from a violent death were used. The parts of tissue were fixed in 10% phosphate-buffered formalin as well as in 4% unbuffered formalin at room temperature. Morphology of tissue was studied using H&E staining. The DNA was isolated 6 h, 1–7 days (every 24 h), 10, 14, 28 days and 2 months after fixation using two different methods: extraction with phenol-chloroform-isoamyl alcohol as well as with PureLink Genomic DNA Kit. Yield and purity of the DNA samples were measured spectrophotometrically at 260 nm and 280 nm. The PCR amplifications of the glycerol-3-phosphate dehydrogenase 1 (GPD1, 150 bp), ß actin (ACTB, 262 bp) and ribosomal protein L4 (RPL4, 407 bp) genes were performed to evaluate the degree of DNA fragmentation. The RPL4 gene was amplified up to 72 h, ACTB gene up to 14 days and GPD1 gene up to 28 days from tissue fixed in phosphate-buffered formalin using phenol-chloroform-isoamylalcohol protocol for DNA isolation. Liver and kidney gave better results of PCR amplification, but statistical significance between tissues was not found. Preserving period, fixative and DNA extracting method are important factors for successful PCR amplification. The healthy tissue, fixed in phosphate-formalin up to 28 days, can be useful source in molecular studies. Tissues fixed in unbuffered formalin are suitable for molecular analysis up to 7 days.

A metagenomic study of the rumen virome in domestic caprids.

This project sought to investigate the domestic caprid rumen virome by developing a robust viral DNA isolation and enrichment protocol (utilizing membrane filtration, ultra-centrifugation, overnight PEG treatment and nuclease treatment) and using RSD-PCR and high throughput sequencing (HTS) techniques. 3.53% of the reads obtained were analogous to those of viruses denoting Siphoviridae, Myoviridae, Podoviridae, Mimiviridae, Microviridae, Poxviridae, Tectiviridae and Marseillevirus. Most of the sequenced reads from the rumen were similar to those of phages, which are critical in maintaining the rumen microbial populations under its carrying capacity. Though identified in the rumen, most of these viruses have been reported in other environments as well. Improvements in the viral DNA enrichment and isolation protocol are required to obtain data that are more representative of the rumen virome. The 102,130 unknown reads (92.31%) for the goat and 36,241 unknown reads (93.86%) for the sheep obtained may represent novel genomes that need further study.

Method for improving the quality of genomic DNA obtained from minute quantities of tissue and blood samples using Chelex 100 resin

BackgroundAlthough genomic DNA isolation using the Chelex 100 resin is rapid and inexpensive, the DNA obtained by this method has a low concentration in solution and contains suspended impurities. The presence of debris in the DNA solution may result in degradation of DNA on long term storage and inhibition of the polymerase chain reaction. In order to remove impurities and concentrate the DNA in solution, we have introduced modifications in the existing DNA isolation protocol using Chelex-100. We used ammonium acetate to precipitate proteins and a sodium acetate- isopropanol mixture to pellet out DNA which was washed with ethanol.ResultsA pure DNA pellet that can be dissolved in water or Tris-EDTA buffer and stored for a long time at − 80 °C was obtained. We also observed a 20-fold change in the DNA concentration following precipitation and re-dissolution.ConclusionOur method is different from other extraction methods since it uses non-toxic, easily available and inexpensive reagents as well as minimal amounts of blood or tissue samples for the DNA extraction process. Besides its use in sex determination and genotyping in lab animals as described in this paper, it may also have applications in forensic science and diagnostics such as the easy detection of pathogenic DNA in blood.

Isolation of high molecular weight and humic acid-free metagenomic DNA from lignocellulose-rich samples compatible for direct fosmid cloning.

Activity-based screening of metagenomic DNA libraries is a promising approach to fish out genes encoding novel bioactive compounds/enzymes of industrial importance. The starting point of such functional screening in fosmid vectors is isolation of high molecular weight (HMW) DNA of sufficient purity from diverse environments. Metagenomic DNA isolation protocols mostly employ mechanical cell lysis that yields fragmented DNA. Those established for HMW DNA using enzymatic lysis have not considered samples with high lignocellulose or humic acid content. Enzymes from such environments are in great demand for bioenergy, paper, and related industries. Thus, an improved method was standardized that has three key features, i.e., use of harvested microbial biomass instead of raw samples, removal of humic substances prior to cell lysis by aluminum sulfate flocculation, and enzymatic/chemical lysis of cells with a lysozyme, mutanolysin, proteinase K, and SDS cocktail followed by phenol-chloroform extraction and precipitation of DNA by polyethylene glycol and NaCl. HMW DNA (~ 40kb) was efficiently isolated from garden and forest soils, rice straw compost, and degrading wood from a hypersaline lake. The humic acid removal efficiency across samples was 96-98%. The isolated DNA was of high quality/purity and could be successfully used in downstream applications like PCR, ligation, and fosmid cloning. In fact, the DNA was directly used without any size selection, for fosmid library preparation with 70-90% efficiency as compared to the control insert. Thus, the method could suitably be used for HMW DNA isolation for the functional screening of enzymes from diverse humic acid-/lignocellulose-rich environments.

An Improved Protocol for Genomic DNA Isolation from Bryophyte Species

Genetic diversity studies of plants depend upon the quality and quantity of isolated DNA sample. For many lower groups of plants, apart from usual steps involved for DNA isolation, several additional steps are invariably required specific to the particular plant group. In bryophytes, nucleic acid isolation has always been a difficult task. The Bryophytes possess a substantial quantity of polyphenols and polysaccharides that are known to interfere with DNA isolation and Polymerase chain reaction amplification. In order to overcome these problems, an improved protocol is developed for DNA isolation by optimizing important components of the cetyltrimethylammonium bromide (CTAB) extraction protocol by varying concentrations of CTAB (2–4%), polyvinylpolypyrrolidone (PVP) (0–4%) and β-mercaptoethanol (0–1.5%). It was observed that CTAB (3%), PVP (2%), and β-mercaptoethanol (1%) is optimum with a DNA yield of 900–1582 µg/0.5 gm leaf sample. DNA concentration (25 ng/µl), MgCl2 (1.5 mM), primer (1 µm), annealing temperature (48–52 °C) and a number of cycle set at 35 is optimum for the reproducible and high-intensity Simple sequence repeats amplification. The efficacy of this protocol has been validated in 6 moss taxa and 2 liverwort taxa.

Comparison of detergent and CTAB method for isolation of DNA from Salak ( Salacca zalacca (Gaert.) Voss. ‘Pondoh’)

This study conducted in Laboratory of Molecular Biology, Balai Besar Bioteknologi dan Sumberdaya Genetik Pertanian (BB- Biogen) Bogor. The aims of this study are to determine and comparing the quantity, quality and the efficiency of DNA isolation result using detergent method and CTAB method. The parameters observed in this study are the value of DNA concentration, purity, and visualization result using gel electrophoresis. The samples are the leaves of Salak ‘Pondoh’ (Salacca zalacca (Gaert.) Voss.). Detergent method is a method which was developed by Faculty of Biology UGM, it has simple method and relatively affordable cost. Meanwhile, CTAB method is one of the commonly used methods of DNA isolation protocol with relatively expensive cost. Detergent method used detergent in the cell wall separation and protein removal in the sample. The CTAB method used Cetyltrimethyl ammonium bromide (CTAB) for cell membrane separation in the sample. The research methods included DNA isolation with detergent and CTAB methods, PCR analysis and electrophoresis. Data analysis was done quantitatively using spectrophotometric method and qualitative used electrophoresis method. The result of the study showed that DNA isolation using CTAB method showed higher purity compared with detergent method with the purity values ranging from 1,3- 1,4 . Meanwhile, the concentration of DNA in the detergent method was higher than that of CTAB with the highest concentration of 1730 µg/ml. There is no difference between the quality of genomic DNA isolated by CTAB and detergent methods.

Aerobic microbial taxa dominate deep subsurface cores from the Alberta oil sands.

Little is known about the microbial ecology of the subsurface oil sands in Northern Alberta, Canada. Biodegradation of low molecular weight hydrocarbons by indigenous microbes has enriched high molecular weight hydrocarbons, resulting in highly viscous bitumen. This extreme subsurface environment is further characterized by low nutrient availability and limited access to water, thus resulting in low microbial biomass. Improved DNA isolation protocols and increasingly sensitive sequencing methods have allowed an in-depth investigation of the microbial ecology of this unique subsurface environmental niche. Community analysis was performed on core samples (n = 62) that were retrieved from two adjacent sites located in the Athabasca Oil Sands at depths from 220 to 320 m below the surface. Microbial communities were dominated by aerobic taxa, including Pseudomonas and Acinetobacter. Only one core sample microbial community was dominated by anaerobic taxa, including the methanogen Methanoculleus, as well as Desulfomicrobium and Thauera. Although the temperature of the bitumen-containing subsurface is low (8°C), two core samples had high fractions of the potentially thermophilic taxon, Thermus. Predominance of aerobic taxa in the subsurface suggests the potential for in situ aerobic hydrocarbon degradation; however, more studies are required to determine the functional role of these taxa within this unique environment.

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens.

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

An Alternative Method for Extracting Plasmodium DNA from EDTA Whole Blood for Malaria Diagnosis.

Molecular techniques have been introduced for malaria diagnosis because they offer greater sensitivity and specificity than microscopic examinations. Therefore, DNA isolation methods have been developed for easy preparation and cost effectiveness. The present study described a simple protocol for Plasmodium DNA isolation from EDTA-whole blood. This study demonstrated that after heating infected blood samples with Tris–EDTA buffer and proteinase K solution, without isolation and purification steps, the supernatant can be used as a DNA template for amplification by PCR. The sensitivity of the extracted DNA of Plasmodium falciparum and Plasmodium vivax was separately analyzed by both PCR and semi-nested PCR (Sn-PCR). The results revealed that for PCR the limit of detection was 40 parasites/μl for P. falciparum and 35.2 parasites/μl for P. vivax, whereas for Sn-PCR the limit of detection was 1.6 parasites/μl for P. falciparum and 1.4 parasites/μl for P. vivax. This new method was then verified by DNA extraction of whole blood from 11 asymptomatic Myanmar migrant workers and analyzed by Sn-PCR. The results revealed that DNA can be extracted from all samples, and there were 2 positive samples for Plasmodium (P. falciparum and P. vivax). Therefore, the protocol can be an alternative method for DNA extraction in laboratories with limited resources and a lack of trained technicians for malaria diagnosis. In addition, this protocol can be applied for subclinical cases, and this will be helpful for epidemiology and control.

Evaluating Protocols for Porcine Faecal Microbiome Recollection, Storage and DNA Extraction: from the Farm to the Lab.

There is a growing interest in understanding the role of the gut microbiome on productive and meat quality-related traits in livestock species in order to develop new useful tools for improving pig production systems and industry. Faecal samples are analysed as a proxy of gut microbiota and here the selection of suitable protocols for faecal sampling and DNA isolation is a critical first step in order to obtain reliable results, even more to compare results obtained from different studies. The aim of the current study was to establish in a cost-effective way, using automated ribosomal intergenic spacer analysis technique, a protocol for porcine faecal sampling and storage at farm and slaughterhouse and to determine the most efficient microbiota DNA isolation kit among those most widely used. Operational Taxonomic Unit profiles were compared from Iberian pig faecal samples collected from rectum or ground, stored with liquid N2, room temperature or RNAlater, and processed with QIAamp DNA Stool (Qiagen), PowerFecal DNA Isolation (Mobio) or SpeedTools Tissue DNA extraction (Biotools) commercial kits. The results, focused on prokaryote sampling, based on DNA yield and quality, OTU number and Sørensen similarity Indexes, indicate that the recommended protocol for porcine faecal microbiome sampling at farm should include: the collection from porcine rectum to avoid contamination; the storage in liquid N2 or even at room temperature, but not in RNAlater; and the isolation of microbiota DNA using PowerFecal DNA Isolation kit. These conditions provide more reliable DNA samples for further microbiome analysis.

Plant Genomic DNA Isolation-the Past and the Present, a Review

Successful isolation of a good quality of plant genomic DNA in sufficient quantity is a necessary prerequisite for its downstream applications, that are aimed to examine and analyze the molecular intricacies of the plant kingdom. The quantity and quality of isolated genomic DNA often determines the accuracy and robustness of results derived from many DNA based applications. The choice of the method adopted for DNA isolation depends on a large number of factors like the amount, nature and age of the starting plant tissue, the infrastructure available in terms of laboratory facilities, ready availability of required laboratory chemicals, time at hand and the downstream applications of the obtained DNA. A large number of modifications have been suggested to optimize DNA isolation from a particular plant species or tissue. There cannot be a universal protocol for DNA isolation across all plant species or tissues owing to the highly heterogeneous nature of plant cells. Hence, efforts need to be made to carry out suitable variations in the extraction method, so as to develop a protocol which is best suited to a particular plant species or tissue or application. So far, a large number of DNA isolation protocols from plants have been published, but there is no comprehensive report on suitability of these methods over each other and the different modifications which have been attempted to optimize the yield and quality of DNA. Many review papers have described various DNA isolation protocols and their underlying principles, that have been adopted in order to optimize DNA isolation from different plant species and tissues (Varma et al., 2007; Tan and Yiap 2009; Kumari et al., 2012). However, there is no comprehensive study which gives details of modifications at different steps of the plant genomic DNA isolation protocol, that have been attempted by researchers to overcome the specie and tissue specific limitations. This review gives a detailed account of the steps involved in genomic DNA isolation from various plant species and tissues and how each of these steps have been modified to overcome and eliminate the problem of contaminants in the extracted DNA so as to obtain a good quality of DNA amenable to downstream molecular biology applications.

Glutathione S-Transferase M1 and T1 Gene Deletions and Susceptibility to Acute Lymphoblastic Leukemia (ALL) in adults.

Objective:Biotransformation of xenobiotics are critical for their metabolism and removal from the body which is carried out by xenobiotic metabolizing enzymes. Individuals carrying variants of genes that encode these enzymes have an altered ability to metabolize xenobiotics which may lead to an increased risk of acute lymphoblastic leukemia. The current study aimed to investigate the impact of GSTM1 and GSTT1 gene deletions in causing predisposition to adult ALL.Methods:The current case-control study involved 62 adult ALL patients and 62 age and gender matched healthy controls. Whole blood samples processed with standard phenol chloroform protocol for DNA isolation were genotyped using multiplex PCR approach for simultaneous identification of GSTM1 and GSTT1 deletions. The genotype frequency obtained for patients was compared to controls using odds ratio and chi-square.Results:The null genotype frequency of GSTM1 and GSTT1 in a group of adult ALL patients from Pakistan were 47% and 11% respectively. Deletion of GSTM1 and GSTT1 did not show statistically significant association with adult ALL (p=0.86 and p=0.35 respectively). The combined GSTM1/GSTT1 deletion was observed in 2% patients and was not significantly associated with ALL in adults (p=0.85).Conclusions:The results reveal that homozygous null polymorphism of GSTM1 and GSTT1genes does not influence ALL susceptibility among adult patients. Cancer susceptibility associated with GST polymorphism varies with ethnic and geographic differences. Therefore, further investigation on different populations is needed to understand the role of these genetic variations in modifying adult ALL risk.

Profiling soil microbial communities with next-generation sequencing: the influence of DNA kit selection and technician technical expertise.

Structure and diversity of microbial communities are an important research topic in biology, since microbes play essential roles in the ecology of various environments. Different DNA isolation protocols can lead to data bias and can affect results of next-generation sequencing. To evaluate the impact of protocols for DNA isolation from soil samples and also the influence of individual handling of samples, we compared results obtained by two researchers (R and T) using two different DNA extraction kits: (1) MO BIO PowerSoil® DNA Isolation kit (MO_R and MO_T) and (2) NucleoSpin® Soil kit (MN_R and MN_T). Samples were collected from six different sites on Okinawa Island, Japan. For all sites, differences in the results of microbial composition analyses (bacteria, archaea, fungi, and other eukaryotes), obtained by the two researchers using the two kits, were analyzed. For both researchers, the MN kit gave significantly higher yields of genomic DNA at all sites compared to the MO kit (ANOVA; P < 0.006). In addition, operational taxonomic units for some phyla and classes were missed in some cases: Micrarchaea were detected only in the MN_T and MO_R analyses; the bacterial phylum Armatimonadetes was detected only in MO_R and MO_T; and WIM5 of the phylum Amoebozoa of eukaryotes was found only in the MO_T analysis. Our results suggest the possibility of handling bias; therefore, it is crucial that replicated DNA extraction be performed by at least two technicians for thorough microbial analyses and to obtain accurate estimates of microbial diversity.

A Cost-Efficient and Simple Plant Oil DNA Extraction Protocol Optimized for DNA-Based Assessment of Product Authenticity

DNA-based assays offer precision in ascertaining the species/cultivar origin of agro-food products. Yet, obtaining DNA of sufficient quality and quantity is the main challenge while performing DNA-based food authentication analyses. The aim of the present work was to standardize a cost-efficient, easy-to-apply, yet effective plant oil DNA isolation protocol that allows reliable downstream PCR-based analyses. Because capillary electrophoresis (CE) separation of species/cultivar discriminating genomic fragments is a widely adopted approach in food genomics, a CE system was utilized in order to assess the performance of the proposed cetyl trimethyl ammonium bromide (CTAB)-based protocol. A plastid intergenic spacer and a nuclear olive gene were used as targets in order to evaluate the amplificability of DNA extracted with the CTAB-based protocol. The plastid barcode not only allowed assessing the reproducibility of PCR amplifications from the extracted oil DNA samples (olive, hazelnut, corn, rapeseed, cottonseed, and soybean oils) but also proved successful in discriminating all tested oil crop species based on amplified fragment length polymorphisms. Moreover, the barcode assay proved successful in correctly identifying the tested olive oil: cottonseed oil blends as admixtures of the two oil species. Thus, it was also feasible to demonstrate the potential of the barcode sequence as a discriminatory analyte to detect adulteration in plant oils. In addition, application of a CAPS (cleaved amplified polymorphic sequence) assay designed to genotype a nuclear SNP (single nucleotide polymorphism) marker resulted in the successful identification of the two single-cultivar olive oils included in the study. As a result of the present work, it was feasible to standardize a reliable and cost-efficient DNA extraction protocol that works well with both unrefined (olive and hazelnut) and refined (corn, rapeseed, cottonseed, and soybean) oils.

Rapid and affordable size-selected PacBio single-molecule real-time sequencing template library construction using the bead-beating DNA extraction method.

This study demonstrated that bead-beating method facilitates a simple and rapid protocol for genomic DNA isolation for Pacific BioSciences (PacBio) sequencing with library construction of sufficient length. The protocol may also be beneficial for inactivating pathogens by simultaneous and instant DNA fragmentation, with no special equipment required to obtain large DNA fragments. This protocol was comparable in terms of quality to the standard protocol suggested by PacBio and represents an alternative, rapid shortcut for performing accurate PacBio sequencing.

A Standardized Protocol for Genomic DNA Isolation from the Species of Plumbago Linn

A Standardized Protocol for Genomic DNA Isolation from the Species of Plumbago Linn

A rapid and cost effective protocol for plant genomic DNA isolation using regenerated silica columns in combination with CTAB extraction

AbstractIsolation of high quality DNA from multiple samples can be both time-consuming and expensive. We have developed a combined protocol to reduce the time component of the hexadecyltrimethylammonium bromide (CTAB) extraction method and reduced costs by regenerating the silica columns used to purify genomic DNA. We present data that shows, by increasing the temperature used during the CTAB method, the time required to extract crude genomic DNA can be reduced. We show that silica columns can be regenerated using HCl and still maintain their DNA-binding capacity. Furthermore, we show both spectrophotometrically, and by restriction enzyme cutting, that the quality of the eluted DNA is high. Critically, using both genomic DNA from pea and perennial ryegrass we demonstrate, using species-specific PCR primers, that there is no carry-over of DNA from repeated use of a single column. The main advantages of the method are high yield, high quality, cost effectiveness and time-saving. This method could satisfy demand when large numbers of plant genomic DNA samples are required, for example from targeting induced local lesions in genomes (TILLING) populations.

DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation.

Potential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin columns under different conditions, and salting-out. We first analysed DNA using array CGH, which revealed a striking wave pattern suggesting primarily GC-rich cerebellar losses, even against matched frontal cortex DNA, with a similar pattern on a SNP array. The aCGH changes varied with the isolation protocol. Droplet digital PCR of two genes also showed protocol-dependent losses. Whole genome sequencing showed GC-dependent variation in coverage with spin column isolation from cerebellum. We also extracted and sequenced DNA from substantia nigra using salting-out and phenol / chloroform. The mtDNA copy number, assessed by reads mapping to the mitochondrial genome, was higher in substantia nigra when using phenol / chloroform. We thus provide evidence for significant method-dependent bias in DNA isolation from human brain, as reported in rat tissues. This may contribute to array “waves”, and could affect copy number determination, particularly if mosaicism is being sought, and sequencing coverage. Variations in isolation protocol may also affect apparent mtDNA abundance.

OPTIMIZED DNA ISOLATION PROTOCOL ENABLES AN INSIGHT INTO MOLECULAR GENETIC BACKGROUND OF ENDEMIC Moltkia petraea (Tratt.) Griseb. FROM BOSNIA AND HERZEGOVINA

The Dinaric endemic plant species Moltkia petraea (Tratt.) Griseb. is often called a "living fossil" of ancient Tertiary flora, with great importance for Bosnia and Herzegovina’s biodiversity. Considering its narrow and limited distribution range, insufficient data on the molecular background of this species is given so far. Due to the presence of various secondary metabolites that interfere with the DNA, isolation of nucleic acids from plant cells is known to be challenging. Even in closely related species it is necessary to optimize DNA isolation protocol in order to obtain high quality PCR amplifiable DNA. We collected 91 samples from five populations in Herzegovina. Doyle and Doyle (1987) CTAB protocol was modified by adding vitamin C (ascorbic acid) to the cell lysis buffer to improve DNA yield and quality. trnL(UAA) intron and nrDNA (ITS1, ITS2) molecular markers were applied to demonstrate amplifiability of isolated DNA and elucidate the intra- and interpopulation genetic diversity. Our results suggest a successful PCR amplification for 81% of the analyzed samples. PCR-RFLP analysis of trnL(UAA) revealed that all individuals in five populations have the same haplotype based on the obtained enzymatic profile for three enzymes (TaqI, HinfI, HindII). Alignment and comparison of ITS sequences didn’t reveal any hypervariable portion that could be informative in elucidating the genetic diversity of M. petraea populations. Further studies with additional application of microsatellite loci, RAPD and AFLP methods are necessary in an attempt to get insights into the genetic diversity of M. petraea.