Amplification Of DNA Sequences Research Articles

DNA barcoding in concentrated Chinese medicine granules using adaptor ligation-mediated polymerase chain reaction

The use of DNA barcodes for species identification is a common laboratory practice. However, PCR amplification of full-length DNA barcode in processed material is difficult because of severe DNA fragmentation. In this study, an adaptor ligation-mediated PCR protocol was derived to amplify sets of target DNA fragments isolated from two CCMG products. The specially designed adaptor with asymmetric strands and terminal modification avoids amplification of non-target DNA sequences. DNA extracted from Angelica sinensis and Panax notoginseng CCMG were ligated with the adaptors and amplified by an adaptor primer and a single universal barcode primer to obtain partial ITS2 sequence. Results showed that various length of DNA fragments within the ITS2 region were amplified and could be used to identify the concerned species. The adaptor ligation-mediated PCR is therefore a promising universal method for species identification in highly processed herbal products.

New Insights into Phasmatodea Chromosomes.

Currently, approximately 3000 species of stick insects are known; however, chromosome numbers, which range between 21 and 88, are known for only a few of these insects. Also, centromere banding staining (C-banding) patterns were described for fewer than 10 species, and fluorescence in situ hybridization (FISH) was applied exclusively in two Leptynia species. Interestingly, 10–25% of stick insects (Phasmatodea) are obligatory or facultative parthenogenetic. As clonal and/or bisexual reproduction can affect chromosomal evolution, stick insect karyotypes need to be studied more intensely. Chromosome preparation from embryos of five Phasmatodea species (Medauroidea extradentata, Sungaya inexpectata, Sipyloidea sipylus, Phaenopharos khaoyaiensis, and Peruphasma schultei) from four families were studied here by C-banding and FISH applying ribosomal deoxyribonucleic acid (rDNA) and telomeric repeat probes. For three species, data on chromosome numbers and structure were obtained here for the first time, i.e., S. inexpectata, P. khaoyaiensis, and P. schultei. Large C-positive regions enriched with rDNA were identified in all five studied, distantly related species. Some of these C-positive blocks were enriched for telomeric repeats, as well. Chromosomal evolution of stick insects is characterized by variations in chromosome numbers as well as transposition and amplification of repetitive DNA sequences. Here, the first steps were made towards identification of individual chromosomes in Phasmatodea.

Array-based sequencing of filaggrin gene for comprehensive detection of disease-associated variants

The filaggrin gene (FLG) is essential for skin differentiation and epidermal barrier formation with links to skin diseases, however it has a highly repetitive nucleotide sequence containing very limited stretches of unique nucleotides for precise mapping to reference genomes. Sequencing strategies using polymerase chain reaction (PCR) and conventional Sanger sequencing have been successful for complete FLG coding DNA sequence amplification to identify pathogenic mutations but this time-consuming, labour intensive method has restricted utility. Next-generation sequencing (NGS) offers obvious benefits to accelerate FLG analysis but standard re-sequencing techniques such as oligoprobe-based exome or customized targeted-capture can be expensive, especially for a single target gene of interest. We therefore designed a protocol to improve FLG sequencing throughput using a set of FLG-specific PCR primer assays compatible with microfluidic amplification, multiplexing and current NGS protocols. Using DNA reference samples with known FLG genotypes for benchmarking, this protocol is shown to be concordant for variant detection across different sequencing methodologies. We applied this methodology to analyze cohorts from ethnicities previously not studied for FLG variants and demonstrate usefulness for discovery projects. This comprehensive coverage sequencing protocol is labour-efficient and offers an affordable solution to scale up FLG sequencing for larger cohorts. Robust and rapid FLG sequencing can improve patient stratification for research projects and provide a framework for gene specific diagnosis in the future.

Multiplex-PCR As a Rapid and Sensitive Method for Identification of Meat Species in Halal-Meat Products.

Species identification and authentication in meat products are important subjects for ensuring the health of consumers. The multiplex-PCR amplification and species- specific primer set were used for the identification of horse, donkey, pig and other ruminants in raw and processed meat products. Oligonucleotid primers were designed and patented for amplification of species-specific mitochondrial DNA sequences of each species and samples were prepared from binary meat mixtures. The results showed that meat species were accurately determined in all combinations by multiplex-PCR, and the sensitivity of this method was 0.001 ng, rendering this technique open to and suitable for use in industrial meat products. It is concluded that more fraud is seen in lower percentage industrial meat products than in higher percentage ones. There was also more fraud found in processed products than in raw ones. This rapid and useful test is recommended for quality control firms for applying more rigorous controls over industrial meat products, for the benefit of target consumers.

Discovery of a Covalent Kinase Inhibitor from a DNA-Encoded Small-Molecule Library × Protein Library Selection.

We previously reported interaction determination using unpurified proteins (IDUP), a method to selectively amplify DNA sequences encoding ligand:target pairs from a mixture of DNA-linked small molecules and unpurified protein targets in cell lysates. In this study, we applied IDUP to libraries of DNA-encoded bioactive compounds and DNA-tagged human kinases to identify ligand:protein binding partners out of 32 096 possible combinations in a single solution-phase library × library experiment. The results recapitulated known small molecule:protein interactions and also revealed that ethacrynic acid is a novel ligand and inhibitor of MAP2K6 kinase. Ethacrynic acid inhibits MAP2K6 in part through alkylation of a nonconserved cysteine residue. This work validates the ability of IDUP to discover ligands for proteins of biomedical relevance.

Combining aptamer-modified gold nanoparticles with barcode DNA sequence amplification for indirect analysis of ethanolamine

An indirect analysis strategy for small organic molecules is presented herein, combining aptamer-modified gold nanoparticles (AuNPs) with a designed barcode sequence for amplification. Ethanolamine, which could not be analyzed directly through liquid chromatography coupled with mass spectrometry (LC/MS), was used as a model compound to demonstrate the feasibility of the proposed scheme. A poly-adenosine barcode sequence was part of the designed cDNA A1 (cDNA+barcode sequence) to provide a large number of adenine bases for signal amplification. In the presence of fixed amounts of the aptamer and cDNA A1, the concentration of ethanolamine could be estimated indirectly through measurement of the concentration of the hydrolyzed bases of the unbound cDNA A1. The thiol-terminated aptamer was immobilized on the AuNPs through Au–S self-assembly. The aptamer-immobilized AuNPs were removed, with their bound ethanolamine and cDNA A1, through centrifugation. The unbound cDNA A1 remaining in the supernatant was hydrolyzed and analyzed. Through indirect analysis and amplification of the cDNA A1, the concentration of ethanolamine could be analyzed with a linear range (on a logarithmic scale) between 5 and 5000nM (detection limit: 1.2nM). This developed method might be applied as a general platform for indirect detection using LC/MS analysis.

Gene Overexpression in Streptomyces hygroscopicus Associated with DNA Amplification.

The genetics of the Streptomyces hygroscopicus strain 10-22 is of interest due to the ability of this strain to produce antifungal compounds. Strain T110 was obtained through insertional mutagenesis of strain 10-22 and was found to have undergone DNA amplification, as determined by both conventional and pulsed-field gel electrophoresis (PFGE). pIJ702, the vector used for insertional mutagenesis, was shown to have integrated into and co-amplified with the chromosomal DNA sequence of T110, as pIJ702 hybridized predominantly with two of the three amplified BamHI fragments. The amplified DNA sequence in T110 is 10.8kb in length and consists of 5.18kb of Streptomyces chromosomal DNA and the entire 5.62kb pIJ702 sequence. Sequence analysis of the 5.18kb chromosomal sequence revealed two open reading frames, one encoding a putative IS5 family transposase and the other encoding a putative dihydroxy-acid dehydratase. Real-time PCR analysis showed that expression of the putative dehydratase gene in T110 is about 50 times greater than in the wild-type strain, consistent with the high level of amplification of this DNA region, and therefore this system has the potential for producing economically or clinically important molecules.

DIVERSITY OF IRANIAN STRAINS OF PSEUDOMONAS SYRINGAE pv. SYRINGAE AND PSEUDOMONAS SYRINGAE pv. LACHRYMANS

Strains of Pseudomonas syringae pv. syringae (Pss), Pseudomonas syringae pv. lachrymans (Psl) and Pseudomonas tolaasii (P. tolaasii) were analyzed for genetic diversity. For molecular identification of the strains, three primer pairs were used. Molecular analysis was done with random amplified polymorphic DNA (RAPD) and Insertion Sequence (IS50) primers. IS50-PCR and RAPD-PCR analysis divided 51 strains into eight sub clusters that were grouped in three clusters. Cluster I comprised all Pss with four sub clusters from stone fruits, pome fruit and cereal hosts, cluster II contained Psl strains from cucumber with two sub clusters and cluster III contained three strains of P. tolaasii with two sub clusters from mushroom. These results indicated that a combination of IS50-PCR and RAPD-PCR fingerprinting can be used as a high resolution genomic fingerprinting methods for differentiates species of Pseudomonas and pathovars of P. syringae strains.

Cloning of Novel Leukocytic X5 ARHGEF18 Proteoform for Functional Characterization

Rho GTPases are bio‐molecular switches in cells with activation depending on guanine nucleotide exchange factors (ARHGEFs) that facilitate GDP (guanosine diphosphate) release so GTP (guanosine triphosphate) can bind to the GTPase. When GTP is bound, GTPases are activated and contribute to cell signaling which includes regulating diverse responses such as gene transcription, cytoskeletal rearrangements, cell growth and motility. Eosinophils undergo a rapid and profound morphological change after cytokine stimulation. ARHGEF18 is abundant in eosinophils, and based on its activity in other cell types could be responsible for the formation of actin stress fibers allowing for the cell's dramatic polarization. However, our data indicates that eosinophil ARHGEF18 has an N‐terminal extension of 349 residues (part of LOC100996504 (LOC1009), which is annotated in UniProt as a unique protein) to comprise a 1364‐residue protein that has never been characterized. This project encompasses the cloning, expression, purification, and characterization of LOC1009‐ARHGEF18 (LOCGEF) and other constructs as a precursor to assessing the functional significance of LOCGEF for cytoskeleton rearrangement. The sequence of interest was created using PCR amplification of the complementary DNA sequences created from the eosinophil cellular mRNA with reverse transcription. Four separate constructs were created, one containing the entire sequence (LOCGEF), canonical ARHGEF18 without the LOC1009 sequence (p114; 1015 residues), and two sequences, one composed of LOC1009 exons 1–10 (LOC ex10; 322 residues), the other LOC1009 exons 1–11 (LOC ex11; 346 residues). The PCR product was restricted and ligated into a bacterial plasmid with a histidine tag encoded at the N‐terminus. E. coli were transformed with these plasmids using the heat shock method and plated onto kanamycin containing media. Those with the ability to grow on the media contained plasmids with the construct sequences. Bacterial expression of LOC ex10 and LOC ex11 was induced with Isopropyl β‐D‐1‐thiogalactopyranoside and the resulting proteins purified using a Ni‐NTA column. Purified LOC ex10 protein was tested in ELISA and detected using rabbit antibodies raised against the LOC1009 portion of the construct (anti‐LOC100996504 IgG; Abcam 185114). LOC ex11 has yet to be tested in ELISA. Plasmids for p114 and LOCGEF also have been created but protein yield for p114 and LOCGEF was poor in the E. coli expression system. The next step is to express these proteins in a eukaryotic system. The characterization of LOCGEF is important in understanding eosinophil cytoskeletal rearrangement needed for immune response. The function of the N‐terminal extension of LOCGEF and how it correlates with functions of the shorter isoform of ARHGEF18 previously studied in other cell types are unknown. The creation of these proteins and constructs will enable structural characterization, antibody binding, and discovery of binding partners in pull‐down assays.Support or Funding InformationNIH P01 HL088594 (Role of Eosinophils in Airway Inflammation and Remodeling)1R01AI125390‐1 (Proteomics of Eosinophil activation)

Comparing reliability of skin microfilarial load with first internal transcribed spacer primers DNA amplification in monitoring onchocerciasis treatment control

Treatment compliance is a serious challenge to community-directed treatment intervention (CDTI) targeting onchocerciasis eradication. Isolated cases of active infections could jeopardise decision to stop mass drug distribution. Onchocerciasis co-endemicity with lymphatic filariasis in the study-area informed the use of primers capable of differentiating sympatric infections in a single assay. The study-population was hitherto found to be negative for presence of skin microfilaria. DNA extracted from skin snips of patients (n=63) comprised of males, n=21 and females, n=42 were analysed using 18S first internal transcribed spacer (ITS1) in polymerase chain reaction (PCR) assay. Amplified DNA sequences were subjected to basic local alignment search tool. Spectrophotometric analysis of DNA concentrations ranged between 1.5- and 24.5 Nano gram (ng) and the two positive controls (adult worm DNA extract) were 10.5 and 105 ng at 260 nanometer (nm) wave length. The 260/ 280 nm ratios were between 1.44 and 1.68. Four samples (6.35%), two males and two females, and the two positive controls showed PCR amplification products. Two of the positive samples indicated double bands in an agarose gel electrophoregram with molecular weight of 350 and 500 base pairs (bp), while two had single band of 350bp, which is within the expected Mw range of 344 bp for Onchocerca volvulus. The double bands may not be due to unspecific amplification. Moreover, the DNA sequence of a sample had 69.1% homology with control sequence but not with O. volvulus sequences available in the GenBank database. This study underscored the need to assess effectiveness of CDTI with highly sensitive test and not depend on microscopy of emerged microfilaria from skin snips. Whether the DNA was from dead or living microfilaria remained a conjecture. Keywords : Skin microfilaria; DNA sequences.

VARIASI SEKUENS DNA YANG DIAMPLIFIKASI MENGGUNAKAN PRIMER atpB-rbcL PADA BEBERAPA KULTIVAR KACANG TANAH

Peanut is one of food crops commonly consumed in Indonesia. This species comprises several cultivars such as Kancil, Bison, Jerapah, Talam, and Tuban, each of which has its individual advantages and disadvantages. The vast variation among peanut cultivars leads to the need of study on genetic diversity and relationship among them using particular molecular marker. This study aims to see whether variation on DNA sequences among some peanut cultivars amplified with atpB-rbcL primers exists or not and to know the relationship among the cultivars based on the amplicon sequences. The method involves some sequential steps, i.e. genomic DNA isolation using CTAB protocol, amplification of DNA sequence using atpB-rbcL primers and sequencing of the amplification products. Data on sequences were edited manually using Bioedit version 7.0.4.1. Sequence alignment was performed using ClustalW, which is also implemented in Bioedit version 7.0.4.1. Arlequin 2.0 was used to calculate nucleotide diversity p. Phylogenetic analysis was performed using Maximum Parsimony in MEGA 5.0. The results showed that considerably high variation in DNA sequences of some peanut cultivars amplified with atpB-rbcL primers are observed. On the other hands, very close genetic relationship among cultivars is found.

Specific and Sensitive Isothermal Electrochemical Biosensor for Plant Pathogen DNA Detection with Colloidal Gold Nanoparticles as Probes

Developing quick and sensitive molecular diagnostics for plant pathogen detection is challenging. Herein, a nanoparticle based electrochemical biosensor was developed for rapid and sensitive detection of plant pathogen DNA on disposable screen-printed carbon electrodes. This 60 min assay relied on the rapid isothermal amplification of target pathogen DNA sequences by recombinase polymerase amplification (RPA) followed by gold nanoparticle-based electrochemical assessment with differential pulse voltammetry (DPV). Our method was 10,000 times more sensitive than conventional polymerase chain reaction (PCR)/gel electrophoresis and could readily identify P. syringae infected plant samples even before the disease symptoms were visible. On the basis of the speed, sensitivity, simplicity and portability of the approach, we believe the method has potential as a rapid disease management solution for applications in agriculture diagnostics.

Comparison of Polymerase Chain Reaction and Immunohistochemistry Assays for Analysing Human Papillomavirus Infection in Oral Squamous Cell Carcinoma.

Polymerase Chain Reaction (PCR) and Immunohistochemistry (IHC) are two well-known techniques used for the diagnoses of genetic diseases, tumours and different pathogens. PCR basically amplify regions of DNA within a single molecule which may have etiologic significance, it is a method for in vitro amplification of specific DNA or RNA sequences, whereas IHC is used to verify tissue constituents (the antigens) with the utilization of specific antibodies that can be visualized through staining. To compare and analyse PCR and IHC for their sensitivity to detect Human Papillomavirus (HPV) in Oral Squamous Cell Carcinoma (OSCC). This study was based on samples retrospectively collected from 47 patients with primary OSCC who were diagnosed and treated at The Aga Khan University Hospital, Karachi, Pakistan, during the period of January 2010 to December 2013. Inclusion criteria were complete clinicopathologic data, adequate clinical follow up and availability of sufficient paraffin- embedded tumour material. HPV general and type specific 16 and 18 were investigated by means of PCR. HPV immunoreactivity was further investigated by means of IHC. Among the 47 evaluated patients, 32 (68.1%) were male and 15 (31.9%) were female, PCR detected the presence of HPV in 32 (68.1%) patients while IHC showed no positive test results. p53 was positive in 32 (68.1%) patients and negative in 15 (31.9%). HPV type 16 being most prevalent showing positivity in 27 (57.4%) patients whereas, type 18 was positive in only 1 (2.1%) patient. We concluded that PCR is more sensitive and reliable when diagnosing and detecting HPV for OSCC rather than IHC as results from IHC were all negative and insignificant, hence PCR should be the first initial diagnostic test for detecting HPV due to its better sensitivity and successful detection of HPV.

Improving the PCR protocol to amplify a repetitive DNA sequence.

Although PCR-based techniques have become an essential tool in the field of molecular and genetic research, the amplification of repetitive DNA sequences is limited. This is due to the truncated nature of the amplified sequences, which are also prone to errors during DNA polymerase-based amplification. The complex structure of repetitive DNA can form hairpin loops, which promote dissociation of the polymerase from the template, impairing complete amplification, and leading to the formation of incomplete fragments that serve as megaprimers. These megaprimers anneal with other sequences, generating unexpected fragments in each PCR cycle. Our gene model, MaSp1, is 1037-bp long, with 68% GC content, and its amino acid sequence is characterized by poly-alanine-glycine motifs, which represent the repetitive codon consensus. We describe the amplification of the MaSp1 gene through minor changes in the PCR program. The results show that a denaturation temperature of 98°C is the key determinant in the amplification of the MaSp1 partial gene sequence.

The presence of mycobacterial antigens in sarcoidosis associated granulomas.

Background: Sarcoidosis is a multi-organ disorder with unknown etiology. The role of bacteria in pathogenesis of sarcoidosis is still controversial. This study analyses new aspects of Mycobacterium Tuberculosis (MTB) presence in sarcoidosis diseases. Objectives: To find MTB in paraffin embedded tissues of sarcoidosis patients, samples of 10 sarcoidosis, 12 confirmed pulmonary tuberculosis (PTB) and 5 controls associated with granulomatous tissues were analysed. Methods: The paraffin embedded tissue specimens of the selected patients from the pathology archive of a subspecialty pulmonary hospital in IRAN were evaluated by Real Time PCR for MTB DNA using IS6110. Immunohistochemistry (IHC) method using MTB purified protein derivative (PPD) antibody was used to detect mycobacterial antigens. Results: All sarcoidosis patients had negative MTB DNA results in Real time PCR analysis. This analysis resulted in 10 (83.3%) positive cases for TB patients. The IHC analysis for MTB anti-PPD antibody showed positive diffused cytoplasmic staining for all TB patients whereas this staining was positive for 3 sarcoidosis patients (30%). Conclusion: Amplification of the IS6110 DNA sequence that is the most common target used for MTB diagnosis is not sensitive method to detect MTB in sarcoidosis granuloma. However, tissue IHC for anti-PPD antibody shows higher performance to detect MTB in sarcoidal granulomas reveals a mycobacterial signature in sarcoidosis tissue with negative IS6110 assay. This finding supports Mycobacterium tuberculosis may have an etiologic role in sarcoidosis. (Sarcoidosis Vasc Diffuse Lung Dis 2017; 34: 236-241)

Synthesis of Fusion Genes for Cloning by Megaprimer-Based PCR.

The polymerase chain reaction (PCR) is the technique of choice used to obtain DNA for cloning, because it rapidly provides high amounts of desired DNA fragments and allows the easy introduction of extremities adequate for enzyme restriction or homologous recombination, and of artificial, native, or modified sequence elements for specific applications. In this context, the use of megaprimer-based PCR strategies allows the versatile and fast assembly and amplification of tailor-made DNA sequences readily available for cloning.In this chapter, we describe the design and use of a megaprimer-based PCR protocol to construct customized fusion genes ready for cloning into commercial expression plasmids by restriction digestion and ligation.

The first detection of potentially toxic Microcystis strains in two Middle Atlas Mountains natural lakes (Morocco).

Aguelmam Azizgza (LAZ) and Dayet Afourgah (DAF) are two Moroccan natural lakes located in a humid hydrographic basin of the Middle Atlas Mountains. Both are considered important reservoirs of plant and animal biodiversity. In addition, they are extensively used for recreational and fishing activities and as a water source for irrigation of agricultural crops. Recurrent cyanobacteria scum episodes in the two water bodies have been reported, Microcystis being the main genus in the scums. Here, we report on the toxic potential of three Microcystis aeruginosa strains isolated from those lakes: Mic LAZ and Mic B7 from LAZ and Mic DAF isolated from DAF. The toxic potential was checked by their microcystin (MC) content and the presence of mcy genes involved in MC synthesis. The identification and quantification of MC variants were performed by high-performance liquid chromatography-photo-diode array. The detection of mcy genes was achieved by whole-cell multiplex PCR that allowed the simultaneous amplification of DNA sequences corresponding to specific mcy regions. MC content of cultured cells, as MC-LR equivalents per gram cell biomass, was slightly higher in Mic LAZ (ca. 860) than in Mic B7 (ca. 700) and Mic DAF (ca. 690). Four MC variants were identified in the three isolates: MC-WR, MC-RR, MC-DM-WR, and MC-YR. The presence of toxic Microcystis strains in the two studied lakes may be regarded as an environmental and health hazard, especially during periods of bloom proliferation. It would be recommended the use of two complementary techniques, as those utilized herein (HPLC and mcy detection) to alert on highly probable toxicity of such lakes.

Genetic identification of new microalgal species from Epe Lagoon of West Africa accumulating high lipids

The green microalgal strains are favorable alternatives for producing biodiesel. In spite of the worldwide awareness on microalgae-derived oil to potentially replace the conventional fossil fuel, information on potential microalgal strains from Nigerian lagoons is scarce. In view of this, environmental samples were collected from the Epe Lagoon, Nigeria to assess the biodiversity of microalgae and identify robust strains for biodiesel production. The field samples were collected and genomic DNA was isolated from the monoclonal cultures. Microalgal species were identified based on the PCR amplified DNA sequences of 18S ribosomal DNA. The sequences of PCR products from the unknown samples were compared with the DNA database. Using the phylogenetic maximum-likelihood trees, the green algal strains Desmodesmus sp., Desmodesmus costato-granulatus, Tetradesmus sp. and Chlorella sp. were identified, while two strains belonging to the Chlorococcaceae and Family Selenastraceae need further validation. Microalgal species morphology was validated using electron microscopy. Lipid analysis was carried out using Fatty Acid Methyl Ester (FAMEs) analysis, Gas Chromatography and Mass Spectrometry (GC–MS). The strain of the Family Selenastraceae produced the maximum amount of poly-unsaturated fatty acids (56%), which has potential for food processing and other bio-based products; while Tetradesmus sp. with 35.16mg (FAMEs per gram of dry cell weight) had a balanced lipid composition (44% saturated fatty acid, 39% mono-unsaturated fatty acid, 15% poly-unsaturated fatty acids) making it a good candidate for exploitation as a biodiesel feedstock.

DNA G-Quadruplex-Based Assay of Enzyme Activity.

DNA G-quadruplexes are special three-dimensional (3D) DNA nanostructures formed by specific G-rich DNA sequences. These 3D DNA nanostructures can bind with hemin and significantly improve the intrinsic peroxidase activity of hemin. Besides this function, they also enhance the fluorescence intensity of some G-quadruplex-specific dyes. Owing to these features, G-quadruplexes possess several superiorities in the detection of enzymes involved in nucleic acid metabolism, including facile probe fabrication without labeling, simple detection process without washing or separation steps, rapid observation by naked eyes, and easy integration with nucleic acid amplification strategies to amplify signals. Herein, we describe two strategies for label-free detection of enzyme activity based on DNA G-quadruplexes. To increase sensitivity, template-dependent and template-independent DNA amplifications were introduced for the amplification of G-rich DNA sequences. DNA methyltransferase and terminal deoxynucleotidyl transferase were detected as two model analytes, respectively.

RETRACTED ARTICLE: Peroxidase Gene-Based Estimation of Genetic Relationships and Population Structure Among Wild Pistacia Species Populations.

Plant peroxidases belong to a multigene family and possess highly conserved domains allowing designing oligonucleotide primers to amplify DNA sequences coding for peroxidases from plants with unsequenced genomes. Peroxidase gene-based polymorphism among wild Pistacia species, and correlation between peroxidase markers were deduced in this study. Wild Pistacia germplasm was evaluated using 30 peroxidase-specific primers. Targeted-PCR amplification of genomic DNA from 100 wild accessions of Pistacia species produced polymorphisms giving a similarity range from 0.29 to 1.00 with a mean of 0.65. Based on the POGP gene polymorphism, two distinct clusters were detected among the wild accession of Pistacia species, suggesting different evolutionary pathways. Correlation estimates as an indication of linkage disequilibrium between POGP markers ranged from 0.11 to 0.90, suggesting that few POGP markers were clustered and the remaining POGP markers evenly distributed throughout the Pistacia genome. These results demonstrate that primers targeting the peroxidase gene family can be used to study genotypic diversity and evolutionary relationships on an intra- and inter-specific basis.