Cloning Of Polymerase Chain Reaction Products Research Articles

Polymorphisms in exon 11 of the mptl-1 gene and monepantel resistance in Haemonchus contortus.

Chemical treatments are the main strategy to control gastrointestinal nematodes in sheep,and the emergence of anthelmintic resistance, as consequence, results in control failures and leads to economic losses. Thus, molecular tests may constitute an excellent tool for the early detection of anthelmintic resistance-related mutations. Thus, a polymerase chain reaction (PCR)-based genotyping assay followed by polyacrylamide gel electrophoresis (PAGE) was developed to detect polymorphisms in exon 11 of the acetylcholine receptor monepantel-1 gene (mptl-1) that were previously associated with monepantel resistance through a genome-wide study in Haemonchus contortus. DNA samples recovered from individual and pooled third-stage larvae from two susceptible field-derived isolates and five (three in vivo-derived and two field-derived) resistant populations were used. New polymorphisms, including a 6-bp deletion and a 3-bp insertion, were detected in resistant individuals. These indels, confirmed using sequencing of cloned PCR products, are predicted to result in amino acid changes in transmembrane domain 2 (TMD2) of the MPTL-1 protein. The two susceptible isolates showed only the presence of the wild-type allele (100%), whereas lower frequencies of the wild-type allele were detected in monepantel-resistant populations (11.1 to 66.7%). These findings report new polymorphisms in the mptl-1 gene, validate the results obtained through genomic mapping for monepantel resistance, and provide a PCR-based assay to genotype indels located in exon 11 of mptl-1 in H. contortus.

Cloning Polymerase Chain Reaction Products: Addition of Restriction Sites to the Termini of Amplified DNA.

To generate polymerase chain reaction (PCR) products that can be directionally cloned into a vector, different restriction sites are built into the forward and reverse primers that are used in the PCR. After PCR, the amplified product is purified, cleaved with the appropriate restriction enzymes, ligated into a vector with compatible cohesive ends, and used to transform E. coli.

The Length Limit of 5′ Nucleotide Additions to PCR Primers

The addition of nucleotides to polymerase chain reaction (PCR) primers has become a widely used technique to facilitate the cloning of PCR products. A long fragment mismatched with template, such as an epitope-encoding sequence, needs to be added to the 5′ end of the primer for identification and purification of the expressed protein. However, the length limit of added sequences has not yet been determined. Under our condition, the maximum length for 5′ nucleotide additions was found to be 108, when a fully matched forwards primer and partially matched reverse primer contained 44.4–55.6% GC in the matched region with template are used. The results shown here are very useful for scientists who want to add a long mismatched sequence with template, such as an epitop/a signal peptide encoding sequence and/or the multiple cloning sites.

Geographical Distribution and Diversity of Gut Microbial NADH:Ubiquinone Oxidoreductase Sequence Associated with Alzheimer's Disease.

Earlier we reported induction of neurotoxicity and neurodegeneration by tryptophan metabolites that link the metabolic alterations to Alzheimer's disease (AD). Tryptophan is a product of Shikimate pathway (SP). Human cells lack SP, which is found in human gut bacteria exclusively using SP to produce aromatic amino acids (AAA). This study is a first attempt toward gene-targeted analysis of human gut microbiota in AD fecal samples. The oligonucleotide primers newly-designed for this work target SP-AAA in environmental bacteria associated with human activity. Using polymerase chain reaction (PCR), we found unique gut bacterial sequence in most AD patients (18 of 20), albeit rarely in controls (1 of 13). Cloning and sequencing AD-associated PCR products (ADPP) enables identification of Na(+)-transporting NADH: Ubiquinone reductase (NQR) in Clostridium sp. The ADPP of unrelated AD patients possess near identical sequences. NQR substrate, ubiquinone is a SP product and human neuroprotectant. A deficit in ubiquinone has been determined in a number of neuromuscular and neurodegenerative disorders. Antibacterial therapy prompted an ADPP reduction in an ADPP-positive control person who was later diagnosed with AD-dementia. We explored the gut microbiome databases and uncovered a sequence similarity (up to 97%) between ADPP and some healthy individuals from different geographical locations. Importantly, our main finding of the significant difference in the gut microbial genotypes between the AD and control human populations is a breakthrough.

Ancient Ascaris DNA Sequences of Cytochrome B, Cytochrome C Oxidase Subunit 1, NADH Dehydrogenase Subunit 1, and Internal Transcribed Spacer 1 Genes from Korean Joseon Mummy Feces.

We analyzed Ascaris ancient DNA of cytochrome b, cytochrome c oxidase subunit 1, NADH dehydrogenase subunit 1, and internal transcribed spacer 1 genes extracted from the feces or precipitates of 15- to 18th-century Korean mummies. After multiple Ascaris genes in ancient samples were successfully amplified by polymerase chain reaction (PCR), consensus sequences could be determined by the alignment of the sequences of cloned PCR products. The obtained sequences of each gene were highly similar to those of Ascaris spp. reported thus far but were genetically distinct from Baylisascaris, Parascaris, and Toxascaris spp. The current report establishes that the genetic characteristics of the Ascaris spp. infecting pre-modern Korean societies were not uniform but were diverse to some degree.

RT PCR Data Plagiarism Their Ignorance and Impact

Polymerase Chain Reaction (PCR) is a technique based on the ability of DNA polymerase to synthesize a new strand of DNA which is complementary to the template strand offered and amplifying this strand to billions of amplicons. There are different types of PCR reactions for different types of experiments but reverse transcription-polymerase chain reaction (RT-PCR) is most commonly used PCR reactions in the field of genomics and proteomics basically at mRNA level. A wide range of procedures have been devised for studying PCR products like gel electrophoresis, cloning and sequencing of PCR products, etc. The PCR products from gel electrophoresis are in the form of images, therefore image processing techniques are commonly used to analyze these images in three main steps i.e. band detection, band matching, and data quantification. Image processing sometimes alters the intensity of image which can at points improve the visualization of the data. But if that image alteration mischaracterizes the data, one has gone too far and this is known as plagiarism. Therefore, it is important to safeguard and protect the original, unaltered image in order to avoid the accusations of misconduct which will stand or fall on the basis of whether or not the original image is available to be compared with its altered copies. For example, investigators whose work fall under the food and drug administration’s (FDA’s) “Final Rule on Electronic Records and Electronic Signatures” must maintain the integrity of the original image. Similarly, industries whose work products are used in forensic activities or in health insurance portability and accountability act (HIPAA) related aspects of health care might be required to maintain an original image. Therefore, the plagiarism of RT-PCR data has tremendous impact on research if left ignored as seen through the above examples and therefore should be taken care.

Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering.

BackgroundOver the last decades, molecular cloning has transformed biological sciences. Having profoundly impacted various areas such as basic science, clinical, pharmaceutical, and environmental fields, the use of recombinant DNA has successfully started to enter the field of cellular engineering. Here, the polymerase chain reaction (PCR) represents one of the most essential tools. Due to the emergence of novel and efficient PCR reagents, cloning kits, and software, there is a need for a concise and comprehensive protocol that explains all steps of PCR cloning starting from the primer design, performing PCR, sequencing PCR products, analysis of the sequencing data, and finally the assessment of gene expression. It is the aim of this methodology paper to provide a comprehensive protocol with a viable example for applying PCR in gene cloning.ResultsExemplarily the sequence of the tdTomato fluorescent gene was amplified with PCR primers wherein proper restriction enzyme sites were embedded. Practical criteria for the selection of restriction enzymes and the design of PCR primers are explained. Efficient cloning of PCR products into a plasmid for sequencing and free web-based software for the consecutive analysis of sequencing data is introduced. Finally, confirmation of successful cloning is explained using a fluorescent gene of interest and murine target cells.ConclusionsUsing a practical example, comprehensive PCR-based protocol with important tips was introduced. This methodology paper can serve as a roadmap for researchers who want to quickly exploit the power of PCR-cloning but have their main focus on functional in vitro and in vivo aspects of cellular engineering.Electronic supplementary materialThe online version of this article (doi:10.1186/1754-1611-9-2) contains supplementary material, which is available to authorized users.

Improvement of TA Cloning Method to Facilitate Direct Directional Cloning of PCR Products

Directional cloning is a prerequisite for the construction of expression vectors in molecular biology laboratories. Although TA cloning is widely used to clone unmodified PCR (polymerase chain reaction) products, a major disadvantage of this technique is that cloning is not directional. Here we reported a novel PCR products cloning vector with one deoxythymidine overhang and one deoxycytidine overhang at two 3'-ends respectively. With the choice of nucleotides of 5'-ends of PCR primers, PCR products can be cloned to this vector both directly and directionally. The feasibility and efficacy of this cloning method were confirmed by using a pET-17b derivative vector and a green fluorescent protein gene (EGFP) and a red fluorescent protein reporter (Ds-Red) gene. This cloning strategy may be useful in the high-throughput construction of expression vectors and could be viewed as an interesting improvement of existing TA cloning method.

EGFR mutations detected on cytology samples by a centralized laboratory reliably predict response to gefitinib in non–small cell lung carcinoma patients

Epidermal growth factor receptor (EGFR) mutations are reliably detected by referral laboratories, even if most lung cancer cytology specimens sent to such laboratories contain very few cells. However, EGFR mutations may be distributed heterogeneously within tumors, thereby raising concerns that mutations detected on cytology are not representative of the entire tumor and, thus, are less reliable in predicting response to tyrosine kinase inhibitor (TKI) treatment than mutations detected on histology. To address this issue, the authors reviewed their clinical practice archives and compared the outcome of TKI treatment among patients who were selected by cytology versus patients who were selected by histology. From July 2010 to July 2012, 364 cytology samples and 318 histology samples were received. Exon 19 deletions and the L858R point mutation in exon 21, detected by fragment assay and TaqMan assay, respectively, were confirmed by direct sequencing; discrepancies were resolved by cloning polymerase chain reaction products. The response rate (RR) and progression-free survival (PFS) at 12 months (range, 3-34 months) were evaluable in 13 EGFR-mutated patients who were selected for treatment by cytology and 13 patients who were selected by histology. The mutation rate was similar in histology samples (8.5%) and cytology samples (8.8%). The RR (54%) and PFS (9.2 months) were similar in histologically selected patients and cytologically selected patients (RR, 62%; PFS, 8.6 months; P = .88). The disease control rate (responsive plus stable disease) was 92% in histologically selected patients and 100% in cytologically selected patients. EGFR mutations detected on cytology specimens by a centralized laboratory can predict TKI treatment response equally well as mutations identified on histology samples.

Detection of human papillomavirus type 16 in Bowen’s carcinoma of the toe

Human papillomavirus (HPV) is known to cause cervical cancer. Because it has been detected in lesions of Bowenoid papulosis, Bowen's disease, and Bowen's carcinoma, HPV infection has been implicated in the pathogenesis of these diseases. A 44-year-old man was diagnosed clinicopathologically with Bowen's carcinoma of the right great toe. He developed multiple organ metastases and died. We examined HPV DNA in skin biopsy specimens from the primary and skin metastatic lesions by polymerase chain reaction (PCR) and in situ hybridization (ISH). The PCR assay was carried out using primer sets specifically designed for detecting the E6 and E7 genes of the HPV types associated with malignancy. Purified and cloned PCR products were subjected to DNA sequence analysis. The ISH studies used INFORM(®) HPV III probes. We found HPV DNA in specimens from both the primary and the skin metastatic lesions. DNA sequencing detected HPV type 16. We compared the base sequences of viral DNA from the primary and metastatic lesions. Point mutations of the base sequences of the E6 and E7 genes were observed in viral DNA from metastases but not in that from primary lesions. The E6 gene had mutated from G to A in the 383rd base sequence, causing a Glu-to-Lys amino acid change. Results of ISH showed punctuate signals in the nuclei of tumor cells. We suspect an association between HPV 16 infection and the development of this malignant occurrence.

Reappraisal of KIT mutation in adenoid cystic carcinomas of the salivary gland

While overexpression of KIT protein has been well documented in adenoid cystic carcinomas (ACCs), mutation of KIT gene has been a controversial issue. We wanted to evaluate clinical value of the KIT mutation and protein expression in ACC. We analyzed 33 cases of ACC. Gene mutations in KIT exons 9, 11, 13, and 17 were analyzed using paraffin-embedded tissue, and two different sets of primers with direct sequencing after polymerase chain reaction (PCR) for exon 9, 11, 13, and 17, and cloning of PCR products for exon 11. KIT protein expression was assessed by immunohistochemistry. The correlation between clinicopathological findings and these biomarkers was analyzed. No KIT mutation was observed in all of the 33 cases. With one primer set, KIT mutation was found in nine of 33 cases (27.3%). However, these mutations were not reproducible in the experiment using another primer set. KIT protein overexpression was detected in 22 of 33 patients (66.7%). KIT protein expression was not statistically correlated with either clinicopathological factors or survival. Patients with metastasis showed a tendency of longer progression-free survival (P = 0.052) and overall survival (P = 0.080) when the tumor overexpressed KIT protein. This study supports that mutational study using paraffin-embedded tissue should be interpreted with great caution. KIT gene mutation is very rare in ACC, and gene mutation is not the cause of protein overexpression. KIT protein expression may have a potential value for better prognostic factor in patients with metastasis.

Inverse association between methylation of human papillomavirus type 16 DNA and risk of cervical intraepithelial neoplasia grades 2 or 3.

BackgroundThe clinical relevance of human papillomavirus type 16 (HPV16) DNA methylation has not been well documented, although its role in modulation of viral transcription is recognized.MethodsStudy subjects were 211 women attending Planned Parenthood clinics in Western Washington for routine Papanicolaou screening who were HPV16 positive at the screening and/or subsequent colposcopy visit. Methylation of 11 CpG dinucleotides in the 3′ end of the long control region of the HPV16 genome was examined by sequencing the cloned polymerase chain reaction products. The association between risk of CIN2/3 and degree of CpG methylation was estimated using a logistic regression model.ResultsCIN2/3 was histologically confirmed in 94 (44.5%) of 211 HPV16 positive women. The likelihood of being diagnosed as CIN2/3 increased significantly with decreasing numbers of methylated CpGs (meCpGs) in the 3′ end of the long control region (P for trend = 0.003). After adjusting for HPV16 variants, number of HPV16-positive visits, current smoking status and lifetime number of male sex partners, the odds ratio for the association of CIN2/3 with ≥4 meCpGs was 0.31 (95% confidence interval, 0.12–0.79). The proportion of ≥4 meCpGs decreased appreciably as the severity of the cervical lesion increased (P for trend = 0.001). The inverse association remained similar when CIN3 was used as the clinical endpoint. Although not statistically significant, the ≥4 meCpGs-related risk reduction was more substantial among current, as compared to noncurrent, smokers.ConclusionResults suggest that degree of the viral genome methylation is related to the outcome of an HPV16 cervical infection.

Oral and Cervical Human Papillomavirus Infection among Female Sex Workers in Japan

It has been reported recently that oral human papillomavirus (HPV) infection is associated with oropharyngeal squamous cell carcinomas. The aim of this study was to determine the prevalence of HPV infection and HPV types in the oral cavity and cervix of female sex workers in Japan. Oral and cervical swabs were taken from 196 female sex workers who visited a clinic for regular medical checkups in 2007, and genomic DNA was extracted from those specimens. The HPV L1 gene was amplified by polymerase chain reaction (PCR) using original and modified GP5(+)/6(+) primers, and genotyping was performed using the Kurabo GeneSquare Microarray or by sequencing cloned PCR products. HPV DNA was detected in the oral cavity of 12 (6.1%) women, with HPV-56 being the most common type (7/12). Likewise, HPV DNA was detected in the cervix of 103 (52.6%) women, with HPV-52 (30/103, 29.1%), followed by HPV-16 (24.3%) and HPV-56 (18.4%), being the most common. Of the 12 women with oral HPV infection, only two were infected with the concordant HPV genotype in the cervix. These findings suggest that oral HPV infection occurs independently of cervical HPV infection in this population, and that oral HPV infection may play a role in HPV transmission in Japan.

Subepithelial dendritic B cells in orofacial granulomatosis

Orofacial granulomatosis (OFG) is a chronic, disfiguring, granulomatous inflammation of the lips and oral mucosa. The pathogenesis is unknown, but it has been linked previously to Crohn's disease (CD) and more recently to dietary sensitivity. The oral mucosa is an immunologically responsive site associated with the generation of protective mucosal and systemic immune responses to vaccination and also hyperresponsiveness to allergens in some individuals. Classically, immune responses in oral mucosa are considered to be mediated by mucosa-associated lymphoid tissues (MALT), secondary lymphoid follicles that are intimately associated with epithelia. Immunohistochemistry was used to investigate the inflammatory infiltrate in OFG and control tissue samples. Polymerase chain reaction (PCR), cloning of PCR products, and sequencing were used to characterize the local immunoglobulin gene profile in OFG. We describe large, active, dendritic B cells in oral mucosa that were not associated with any organized lymphoid tissues in the local subepithelial microenvironment. They express activation induced cytidine deaminase, which is essential for immunoglobulin gene diversification by somatic hypermutation and class switch recombination. IgE is also expressed by these B cells. They do not align with any other previously described B-cell subset in secondary lymphoid tissues in terms of morphology, proliferative activity, or phenotype. These subepithelial dendritic B cells may contribute to the immune responsiveness of the oral mucosa, including IgE-mediated allergic responses. In patients with OFG, further understanding of the role these cells play in oral immunity may lead to novel therapeutic possibilities.

Strategies for Cloning PCR Products

INTRODUCTIONCloning polymerase chain reaction (PCR)-amplified fragments into plasmids offers several advantages. Bacteria containing plasmids can be frozen, providing a ready supply of amplified material. Because of the variety of available plasmids with different promoters and selectable markers, cloning is also useful when mutations are to be introduced into the fragment before expression, or when sequence tags encoded in the vector are to be added in-frame. The ease with which nucleotide sequences can be added to the ends of PCR products has led to the development of a variety of cloning strategies. Because such cloning is typically the first step for generating a reagent that will be used to achieve a specific experimental goal, the efficiency of the cloning procedure is an important consideration: Cloning strategies should be simple in design and execution, requiring a minimum of enzymatic steps. Toward this goal, many companies market and continue to develop reagent kits that improve the ease and rapidity of cloning PCR products. This article focuses on some common and efficient cloning strategies, such as those that use DNA ligase or vaccinia virus topoisomerase I (TOPO), as well as techniques for in vitro and in vivo recombination of PCR products and vectors having homologous duplex ends. Also covered is the production of linear PCR products with defined 5′ and 3′ functional elements, which enable direct mammalian cell expression or in vitro transcription/translation. We present an overview of these strategies, their molecular basis, and their advantages and disadvantages for specific applications.

Haplorchis pumilio and H. taichui in Vietnam discriminated using ITS-2 DNA sequence data from adults and larvae

Samples of Haplorchis taichui and Haplorchis pumilio of different life-stages (cercariae, metacercariae and adults) and from different host species (snail, fish, dog, cat and human) were collected in Nghe An and Nam Dinh Provinces in Vietnam. Samples from Thailand were available for comparison. All adults and metacercariae were initially identified using morphological criteria. Polymerase chain reaction (PCR) assays were developed for discriminating between the species. The complete sequence for the nuclear ribosomal internal transcribed spacer-2 (ITS-2) was obtained from one adult and one metacercaria of H. taichui and three adults and three metacercariae of H. pumilio from Vietnam. Sequences from cercariae from three different snails clustered with those of H. pumilio. Intra-individual variation in the ITS-2 region was detected by sequencing of cloned PCR products. These are the first sequences from Vietnamese Haplorchis spp. to be reported and demonstrate that H. taichui and H. pumilio can be identified unambiguously from any life-stage, including the cercarial stage that is difficult to identify using morphology. Discrepancies in the literature are discussed and examples of apparent misidentification highlighted. The data provide a resource to assist in taxonomic studies on heterophyids, in the design of probes for diagnosis and for field surveys to identify infection in snails.

Fern-associated arbuscular mycorrhizal fungi are represented by multiple Glomus spp.: do environmental factors influence partner identity?

Symbioses involving arbuscular mycorrhizal fungi (AMF) are among the most important ecological associations for many plant species. The diversity of AMF associated with ferns, however, remains poorly studied. Using recently designed Glomus-specific primers, we surveyed the AMF community associated with ferns from deciduous, broad-leaved second-growth forest habitats at the eastern edge of the piedmont region of central Virginia, USA. Results indicate that this molecular approach may be a useful tool for detecting AMF in ferns compared to traditional techniques based on morphology. Over 30 potential fungal ribotypes were identified from eight fern species using denaturing gradient gel electrophoresis. Fungal ribotypes were found to differ widely in terms of (1) the number of fern partners with which they interact and (2) their relative frequency within each fern. Sequence analysis of fungal isolates from three species of fern indicated that the primers were generally highly specific for Glomus species but some non-target DNA was also amplified. Cloned polymerase chain reaction (PCR) products from Polystichum acrostichoides and Osmunda regalis revealed several phylogenetically distinct Glomus species. A single Glomus species was identified in the cloned PCR products from Botrychium virginianum. These findings challenge the hypothesis that the extent or degree of fern-fungal symbiosis is somehow tied to root complexity. Environmental factors appear to influence the suite of AMF that form partnerships with ferns. Some species of fern from similar habitats associated with dissimilar fungal partners (e.g., P. acrostichoides and Athyrium filix-femina var. asplenioides), whereas others harbored uniform fungal communities (e.g., Asplenium platyneuron). The significance of these data in terms of ecological and evolutionary dynamics of the AMF-fern symbiosis is discussed.

High‐risk HPV types in lesions of the uterine cervix of female commercial sex workers in the Philippines

In order to prevent cervical cancer, vaccines against human papilloma virus types 16 (HPV-16) and 18 (HPV-18) have been implemented worldwide. However, the HPV types that cause cancer can differ according to geographical area and ethnicity. In this new era of the HPV vaccine, it is important to elucidate the prevalent HPV types in each area. Therefore, the prevalence of HPV infection and cervical abnormalities among 369 female commercial sex workers in the Philippines were examined. HPV L1 gene was amplified by polymerase chain reaction (PCR) using modified GP5+/6+ primers, and genotyping was performed by sequencing cloned PCR products. HPV DNA was detected in 211 (57.2%) women, among whom 46 HPV types were identified. HPV-52 was most common and multiple-type infection was observed in 44.5%. Among 56 women with abnormal cervical cytology (low- and high-grade squamous intraepithelial lesions and adenocarcinoma in situ), HPV-52 was most common (23.2%), followed by HPV-16 (19.6%), -58 (10.7%), and -67 (10.7%). Only 27% of these women were positive for HPV-16 and -18. Multivariate analysis revealed that HPV-16, -39, -52, -67, and -82 were significantly associated with abnormal cytology. Repeated analysis of HPV-52 single-positive samples using the original GP5+/6+ PCR primers produced negative results in 57% of cases, suggesting that the prevalence of HPV-52 infection may have been underestimated in previous studies, and the current vaccines may not be sufficient for preventing infection and the development of premalignant lesions of the cervix in women in the Philippines.

Community of arbuscular mycorrhizal fungi in a coastal vegetation on Okinawa island and effect of the isolated fungi on growth of sorghum under salt-treated conditions

Community of arbuscular mycorrhizal (AM) fungi in a coastal vegetation on Okinawa island in Japan was examined. A sampling plot was established in a colony of Ipomoea pes-caprae (Convolvulaceae) on the beach in Tamagusuku, Okinawa Pref, in which eight root samples of I. pes-caprae and three root samples each of Vigna marina (Leguminosae) and Paspalum distichum (Poaceae) were collected. Partial 18S rDNA of AM fungi was amplified from the root samples by polymerase chain reaction (PCR) with primers NS31 and AM1. Restriction fragment length polymorphism analysis with HinfI and RsaI for cloned PCR products revealed that two types of Glomus sp., type A and type B, were dominant in the colony. Among them, the fungi of type A were especially dominant near the edge of the colony facing the sea. A phylogenetic analysis showed that the AM fungi of type B are closely related to Glomus intraradices and those of type A are nearly related to type B. From the sequence data, it was also found that type A was further divided into two types, type A1 and A2. One representative strain each of the three types, type A1, A2, and B, propagated from single spore each, was examined for the growth of sorghum (Sorghum bicolor) at three different salinity levels, 0, 100, and 200 mM NaCl. At the non-salt-treated condition, the type B fungus was the most effective on shoot growth enhancement of the host plant, whereas at the salt-treated conditions, the type A2 fungus was the most effective. An efficient suppression of Na + translocation into the shoot by the examined AM fungi was found. These results suggested that the AM fungi dominant near the sea are adapted to salt-stressed environment to alleviate the salt stress of host plants.

Intragenomic Heterogeneity of rDNA Internal Transcribed Spacer 2 in <I>Anopheles messeae</I> (Diptera: Culicidae)

Because Anopheles messeae Falleroni (Diptera: Culicidae) is one of the main vectors of malaria in Russia, studying its genetic markers is important for reliable identification of this species. This species is distributed nearly throughout the Palearctic region, and it exhibits high genetic variability. We investigated polymorphism of the rDNA internal transcribed spacer (ITS) 2 of An. messeae in various regions of Russia, and we found intragenomic heterogeneity of ITS2 copies verified by chromatograms, polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis, and cloning PCR products. In total, we found nine different ITS2 variants. ITS2 variants that were considered specific to An. messeae and Anopheles daciae Linton, Nicolescu & Harbach were simultaneously present in one individual. These findings improve methods of species identification of An. messeae, and they do not support the species status of An. daciae.