Use Of Molecular Genetic Methods Research Articles

Assessment of the occurrence of gene polymorphisms CAPN316 and UoGCAST in the population of cattle

To solve the problem of increasing the rate of breeding, but reduce the breeding work with farm animals, it is necessary to form herds with the desired level of productivity, that are adapted to specific regions of breeding, resistant to various diseases, with a decrease in the time for the breeding process. The active use of molecular genetic methods has contributed to the expansion of the list of DNA markers for farm animals, which are candidate genes for economically useful traits. Among these genes are widely known members of the calpain-calpastatin system, which is associated with postmortem proteolysis and tenderisation of muscles. The calpain system consists of an actively expressed μ-calpain (CAPN1) and m-calpain (CAPN2) and a single endogenous inhibitor, CAPN1 and CAPN2-calpastatin (CAST). The study of polymorphisms of these genes contributes to the expansion of marker characterisation in breeding. DNA samples (n=139) obtained from the blood of cattle were used in the work. Real-time PCRs were carried out using a ANK-32 programmable thermocycler (Synthol, Moscow, Russia). The CAPN316 gene polymorphism was present in 15% of animals tested, with allele G being found in 85% of animals. Similar calculations on the occurrence of the UoGCAST gene polymorphism in this sample of animals found the desirable allele G in 22% of animals and allele C in the remaining 78%. From the analysis of the occurrence of both genes and their polymorphisms in the study population, animals that had a combination of both desirable genotypes (CC*GG*) of CAPN316 and UoGCAST genes were not identified. There were also no animals that had the desired CAPN316 CC genotype and the heterozygous state of the UoGCAST gene (CC*GC). While 7.6% of the animals were CC*GG for the desirable expression of gene CAPN316, they contained the homozygous form of the gene UoGCAST.

Current State and Prospects of DNA Barcoding and DNA Fingerprinting in the Analysis of the Quality of Plant Raw Materials and Plant-Derived Drugs

Abstract—This paper reviews the use of molecular genetic methods (DNA barcoding and DNA fingerprinting) for the authentication of plant raw materials and herbal medicinal products. ITS2 is shown to be the most effective DNA barcode for plants. This marker allows researchers to identify even highly degraded DNA, which is especially important in the case of herbal preparations and biologically active additives. The main problem of traditional DNA barcoding via Sanger sequencing is that it is impossible to read a barcode without preliminary cloning of an amplificate when a plant mix includes a large amount of various adulterants and excipients of plant origin, since species-specific chromatograms are superimposed on each other. Thus, NGS sequencing of ITS2 amplicons is a preferable method for the DNA barcoding of plant materials. This method can be used to analyze highly degraded DNA in multicomponent plant mixes and can be used to identify all their ingredients. The review also covers such DNA fingerprinting techniques as RFLP, RFLP-PCR, RAPD, RAPD-SCAR, AFLP-PCR, and ISSR.

АНАЛИЗ ГЕНЕТИЧЕСКИХ ВЗАИМОСВЯЗЕЙ СОРТОВ РИСА ИЗ РАЗНЫХ ЭКОЛОГО-ГЕОГРАФИЧЕСКИХ ГРУПП С ИСПОЛЬЗОВАНИЕМ SSR МАРКЕРОВ

Microsatellite DNA markers are widely used in genetic research on rice: for studying the genetic diversity of genetic resources collections, iden-tifying microevolutionary processes within the genus Oryza L. and analyzing genetic relationships of gene pools from different regions of the world, as well as in gene mapping and performing marker-assisted selection in breeding. Given the size of the collection held by the All-Russian Rice Research Institute, which has a total of about 7,000 accessions, it is important to analyze its genetic structure and study genetic diversity, including the use of molecular genetic methods. The aim of the work was to clarify the genetic relationships among modern Russian rice varieties and varieties from different regions of Asia (Central Asia or East Asia) representing various ecogeographic groups using microsatellite DNA markers. The material of the study was 32 rice cultivars representing three different ecogeographic groups: Eastern, Central Asian and European. We used 10 SSR markers, which showed a level of polymorphism from 2 (RM11, RM316, RM19) to 8 alleles (RM1). For a total of ten loci, 46 alleles were identified. The average PIC was 0.45. The results of the Bayesian analysis and clustering using the UPGMA method made it possible to make a conclusion about the complex genetic structure of the set of the studied cultivars, which is consistent with the presence of cultivars from different ecogeographic groups (EGG). A number of cultivars that occupy an intermediate position between ecogeographic groups were identified, which obviously is a consequence of the active use of crosses between cultivars belonging to different EGGs in the process of breeding. Russian cultivars, which according to their morphological characteristics belong to the European ecogeographic group, were attributed to the group which also included some varieties from Central Asia and from the Eastern EGG. It was a consequence of the use of cultivars belonging to different ecogeographic groups in domestic breeding programs.

The Assembly and Analysis of a Reference Library Based on the Mitochondrial COI Gene Nucleotide Sequences for Delimitation of Sand Lance Species (Uranoscopiformes: Ammodytidae) from the Northern Hemisphere

Sand lances of the Northern Hemisphere (Uranoscopiformes: Ammodytidae) are shelf-dwelling fish characterized by cryptic diversity at the species level that show paraphyletic relationships at the genus level. Recently, they have drawn the attention of taxonomists due to the widespread use of molecular genetic methods in taxonomy. In the present study, a reference library of mitochondrial COI gene nucleotide sequences for eight sand lance species of the genus Ammodytes and two species of the genus Hyperoplus has been assembled and analyzed using the distance-based (ABGD, comparison of genetic distances) and topological (PTP) criteria. These species identification methods are shown to be reliable for high values of interspecific divergence (five species of Ammodytes and one species of Hyperoplus), whereas for the species that exhibit a low divergence (A. tobianus and H. lanceolatus) the topological criteria exceed the distance-based ones. The species A. personatus has been found in the eastern Sea of Okhotsk for the first time.

Концепция вида у тополей (genus Populus L., Salicaceae) на примере представителей подрода Tacamahaca (Spach) Penjkovsky, произрастающих в России и сопредельных странах

Using the traditional research methods, based on the example of local and cultivated representatives of the Tacamahaca (Spach) Penjkovsky subgenus growing in Russia and neighboring countries, the concept of a species in poplars (genus Populus L., Salicaceae) has been developed. It is suggested that the sections of black poplars (Aigeiros Duby) and balsamic poplars (Tacamahaca Spach) in Eurasia form a single supraspecific system with a common gene pool, which is very similar to syngameon. The taxonomic species that make up such a system are different dynamic states of this system and exist in the equilibrium state between natural selection, which forms and preserves the specifics of each dynamic state, and gene flows from other taxonomic species. Such a system can also be considered as a large Linnaean species represented by many subspecies or even geographical and ecological races. The use of molecular genetic methods for the taxonomic species study in the Populus genus is difficult due to the powerful flow of genes between the species, since the species differ in a small number of genes responsible for adaptive characters. The sections of the subgenus Tacamahaca poplars are ecological. The combination of species in these sections is not so much about the unity of their origin, but about the common growing conditions: black poplars tend to grow in lowlands, while the balsamic poplars tend to grow in the mountains, with which the poplars’ diagnostic characters are associated. In nature, belonging to a certain section is more profitable than the intermediate state, but in cultivation and in the urban environment, the opposite situation is observed.

Використання молекулярно-генетичних методів дослідження для контролю якості та безпеки продукції агропромислового комплексу

Використання молекулярно-генетичних методів дослідження для контролю якості та безпеки продукції агропромислового комплексу

Aphid resistance in potato

The problem of aphid resistance of potato genetic resources is discussed. Generally, resistance to the green peach aphid (Myzus persicae Sulzer) and the potato aphid (Macrosiphum euphorbiae Thomas) is analyzed in all available literature sources. The Solanum tuberosum L. gene pool contains very few resistant forms. Accessions highly resistant to M. persicae were revealed among 36 of 86 examined wild potato species. The largest number of resistant forms was found among S. bulbocastanum Dun., S. tarijense Hawkes, S. infundibuliforme Phil., S. canasense Hawkes and S. stoloniferum Schlechtd. et Bche. The forms highly resistant to M. euphorbiae belong to 24 of 85 studied species, namely to 10 of 18 series; the species S. bulbocastanum, S. stoloniferum and S. demissum Lindl. include the maximum number of resistant forms. Wild species accessions with specific and group resistance to aphids were revealed. Glandular pubescence of leaves and stems in such species as S. polyadenium Greenm., S. tarijense and S. berthaultii Hawkes is considered as the main mechanism of resistance. Hybrids between S. tuberosum and S. berthaultii were widely used in breeding programs; however, the presence of glandular trichomes was linked to a number of undesirable characters. It is expected that association between the trichome conditioned insect resistance of S. berthaultii and undesirable agronomic traits could be overcome with the use of molecular genetic methods. Studies of the antifeedant activity of potato glycoalkaloids and their aglucons produced controversial results. In numerous works the aphid resistance of potato is connected with plant physiological state. Differences among wild potato accessions in the localization of resistance factors (leaf surface, epidermis, mesophyll, floema) were found that were indicative of differences of resistance mechanisms in the studied forms. Works on investigating induced aphid resistance in potato have great theoretical and practical significance.

Mobilization, conservation and study of cultivated and wild potato genetic resources

Nowadays, the world’s largest Potato Genebanks, including VIR Potato Genebank, hold about 98 thousand accessions of potato and its wild relatives. This gene pool, which has been accumulated for almost a century, represents a huge genetic diversity of tuber bearing species of the genus Solanum L., which has a great importance for basic and applied research in general, and for human food security. The genebanks of potato and other crops have three highly important tasks such as permanent replenishment of collection material, ensuring its preservation by various storage and maintaining methods, including seed and vegetative propagation, its all-round use in basic and applied research, as well as the improvement of the efficiency of agricultural production. Analysis of performance of the above tasks in genebanks and research institutes in most countries concerned with storage, study and use of potato germplasm, shows great results and achievements, both in terms of its biology and its improvement as a food crop. However, the above three ways to perform tasks still need to be improved in connection with progress made over past decades, in particular in the field of basic and applied sciences. In this paper, a brief analysis of the status of conservation, study and use of potato genetic resources and its wild relatives in the domestic and foreign breeding, as well as solutions of problems in these areas are provided. A great leap forward today is the use of molecular genetic methods for genotyping collection material in gene banks to identify and itemize collection samples, as well as for using this material in basic and applied research, including breeding programs. Coordination of efforts for the collection of wild species that are not in the collections or are represented by a small number of samples, as well as the exchange of material and information about it, will help expand genetic diversity in genebanks and knowledge of the biological features of the potato.

DETERMINATION OF VIRULENCE PROPERTIES OF PATHOGENIC MICROORGANISMS IN VITRO: STATE-OF-ART

Various methods for evaluation of virulence properties of causative agents of infectious dis- eases in vitro were analyzed: molecular-genetic, cultural-biochemical, immunologic, physiologic. Predominant use of molecular-genetic methods, expediency of a complex approach, relevance of search of novel informative parameters of virulence are noted. Study of biological properties of pathogens in vitro is the first screening stage of evaluation of their virulence.

Genetic alteration and mutation profiling of circulating cell-free tumor DNA (cfDNA) for diagnosis and targeted therapy of gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) have been recognized as a biologically distinctive type of tumor, different from smooth muscle and neural tumors of the gastrointestinal tract. The identification of genetic aberrations in proto-oncogenes that drive the growth of GISTs is critical for improving the efficacy of cancer therapy by matching targeted drugs to specific mutations. Research into the oncogenic mechanisms of GISTs has found that these tumors frequently contain activating gene mutations in either platelet-derived growth factor receptor A (PDGFRA) or a receptor tyrosine protein associated with a mast cell growth factor receptor encoded by the KIT gene. Mutant cancer subpopulations have the potential to disrupt durable patient responses to molecularly targeted therapy for GISTs, yet the prevalence and size of subpopulations remain largely unexplored. Detection of the cancer subpopulations that harbor low-frequency mutant alleles of target proto-oncogenes through the use of molecular genetic methods, such as polymerase chain reaction (PCR) target amplification technology, is hampered by the high abundance of wild-type alleles, which limit the sensitivity of detection of these minor mutant alleles. This is especially true in the case of mutant tumor DNA derived “driver” and “drug-resistant” alleles that are present in the circulating cell-free tumor DNA (cfDNA) in the peripheral blood circulation of GIST patients. So-called “liquid biopsy” allows for the dynamic monitoring of the patients’ tumor status during treatment using minimally invasive sampling. New methodologies, such as a technology that employs a xenonucleic acid (XNA) clamping probe to block the PCR amplification of wild-type templates, have allowed improved molecular detection of these low-frequency alleles both in tissue biopsy samples and in cfDNA. These new methodologies could be widely applied for minimally invasive molecular testing in the therapeutic management of GISTs.

Use of Molecular Genetic Methods for Investigation of Outbreaks of Enterovirus Infection in the Subjects of the Far Eastern Federal District

The results of molecular genetic studies of clinical samples taken from three foci of outbreaks of enterovirus infection, registered on the territory of Khabarovsk Krai and Amur Oblast from 2012 through 2013 were reported. Several strains of enteroviruses were obtained: strains of Coxsackie A-4 and Coxsackie A-16 from De-Kastri rural locality, strains of ECHO-6 from Ust’-Urikma (Amur Oblast) and strains of Coxsackie A-6 from Raichihisnsk (Amur Oblast). Use of molecular genetic methods for investigation of collective morbidity and outbreaks of enterovirus infection help to confirm the identity of enteroviruses, isolated from patients and to identify the imported cases of infection.

Creation of rice breeding material with blast disease resistance gene Pi-b with the use of molecular genetic methods

Introgression of the blast disease resistance gene Pi-b into domestic rice germplasm is accomplished with the use of molecular DNA marking methods. Near-isogenic lines are created on the basis of variety Yantar’ released in Krasnodar krai. New breeding material with the resistance gene is obtained. A series of lines is being prepared for competitive variety trial.

Molecular genetic tools to infer the origin of forest plants and wood.

Most forest tree species exhibit high levels of genetic diversity that can be used to trace the origin of living plants or their products such as timber and processed wood. Recent progress to isolate DNA not only from living tissue but also from wood and wood products offers new opportunities to test the declared origin of material such as seedlings for plantation establishment or timber. However, since most forest tree populations are weakly differentiated, the identification of genetic markers to differentiate among spatially isolated populations is often difficult and time consuming. Two important fields of “forensic” applications are described: Molecular tools are applied to test the declared origin of forest reproductive material used for plantation establishment and of internationally traded timber and wood products. These applications are illustrated taking examples from Germany, where mechanisms have been developed to improve the control of the trade with forest seeds and seedlings, and from the trade with wood of the important Southeast Asian tree family Dipterocarpaceae. Prospects and limitations of the use of molecular genetic methods to conclude on the origin of forest plants, wood, and wood products are discussed.

Comparison of clinical and molecular genetic criteria for diagnosing familial hypercholesterolemia

Familial hypercholesterolemia (FH) is a common autosomal-dominant inherited disorder of lipid metabolism. It is caused by a mutation in the LDL-receptor gene and is characterized by hypercholesterolemia, xanthomas and premature coronary heart disease. However, efficient lipid-lowering drug therapies are available that may normalize the cholesterol levels in FH heterozygotes. FH can be diagnosed by the use of clinical diagnostic criteria or by the use of molecular genetic methods to identify a mutation in the LDL-receptor gene. In this review, we have compared the two diagnostic strategies with respect to their clinical utilities to diagnose FH heterozygotes and to promote therapeutic interventions in those being diagnosed. We conclude, that the clinical diagnostic criteria both have low sensitivity and low specificity, and should, therefore, largely be replaced by molecular genetic methods in order to obtain a specific diagnosis that may form the basis for implementing preventive measures to prevent FH he...

The use of molecular genetic methods for prognosis of aerobic and anaerobic performance in athletes

The distribution of genotypes and alleles of ACE (I/D polymorphism), ACTN3 (R577X), NOS3 (5/4), UCP2 (Ala55Val), and UCP3 (-55C/T) genes, as well as the correlation between the genotype and physiological parameters, was studied in rowers (n = 230) and in a control group (n = 855). The genotypes were determined by analyzing restriction fragment length polymorphism. Physiological parameters were determined with a PM 3 rowing ergometer and a MetaMax 3B gas analyzer. The frequency of the ACE II genotype was significantly higher in elite rowers (n = 107) than in the control subjects. The frequency of the ACTN3 XX genotype, unfavorable for development of speed and strength qualities, was twofold lower in all rowers than in the control subjects. The frequencies of the ACE I, ACTN3 R, UCP2 Val, and UCP3 T alleles increased in the athletes along with an increase in skill, which suggested natural sports selection. In addition, ACE I, NOS3 5, UCP2 Val, and UCP3 T alleles correlated with a high aerobic performance. Thus, the ACE I, NOS3 5, UCP2 Val, and UCP3 T alleles may be regarded as genetic markers associated with enhanced aerobic performance and may be included in a diagnostic system for prognosis of human physical performance.

Genotyping the Butyrylcholinesterase in Patients with Prolonged Neuromuscular Block after Succinylcholine

Succinylcholine remains the standard neuromuscular blocking drug for tracheal intubation in emergency situations. The short duration of action is due to its rapid hydrolytic degradation by butyrylcholinesterase (plasmacholinesterase). Multiple variants of this enzyme are known (A, F, S, H, J, K variants) with different effects on enzyme activity. This study was undertaken to evaluate the use of molecular genetic methods in patients with clinically prolonged neuromuscular block. Nine patients with a neuromuscular block of 14 min to 5 h were selected. All four exons of the butyrylcholinesterase were amplified by polymerase chain reaction and analyzed by automated sequencing. Molecular genetic results were compared with clinical relaxation time and with biochemical test results (total butyrylcholinesterase activity, dibucaine and fluoride inhibition). Seven of nine patients were mutation carriers. Five of these had more than one mutation. The A and K variants were the most frequent variations. Three of four patients who were homozygous for the A variant were also carriers of the K allele. The authors identified one novel mutation (G1294T) introducing a stop codon at amino acid position 432. The duration of neuromuscular block was substantially different between patients with identical butyrylcholinesterase genotypes. Variations in the genetic sequence of butyrylcholinesterase are frequent in patients with prolonged duration of action of succinylcholine. Direct sequencing of the whole butyrylcholinesterase gene is an appropriate method for genotyping and, accordingly, should be used in future clinical studies with drugs metabolized by this enzyme (e.g., succinylcholine, mivacurium).

Quantitative and molecular genetic determination of protein and fat deposition

After 30 years of selection, breeding of the pig breed sus scrofa Piétrain has resulted in reduced backfat thickness (from 3.2 to 1.9 mm) and increased loin muscle area (40 to 60 cm2) which indicates high genetic determination of these body composition traits. The use of sophisticated quantitative genetic methods that include all genetic relationships of large populations has led to a high response to selection of these traits. Selection on feed intake, lean and fat tissue growth using nonlinear functions to optimise these traits during the entire growth period in a biological model offers the opportunity to further improve total genetic potential. Protein and lipid deposition rates during the entire growth period have to be known for this biological model to be applied; thus knowledge of the genetic background of these traits is of high economic value. With the use of molecular genetic methods, such as candidate gene and genome scan approaches, the identification of genes for obesity and growth can be obtained. In sus scrofa, candidate genes associated with obesity and growth include Leptin Receptor, Melanocortin-4 Receptor, Agouti related protein, Heart fatty acid binding protein 3, and Insulin-like growth factor 2. Some of these candidate genes also explain variation in obesity levels in humans. Initial genome-wide scans have identified quantitative trait loci (QTL) on chromosomes 1, 4, 5, 7 and X for obesity and on chromosomes 1, 4, 7, 8, 13 and 18 for growth. Physiological candidate genes and predispositional QTL for obesity are not always located on the same chromosome; this is known the "polygenic paradox". Use of a nonlinear growth function is recommended in order to give more insight into the physiological regulation of obesity traits. Sus scrofa is an excellent model organism to examine the genetic regulation of obesity. The conservation of DNA sequence and chromosomal segments between sus scrofa and homo sapiens will permit easy transfer of results to human studies.

Transcriptional Analysis of Tumor-Specific T-Cell Responses in Cancer Patients

Over the last decade, tumor immunology in general and tumor immunotherapy in particular have made important progress. Thanks to the discovery of tumor-associated antigens (TAA), and the consequent development of active-specific immunization protocols, TAA-specific T-cell responses can now be induced reproducibly in cancer patients. However, clinical responses directly ascribable to TAA-specific T cells occur only occasionally. It is not clear why TAA-specific T cells do not eliminate tumor cells in vivo. This paradoxical coexistence of antigen-bearing tumor cells and antigen-specific T cells constitutes a critical point of current investigation. In recent years, protein-ligand interaction-based methods aimed at the identification and characterization of T cells using antibodies (cell surface phenotyping, intracellular and secreted cytokine detection), or HLA-peptide multimers, have significantly improved the analysis of TAA-specific T cells. These methods, however, seem to have reached the limit of their usefulness. Transcriptional analysis may add sensitivity and resolution and may provide global pictures of the multifactorial requirements for an efficient immune response against tumors. In this review, we describe the use of molecular genetic methods, such as real time qRT-PCR, cDNA microarrays, and TCR repertoire analysis. In addition, we describe techniques for high-fidelity messenger RNA amplification that allow high-throughput analysis of samples obtained from minimal sources, such as HLA/peptide tetramer sorted antigen-specific T cells, laser capture dissection, or fine needle aspirates. Recent work discussed in this review summarizes the complementarity of transcriptional analysis as an essential tool that, in addition to conventional methods, may deepen and broaden the characterization of tumor-specific T cells.

Molecular pathology of soft tissue and bone tumors. A review.

Objective To present recent concepts on the molecular pathogenesis of tumors of soft tissue and bone, and on the use of molecular genetic methods, including their significance as diagnostic markers and prognostic indicators. Data sources and study selection Reports on tumors of bone and/or soft tissue published in the English language literature and observations made using specimens available at the Departments of Pathology at Albany Medical College and Loyola University Medical Center. Data extraction and synthesis Studies on bone and soft tissue tumors containing chromosomal or genetic evaluation were selected for further analysis. Specific chromosomal abnormalities, such as numerical aberrations or translocations with production of fusion genes, were classified according to the tumor of origin. Data were also collected on mutations in tumor suppressor genes, genes coding for growth factors or their receptors, and genes coding for tyrosine kinases. Also noted were mutations of uncertain significance, for which the pathogenic connection between tumor production and mutated gene function is still unclear. Conclusions In general, the mutations reported interfere with the action of peptide growth factors coordinating mesenchyme proliferation and differentiation, although membrane-bound receptors expressing the intracellular signaling modifier, tyrosine kinase activity, have also been involved. Functional types of genes most commonly affected include tumor suppressors, oncogenes, and nuclear transcription factors. Thus, the mutations involved in the pathogenesis of soft tissue and bone tumors have affected multiple genes. Moreover, aberrant fusion gene products may be formed in tumoral tissue and may then act as transcription regulators stimulating cellular proliferation. Cytogenetic studies help at the clinical level by demonstrating aneuploidy and increased ploidy, which may correlate with malignant behavior. Diagnostic tumor-specific chromosomal translocations may be detected with Southern hybridization analysis, polymerase chain reaction, reverse-transcription polymerase chain reaction, or with the fluorescence in situ hybridization technique. Notably, early metastatic disease may be detectable in blood specimens using polymerase chain reaction or reverse-transcription polymerase chain reaction techniques.

Molecular Pathology of Soft Tissue and Bone Tumors

Abstract Objective.—To present recent concepts on the molecular pathogenesis of tumors of soft tissue and bone, and on the use of molecular genetic methods, including their significance as diagnostic markers and prognostic indicators. Data Sources and Study Selection.—Reports on tumors of bone and/or soft tissue published in the English language literature and observations made using specimens available at the Departments of Pathology at Albany Medical College and Loyola University Medical Center. Data Extraction and Synthesis.—Studies on bone and soft tissue tumors containing chromosomal or genetic evaluation were selected for further analysis. Specific chromosomal abnormalities, such as numerical aberrations or translocations with production of fusion genes, were classified according to the tumor of origin. Data were also collected on mutations in tumor suppressor genes, genes coding for growth factors or their receptors, and genes coding for tyrosine kinases. Also noted were mutations of uncertain significance, for which the pathogenic connection between tumor production and mutated gene function is still unclear. Conclusions.—In general, the mutations reported interfere with the action of peptide growth factors coordinating mesenchyme proliferation and differentiation, although membrane-bound receptors expressing the intracellular signaling modifier, tyrosine kinase activity, have also been involved. Functional types of genes most commonly affected include tumor suppressors, oncogenes, and nuclear transcription factors. Thus, the mutations involved in the pathogenesis of soft tissue and bone tumors have affected multiple genes. Moreover, aberrant fusion gene products may be formed in tumoral tissue and may then act as transcription regulators stimulating cellular proliferation. Cytogenetic studies help at the clinical level by demonstrating aneuploidy and increased ploidy, which may correlate with malignant behavior. Diagnostic tumor-specific chromosomal translocations may be detected with Southern hybridization analysis, polymerase chain reaction, reverse-transcription polymerase chain reaction, or with the fluorescence in situ hybridization technique. Notably, early metastatic disease may be detectable in blood specimens using polymerase chain reaction or reverse-transcription polymerase chain reaction techniques.