DNA Profiling Methods Research Articles

Investigation of a DNA Profiling Method Using Only Cementum More Than 70 Years After Death.

Short tandem repeat (STR) typing is widely used not only for blood relationship identification but also for the personal identification of unidentified bodies. However, DNA is susceptible to the effects of environmental factors, consequently leading to reduced DNA yields. Therefore, to maximize the DNA yield required for identification, teeth are generally completely pulverized during DNA extraction. However, this renders subsequent testing after DNA profiling impossible. In this study, we investigated the utility of DNA profiling using only the cementum from teeth that had been left outdoors for long postmortem intervals. We analyzed 90 molars (fresh teeth) that were extracted within six months at a dental clinic and 90 molars (stale teeth) exposed outdoors for over 70 years, and following cementum extraction, the accuracy of STR profiling, optimal site for cementum collection, and minimum amount of cementum required for STR profiling were determined. The results demonstrated that the profiling accuracy of DNA extracted from cementum was comparable to that of DNA from dental pulp and dentin. Furthermore, the collection of cementum from either near the cervical line or from the root apex areas did not show significant differences in DNA profiling accuracy, indicating that securing at least 5 mg of cementum was sufficient to ensure precise DNA profiling. These findings suggest that DNA profiling using only cementum is viable even in teeth that have been subjected to a long postmortem interval.

DNA Profiling, Bioinformatics and Databases in Forensics: Human Identification Purposes

Individual identification is an essential element in establishing truth the forensic investigation process, be it in criminal, medico-legal, or mass disasters case. When ante-mortem data are not available, the only thing that can be used is identification trough DNA. Alec Jeffrey, a geneticist (1984), found that certain regions of DNA contain repetitive sequences and the number of repetitions in individuals differs from one another. This identification method known as DNA profiling. DNA profiling is described as an important and significant discovery in forensics and has been considered as the standard in modern human identification. Until now, the recommended DNA profiling method must be based on the PCR to analyze degraded DNA and short-sized DNA (Short Tandem Repeats) through PCR amplification. In profiling, the result DNA amplification are analyzed through genetic markers (DNA Markers) and then matched into the database or compared with the collected reference samples. In the human genome, the genetic markers most frequently used in forensics are autosomal STR, Y-STR, mtDNA, X-STR, SNPs, and Amelogenin. Accomplishment of proving the suitability of DNA profiles, an application of bioinformatics in forensics is carried out. Bioinformatics is a combination of molecular biology and computer informatics that aims to manage and analyze data and store biological (genetic) information. DNA database is an application of bioinformatics in the form of special software which has become an important tool for biologists and forensics. This genetic database will predict the similarities between one DNA profile and another.

The present and future of forensic genetics

In this article, we discuss the current state and future directions in the field of forensic genetics. The DNA analysis of biological traces found at a crime scene, which was first used in a criminal investigation in 1987, did revolutionize forensic science. Over the past three decades, significant advances have been made in the recognition capacity, speed, and sensitivity of DNA profiling methods, as well as in their capability of typing increasingly complex patterns. Creation of DNA databases of criminals and crime scenes, as well as population allele frequencies, allows suspects to be identified from crime scene samples and DNA evidence to be statistically processed to verify its reliability. At present, it has become possible to identify even single cells left at a crime scene and to successfully analyze ancient human remains. Forensic DNA profiling can be used to identify not only individuals known to the investigating authorities. Experts are increasingly applying new genetic markers that can expand the scope of DNA profiling methods. Modern developments enable extraction of new types of forensically significant information from biological traces, e.g., using molecular approaches to searching for individuals previously unknown to investigators. New methods have been proposed to identify the relationship between the donors of forensic samples and the crimes committed. Modern advances in the decoding of the human genome, as well as the availability of genome-wide analysis and sequencing techniques, pave the way for new forensic DNA tools capable of enhancing the quality of forensic science in the near future.

Microbial eukaryote community succession over hundreds of years in Chinese lakes

Microbial eukaryotes play critical roles in maintaining the health and stability of lake ecosystems. Consequently, investigating the spatial and temporal evolution of microbial eukaryotic community structures and associated environmental drivers can inform water ecology studies and water environmental protection efforts. In this study, sediment core samples were collected from Lakes Chaohu, Erhai, Lugu and Fuxian in China to reconstruct lake microbial eukaryotic community succession using sediment DNA profiling methods. Mantel tests were used to identify the key factors influencing community succession and explore the mechanisms underlying ecological network changes. Statistical analyses revealed that community structures and diversity in Lakes Chaohu and Erhai shifted in the 2000s and 1980s, respectively, while the communities of Lakes Fuxian and Lugu did not exhibit significant changes during the study period, which may be due to the oligotrophic state of these two lakes. Ciliophora and Chlorophyta were the primary eukaryotic microbial taxa in the four lakes during the study period, with overall relative abundances of 27.2 % and 22.3 %, respectively. The diversities of microbial eukaryotic communities in the lakes were significantly correlated with nutrient enrichment and meteorological factors. Lastly, the ecological niche gap of microbial eukaryotes gradually overlapped after community shifts in Lake Chaohu, while biased interactions strengthened. Overall, this study reveals the impacts of long-term human activities and climate change on lake ecosystems from multiple perspectives of microbial eukaryotic community structure, diversity and ecological network, these findings will contribute to integrate microbial indicators in freshwater environmental monitoring in the future.

Salmon sperm DNA increases sample recovery from cotton swabs

Forensic samples with low DNA template amounts are difficult to analyze and interpret. There is a large body of research demonstrating that adding carrier nucleic acid to storage tubes, solid phase extractions, or filtering devices can improve yields of target DNA. However, the addition of carrier nucleic acid to sampling substrates, like cotton swabs, has not yet been attempted. In this proof-of-concept study, carrier nucleic acids in the form of either Poly (A) RNA or salmon sperm DNA were spotted onto cotton swabs, followed by human genomic DNA, to determine if introducing the carrier prior to sample collection would increase recovery from the swabs post-extraction. Extracts were also evaluated to determine whether adding the carrier nucleic acids to human DNA would interfere with downstream forensic DNA analysis processes such as real-time PCR quantitation, PCR amplification of STR loci, or capillary electrophoresis. The RNA carrier did not improve human sample recovery from cotton swabs. The extraction efficiency of human DNA from cotton swabs was increased when the DNA carrier was applied to the swabs prior to sample deposition, and the scale of the increase depended on the amount of carrier DNA used. When applying the salmon sperm DNA carrier to cotton swabs, with each increase from no carrier to 0.001–1–10 µg, human DNA recovery went from ∼29 % to ∼50 % to ∼75 % to ∼100 %. Additionally, no inhibitory effects from the carrier DNA were observed post-extraction with quantitation or in the DNA profile after amplification. Therefore, salmon sperm DNA carrier will increase human DNA yield from cotton swabs without negative effects on downstream forensic DNA profiling methods, with the optimal carrier amount being 10 µg.

The role of DNA in forensic science: A comprehensive review

Forensic genetics, leveraging molecular tools and scientific applications, has witnessed significant advancements in DNA analysis over the last three decades. These progressions have enhanced the discrimination power, speed, and sensitivity of DNA profiling methods, enabling the analysis of challenging samples. This article explores the significance of forensic genetics in criminal investigations, traces the historical evolution of DNA analysis techniques, and presents recent developments in the field. The article aims to provide a comprehensive understanding of the crucial role of forensic genetics in criminal investigations and sheds light on the latest trends and breakthroughs in this area. The evolution of DNA typing from ABO blood typing to the current standard of short tandem repeat (STR) analysis is discussed, along with alternative DNA analysis methods, such as Y-chromosome analysis and single nucleotide polymorphism (SNP) typing. Massively parallel sequencing (MPS) represents a groundbreaking advancement, enabling whole genome sequencing and addressing complex cases. The article also covers recent innovations, including DNA methylation analysis, body fluid identification, forensic DNA phenotyping, and genetic genealogy, highlighting their potential benefits in forensic investigations. Despite these advancements, standard STR profiling remains the gold standard due to its established protocols and databases. Ethical considerations regarding data privacy and cost implications are crucial as these technologies continue to progress in their pursuit of justice.

Real-world data on renal transplantations from a tertiary-care hospital in North India, in context of Indian regulatory act-transplantation of human organs and tissues act (THOTA): A retrospective analysis

IntroductionRenal transplantation is the treatment of choice for patients suffering from end stage renal disease (ESRD). Indian regulations defined under Transplantation of Human Organs and Tissues Act (THOTA), 2014 restricts organ donations to near-related living donors to curb any malpractices like ‘paid donors’ in living-donor kidney transplantation (LDKT). The aim of our study was to look at real-world data of donor-recipient pairs and to identify relationship of donors (with their respective patients) and the common (or uncommon) DNA profiling methods used for supporting “claimed relationship” in accordance with the regulations. Material and methodsThe donors were categorized and grouped into near-related donor, donors other than near-related donors, swap donors and deceased donors. Claimed relationship was confirmed, commonly by HLA typing, using SSOP method. In few cases, which were uncommon (and infrequent), autosomal DNA analysis, mitochondrial DNA analysis and Y-STR DNA analysis were performed to support the claimed relationship. Data collected included age, gender, relationship, DNA profiling test method. ResultsAmong the 514 donor-recipient pairs evaluated, numbers of female donors out-numbered male donors. The decreasing order of relationships in near-related donor group were wife>mother>father>sister>son>brother>husband> daughter>grandmother. 11.9% of donors were in the category of donors other than near-related donors. In 97.86% cases, the claimed relationship was supported by HLA typing and in just 2.1% cases autosomal DNA analysis>mitochondrial DNA analysis> Y-STR DNA analysis, in this order, were performed to establish relationship. ConclusionThis study brought out gender disparity with women out-numbering men as donors. Among recipients, access to renal transplant was largely restricted to men. As far as relationship of donors to recipients was concerned, mostly near-related family members, like wife, were donors and claimed relationship was almost always (99%) was corroborated by HLA typing.

Forensic DNA Analysis: Development State and Prospects in Ukraine

This research purpose is to summarize information on the main directions of using DNA analysis in the practice of detection and investigation of crimes used in different countries, consider possibilities of these areas and the state of their implementation in domestic investigative and forensic expert practice and formulate the author’s vision of prospects for the further development of theoretical and applied aspects of forensic DNA research in Ukraine.
 Traditional DNA profiling methods based on STR locus analysis and mtDNA SNP research are described, information on the application of the latest technologies for searching relatives by DNA profile bases and determining the age and biogeographical origin of human DNA, as well as methods of mass parallel DNA sequencing and performing rapid DNA profile tests. It was noted that main obstacle to the further development of DNA analysis in Ukraine is the lack of proper legal regulation that hinders formation of effective national database of DNA profiles. Attention is drawn to the obvious lack of scientific support for DNA research in Ukraine.
 Ways to solve existing issues in this area are proposed, in particular: promote relevant knowledge among law enforcement officers to minimize typical errors in handling of traces of biological origin; make changes in the criminal procedural legislation of Ukraine for the purpose of proper adjustment of issues of sampling of biological samples and carrying out rapid analyzes on a DNA profile; take measures to expand the tool base and real capabilities of forensic genetics laboratories.

Genetic relationship of the winter barley varieties assessed by the inter-Primer Binding Site (iPBS) DNA profiling method

ABSTRACT Winter barley (Hordeum vulgare L.) is an important crop with great impact on the nutrition of the world population. Many of the barley varieties used in industry are obtained from private and public seed collection organizations. Understanding the relationships among barley genotypes within these collections is crucial for breeding and maintenance. The objective of this study was to determine the genetic diversity in winter barley from the Kuban State Agrarian University collection by using DNA-profiling polymerase chain reaction (PCR) methods based on a single primer complementary to the primer binding site (PBS) region downstream of the long terminal repeat (LTR) for retrotransposons. In total, 1293 PCR amplicons were identified, of which 646 were polymorphic. Barley genotypes from this collection were mainly divided into two populations based on their geographical origin. Analysis of Molecular Variance (AMOVA) results indicated a high level of genetic diversity among the barley genotypes (83%). Relationships of the studied barley genotypes and their ancestry were established using clustering and Principal coordinate analysis (PCoA) analysis. The results of PCoA and clustering were similar and close genetic relationships of selected accessions were confirmed. Surprising results were obtained for Timofei and Vasya genotypes that are not supposed to be closely related, but were positioned together. Similar unexpected results were shown for Samson and Iosif varieties. Nevertheless, obtained data can be useful for planning of future crossing by breeders using distantly related crossing pairs.

Genetic Diversity and Pathogenicity of Rhizoctonia spp. Isolates Associated with Red Cabbage in Samsun (Turkey).

A total of 132 Rhizoctonia isolates were recovered from red cabbage plants with root rot and wirestem symptoms in the province of Samsun (Turkey) between 2018 and 2019. Based on the sequence analysis of the internal transcribed spacer (ITS) region located between the 18S and 28S ribosomal RNA genes and including nuclear staining, these 124 isolates were assigned to multinucleate Rhizoctonia solani, and eight were binucleate Rhizoctonia. The most prevalent anastomosis group (AG) was AG 4 (84%), which was subdivided into AG 4 HG-I (81%) and AG 4 HG-III (3%), followed by AG 5 (10%) and AG-A (6%), respectively. The unweighted pair group method phylogenetic tree resulting from the data of 68 isolates with the inter-PBS amplification DNA profiling method based on interspersed retrotransposon element sequences confirmed the differentiation of AGs with a higher resolution. In the greenhouse experiment with representative isolates (n = 24) from AGs on red cabbage (cv. Rondale), the disease severity index was between 3.33 and 4.0 for multinucleate AG isolates and ranged from 2.5 to 3.17 for AG-A isolates. In the pathogenicity assay of six red cabbage cultivars, one isolate for each AG was tested using a similar method, and all cultivars were susceptible to AG 4 HG-I and AG 4 HG-III isolates. Redriver and Remale were moderately susceptible, while Rescue, Travero, Integro, and Rondale were susceptible to the AG 5 isolate. The results indicate that the most prevalent and aggressive AGs of Rhizoctonia are devastating pathogens to red cabbage, which means that rotation with nonhost-crops for these AGs may be the most effective control strategy. This is the first comprehensive study of Rhizoctonia isolates in red cabbage using a molecular approach to assess genetic diversity using iPBS-amplified DNA profiling.

Wewnętrzna walidacja RapidHITTM IT200 – systemu zautomatyzowanej identyfikacji człowieka na podstawie analizy DNA

RapidHITTM 200 by IntegenX® (Thermo Fisher Scientific) is an integrated system for fast human identification based on STR analysis, providing a number of advantages over traditional DNA profiling methods in terms of automation, speed of analysis and mobility. However, before rapid DNA technology could be implemented into routine forensic applications, both workflow and its results would have to be rigorously validated for reliability, performance and compatibility with data obtained from capillary electrophoresis, through an internal validation. As we were primarily interested in the RapidHITTM 200’s ability to efficiently generate DNA profiles from samples collected at crime scenes and to upload this data to the CODIS database (Combined DNA Index System), the evaluation study focused mainly on such samples. The results show that RapidHITTM 200 can be a useful tool to complement conventional identification methods used in forensic genetics.

PECULIARITIES WHILE PREPARING TO THE APPOINTMENT OF FORENSIC MOLECULAR GENETICS EXAMINATION IN CRIMINAL PROCEEDINGS INITIATED DUE TO MURDERS

Pre-trial investigation of criminal proceedings initiated due to murders requires to possess special knowledge, take into consideration the peculiarities and procedure for conducting investigative (search) and procedural actions by investigators and prosecutors. The most evidential method is considered to be the method of DNA profiling, which has confirmed its efficiency in the mentioned criminal proceedings.
 The procedure of preparing to the appointment of forensic molecular genetics examinations is a significant stage of forensic examination. It should be carried out at an appropriate level. The errors made by investigators while the detection, collection of biological origin samples, their storage and transportation exclude the possibility of solving identification problems, and sometimes the accomplishment of criminal proceedings tasks, what necessitates the need to develop recommendations aimed at eliminating them.
 The article purpose is to identify the modern capabilities of forensic molecular genetics examinations in the investigation of murders initiated due to murders, determining typical errors, which are made by prosecutors at the stages of preparation and appointment of the indicated examinations and the development of recommendations for their prevention and elimination.
 Ensuring the elimination and prevention of errors that are made by investigators, as well as the prevention of processes of DNA contamination and degradation are possible due to increase in the level of investigators’ special knowledge and the formation on this basis of skills for conducting appropriate investigative (search) and procedural actions. It is possible thanks to the involvement of forensic experts in the field of genetics while carrying out these actions, as well as in the case of appointing a group of investigators on indicated criminal proceedings.
 The indicated and worded recommendations regarding the measures that are expedient to be taken with the purpose of exclusion and elimination of common mistakes are a promising avenue for further scientific developments.

Mitochondrial DNA in human identification: a review.

Mitochondrial DNA (mtDNA) presents several characteristics useful for forensic studies, especially related to the lack of recombination, to a high copy number, and to matrilineal inheritance. mtDNA typing based on sequences of the control region or full genomic sequences analysis is used to analyze a variety of forensic samples such as old bones, teeth and hair, as well as other biological samples where the DNA content is low. Evaluation and reporting of the results requires careful consideration of biological issues as well as other issues such as nomenclature and reference population databases. In this work we review mitochondrial DNA profiling methods used for human identification and present their use in the main cases of humanidentification focusing on the most relevant issues for forensics.

Evaluation of the impact of six different DNA extraction methods for the representation of the microbial community associated with human chronic wound infections using a gel-based DNA profiling method

Infected chronic wounds are polymicrobial in nature which include a diverse group of aerobic and anaerobic microorganisms. Majority of these communal microorganisms are difficult to grow in vitro. DNA fingerprinting methods such as polymerase chain reaction-denaturation gradient gel electrophoresis (PCR-DGGE) facilitate the microbial profiling of complex ecosystems including infected chronic wounds. Six different DNA extraction methods were compared for profiling of the microbial community associated with chronic wound infections using PCR-DGGE. Tissue debris obtained from chronic wound ulcers of ten patients were used for DNA extraction. Total nucleic acid was extracted from each specimen using six DNA extraction methods. The yield, purity and quality of DNA was measured and used for PCR amplification targeting V2–V3 region of eubacterial 16S rRNA gene. QIAGEN DNeasy Blood and Tissue Kit (K method) produced good quality genomic DNA compared to the other five DNA extraction methods and gave a broad diversity of bacterial communities in chronic wounds. Among the five conventional methods, bead beater/phenol–chloroform based DNA extraction method with STES buffer (BP1 method) gave a yield of DNA with a high purity and resulted in a higher DGGE band diversity. Although DNA extraction using heat and NaOH had the lowest purity, DGGE revealed a higher bacterial diversity. The findings suggest that the quality and the yield of genomic DNA are influenced by the DNA extraction protocol, thus a method should be carefully selected in profiling a complex microbial community.

ANALYSIS OF EARPHONE SWAB MITOCHONDRIAL DNA AS AN ALTERNATIVE MATERIAL FOR IDENTIFICATION EXAMINATION

Identification include fingerprint, property, medical, dental, serologic and exclusion methods. In the development, identification methods led to molecular forensics, a new field of science evolving since the 1980s, known as DNA fingerprinting. Specimens widely used in DNA assay for identification are blood spots/bloods, semen spots, vaginal swabs, buccal swabs and bones. In addition to these specimens, the last objects often used by the perpetrators/victims can be used, such as hearing aids (headsets/earphones). In its use, earphones are attached to the outer ear skin; thus, the earwax is suspected to adhere to the device. To date, in Indonesia personal identification is performed through swabs of earphones/headsets using the DNA profiling method. In particular, mitochondrial DNA has not been widely used for identification. The present study was of laboratory experimental. Earphones which have been used for 3 days were placed in room temperature for 1, 7, 14 and 20 days. Results showed that the environmental factor of exposure duration had an effect of a significant decrease in the levels of DNA from day 1 to day 20. Only 126-bp mtDNA (HVS II) was detected on the samples of day 1 and continued with sequencing. Mitochondrial DNA has better durability and relatively higher number of copies than those of nuclear DNA. This leads to greater possibility of success in amplification, given the higher number of mitochondrial DNA copies and the fact that mitochondrial DNA is a single locus that allows recombination.

A rapid nuclear staining test using cationic dyes contributes to efficient STR analysis of telogen hair roots.

Telogen hairs presented in the crime scene are commonly encountered as trace evidence. However, short tandem repeat (STR) profiling of the hairs currently have low and limited use due to poor success rate. To increase the success rate of STR profiling of telogen hairs, we developed a rapid and cost-effective method to estimate the number of nuclei in the hair roots. Five cationic dyes, Methyl green (MG), Harris hematoxylin (HH), Methylene blue (MB), Toluidine blue (TB), and Safranin O (SO) were evaluated in this study. We conducted a screening test based on microscopy and the percentage of loss with nuclear DNA, in order to select the best dye. MG was selected based on its specific nuclei staining and low adverse effect on the hair-associated nuclear DNA. We examined 330 scalp and 100 pubic telogen hairs with MG. Stained hairs were classified into five groups and analyzed by STR. The fast staining method revealed 70% (head hair) and 33.4% (pubic hair) of full (30 alleles) and high partial (18-29 alleles) STR profiling proportion from the lowest nuclei count group (one to ten nuclei). The results of this study demonstrated a rapid, specific, nondestructive, and high yield DNA profiling method applicable for screening telogen hairs.

Authentication of newly established human esophageal squamous cell carcinoma cell line (YM-1) using short tandem repeat (STR) profiling method

Cross-contamination during or early after establishment of a new cell line could result in the worldwide spread of a misidentified cell line. Therefore, newly established cell lines need to be authenticated by a reference standard method. This study was conducted to investigate the authenticity of a newly established epithelial cell line of human esophageal squamous cell carcinoma (ESCC) called YM-1 using short tandem repeat (STR) DNA profiling method. Primary human ESCC epithelial cells were cultured from the fresh tumor tissue of an adult female patient. Growth characteristics and epithelial originality of YM-1 cells were studied. Genomic DNA was isolated from YM-1 cells harvested at passage 22 and ESCC donor tumor sample on two different days to prevent probable DNA contamination. STR profiling was performed using AmpFℓSTR® Identifiler® Plus PCR Amplification Kit. To address whether YM-1 cells undergo genetic alteration as the passage number increases, STR profiling was performed again on harvested cells at passage 51. YM-1 cells grew as a monolayer with a population doubling time of 40.66h. Epithelial originality of YM-1 cells was confirmed using ICC/IF staining of cytokeratins AE1/AE3. The STR profile of the ESCC donor tumor sample was the same with YM-1 cells at passage 22. However, STR profile of the donor tumor sample showed an off-ladder (OL) allele in their D7S820 locus. Also, re-profiling of YM-1 cells at passage 51 showed a loss of heterozygosity (LOH) at D18S51 locus. This suggests that long-term culture of cell lines may alter their DNA profile. Comparison of the DNA fingerprinting results in DSMZ, and ATCC STR profiling databases confirmed unique identity of YM-1 cell line. This study provides an easy, fast, and reliable procedure for authentication of newly established cell lines, which helps in preventing the spread of misidentified cells and improving the reproducibility and validity of experiments, consequently.

DNA profiles from fingermarks.

Criminal investigations would be considerably improved if DNA profiles could be routinely generated from single fingermarks. Here we report a direct DNA profiling method that was able to generate interpretable profiles from 71% of 170 fingermarks. The data are based on fingermarks from all 5 digits of 34 individuals. DNA was obtained from the fingermarks using a swab moistened with Triton-X, and the fibers were added directly to one of two commercial DNA profiling kits. All profiles were obtained without increasing the number of amplification cycles; therefore, our method is ideally suited for adoption by the forensic science community. We indicate the use of the technique in a criminal case in which a DNA profile was generated from a fingermark on tape that was wrapped around a drug seizure. Our direct DNA profiling approach is rapid and able to generate profiles from touched items when current forensic practices have little chance of success.

Allele-specific genome-wide profiling in human primary erythroblasts reveal replication program organization.

We have developed a new approach to characterize allele-specific timing of DNA replication genome-wide in human primary basophilic erythroblasts. We show that the two chromosome homologs replicate at the same time in about 88% of the genome and that large structural variants are preferentially associated with asynchronous replication. We identified about 600 megabase-sized asynchronously replicated domains in two tested individuals. The longest asynchronously replicated domains are enriched in imprinted genes suggesting that structural variants and parental imprinting are two causes of replication asynchrony in the human genome. Biased chromosome X inactivation in one of the two individuals tested was another source of detectable replication asynchrony. Analysis of high-resolution TimEX profiles revealed small variations termed timing ripples, which were undetected in previous, lower resolution analyses. Timing ripples reflect highly reproducible, variations of the timing of replication in the 100 kb-range that exist within the well-characterized megabase-sized replication timing domains. These ripples correspond to clusters of origins of replication that we detected using novel nascent strands DNA profiling methods. Analysis of the distribution of replication origins revealed dramatic differences in initiation of replication frequencies during S phase and a strong association, in both synchronous and asynchronous regions, between origins of replication and three genomic features: G-quadruplexes, CpG Islands and transcription start sites. The frequency of initiation in asynchronous regions was similar in the two homologs. Asynchronous regions were richer in origins of replication than synchronous regions.

English

Bamboos are arborescent grasses belonging to the family Poaceae and are groupedunder the subfamily Bambusoideae. Bambusoideae are differentiated from othermembers of the family by the presence of petiolate blades with parallel venation andstamens are three, four, six or more, gynoecium is with single style with one to threestigmas and caryopsis. Bamboo is regarded as the major resource that meets theneed of common man and also a poverty alleviator due to its multipurpose uses. TheRaigad district, Maharashtra, India, is a good pocket of wild as well as cultivatedbamboo. This district is rich in Bamboo flora specially four species viz. Bambusabambos; Pseudoxytenanthera ritcheyi; Pseudoxytenanthera stocksii andDendrocalamus strictus. To study the genetics of bamboo, one has to faceenormous difficulties simply because of rare flowering and we cannot makehybridizations as easily as like other angiosperms. The randomly amplifiedpolymorphic DNA (RAPD) method is used for genetic characterization of bamboo.This method of DNA profiling is proven excellent for identification, phylogeneticanalysis, population studies and genetic linkage mapping of many plant species. Establishing multilocus markers RAPD analysis of genomes provides a versatile andrapid technology requiring low infrastructural input and use of arbitrary primers fordeveloping plant DNA fingerprinting. In the present study, a comparative geneticanalysis of four species of bamboo B. bambus, P. ritcheyi, P. stocksii and D. strictuscollected from different locations of Raigad district of Maharashtra, India had beencarried out using PCR-RAPD analysis. The present results warrant an extensivesurvey of genetic variation between the given four species of bamboo. The resultswould be helpful in designing sequence characterized amplified regions (SCAR)marker for each species. These SCAR markers can be used as diagnostic marker toauthenticate samples of given four species. Key words: Randomly amplified polymorphic DNA (RAPD), DNA-fingerprinting,sequence characterized amplified regions (SCAR), Genetic characterization, ritcheyi,stocksii, Raigad.