Advances in molecular marker techniques and their applications in plant sciences

Detection and analysis of genetic variation can help us to understand the molecular basis of various biological phenomena in plants. Since the entire plant kingdom cannot be covered under sequencing projects, molecular markers and their correlation to phenotypes provide us with requisite landmarks for elucidation of genetic variation. Genetic or DNA based marker techniques such as RFLP (restriction fragment length polymorphism), RAPD (random amplified polymorphic DNA), SSR (simple sequence repeats) and AFLP (amplified fragment length polymorphism) are routinely being used in ecological, evolutionary, taxonomical, phylogenic and genetic studies of plant sciences. These techniques are well established and their advantages as well as limitations have been realized. In recent years, a new class of advanced techniques has emerged, primarily derived from combination of earlier basic techniques. Advanced marker techniques tend to amalgamate advantageous features of several basic techniques. The newer methods also incorporate modifications in the methodology of basic techniques to increase the sensitivity and resolution to detect genetic discontinuity and distinctiveness. The advanced marker techniques also utilize newer class of DNA elements such as retrotransposons, mitochondrial and chloroplast based microsatellites, thereby revealing genetic variation through increased genome coverage. Techniques such as RAPD and AFLP are also being applied to cDNA-based templates to study patterns of gene expression and uncover the genetic basis of biological responses. The review details account of techniques used in identification of markers and their applicability in plant sciences.     

Improving human forensics through advances in genetics, genomics and molecular biology

Forensic DNA profiling currently allows the identification of persons already known to investigating authorities. Recent advances have produced new types of genetic markers with the potential to overcome some important limitations of current DNA profiling methods. Moreover, other developments are enabling completely new kinds of forensically relevant information to be extracted from biological samples. These include new molecular approaches for finding individuals previously unknown to investigators, and new molecular methods to support links between forensic sample donors and criminal acts. Such advances in genetics, genomics and molecular biology are likely to improve human forensic case work in the near future.     

Review: domestic animal forensic genetics – biological evidence,genetic markers,analytical approaches and challenges

This review highlights the importance of domestic animal genetic evidence sources, genetic testing, markers and analytical approaches as well as the challenges this field is facing in view of the de facto ‘gold standard’ human DNA identification. Because of the genetic similarity between humans and domestic animals, genetic analysis of domestic animal hair, saliva, urine, blood and other biological material has generated vital investigative leads that have been admitted into a variety of court proceedings, including criminal and civil litigation. Information on validated short tandem repeat, single nucleotide polymorphism and mitochondrial DNA markers and public access to genetic databases for forensic DNA analysis is becoming readily available. Although the fundamental aspects of animal forensic genetic testing may be reliable and acceptable, animal forensic testing still lacks the standardized testing protocols that human genetic profiling requires, probably because of the absence of monetary support from government agencies and the difficulty in promoting cooperation among competing laboratories. Moreover, there is a lack in consensus about how to best present the results and expert opinion to comply with court standards and bear judicial scrutiny. This has been the single most persistent challenge ever since the earliest use of domestic animal forensic genetic testing in a criminal case in the mid‐1990s. Crime laboratory accreditation ensures that genetic test results have the courts’ confidence. Because accreditation requires significant commitments of effort, time and resources, the vast majority of animal forensic genetic laboratories are not accredited nor are their analysts certified forensic examiners. The relevance of domestic animal forensic genetics in the criminal justice system is undeniable. However, further improvements are needed in a wide range of supporting resources, including standardized quality assurance and control protocols for sample handling, evidence testing, statistical analysis and reporting that meet the rules of scientific acceptance, reliability and human forensic identification standards.     

Forensic applications of DNA fingerprinting

In many ways, DNA profiling technology is very similar to the conventional techniques used for forensic identification. As with, for example, blood grouping techniques, the molecular characteristics of the scene of crime sample may be determined and compared with those of the scene of reference samples from suspects and victim. If the molecular characteristics of the crime sample and the suspect are different, then they cannot be from the same person, whereas if they match, then the possibility remains that they may be from a single source. Similar material, such as blood or semen stains, may be used for both biochemical and genetic tests, and the main applications of identification and relationship testing are shared by both techniques. At this point, the similarity ends; DNA profiling has the following characteristics:

1. It is more sensitive, being able to generate sound data from only a tiny amount of even partially degraded biological material.
2. It is capable of resolving mixtures of semen or tissue from up to several individuals.
3. It has a far greater power of discrimination between individuals—sometimes up to 1 millionfold higher than conventional techniques.
4. It provides considerably more information on the nature of relationships, particularly in cases of incest.

As such, the technique represents a quantum leap in forensic identification and relationship testing.     

Evaluation of reliability on STR typing at leukemic patients used for forensic purposes

Over the past decades, main advances in the field of molecular biology, coupled with benefits in genomic technologies, have led to detailed molecular investigations in the genetic diversity generated by researchers. Short tandem repeat (STR) loci are polymorphic loci found throughout all eukaryotic genome. DNA profiling identification, parental testing and kinship analysis by analysis of STR loci have been widely used in forensic sciences since 1993. Malignant tissues may sometimes be the source of biological material for forensic analysis, including identification of individuals or paternity testing. There are a number of studies on microsatellite instability in different types of tumors by comparing the STR profiles of malignant and healthy tissues on the same individuals. Defects in DNA repair pathways (non-repair or mis-repair) and metabolism lead to an accumulation of microsatellite alterations in genomic DNA of various cancer types that result genomic instabilities on forensic analyses. Common forms of genomic instability are loss of heterozygosity (LOH) and microsatellite instability (MSI). In this study, the applicability of autosomal STR markers, which are routinely used in forensic analysis, were investigated in order to detect genotypes in blood samples collected from leukemic patients to estimate the reliability of the results when malignant tissues are used as a source of forensic individual identification. Specimens were collected from 90 acute and 10 chronic leukemia volunteers with oral swabs as well as their paired peripheral blood samples from the Oncology Centre of the Department of Hematology at Istanbul University, during the years 2010–2011. Specimens were tested and compared with 16 somatic STR loci (CSFIPO, THO1, TPOX, vWA, D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11 and FGA) widely used in forensic identification and kinship. Only two STR instabilities were encountered among 100 specimens. An MSI in the FGA loci and a LOH in the D2S1338 loci were determined in two individuals separately. Our results demonstrate that the use of the biological samples from leukemia patients in forensic identification and kinship testing is questionable, especially if known microsatellite instability is available. Genetic instabilities may alter the STR polymorphism, leading to potential errors on forensic identification of individuals. Therefore, typing of autosomal STRs from leukemia patients should be performed with both healthy and malignant tissue samples of individual as references.     

Genetic identification of missing persons: DNA analysis of human remains and compromised samples

Human identification has made great strides over the past 2 decades due to the advent of DNA typing. Forensic DNA typing provides genetic data from a variety of materials and individuals, and is applied to many important issues that confront society. Part of the success of DNA typing is the generation of DNA databases to help identify missing persons and to develop investigative leads to assist law enforcement. DNA databases house DNA profiles from convicted felons (and in some jurisdictions arrestees), forensic evidence, human remains, and direct and family reference samples of missing persons. These databases are essential tools, which are becoming quite large (for example the US Database contains 10 million profiles). The scientific, governmental and private communities continue to work together to standardize genetic markers for more effective worldwide data sharing, to develop and validate robust DNA typing kits that contain the reagents necessary to type core identity genetic markers, to develop technologies that facilitate a number of analytical processes and to develop policies to make human identity testing more effective. Indeed, DNA typing is integral to resolving a number of serious criminal and civil concerns, such as solving missing person cases and identifying victims of mass disasters and children who may have been victims of human trafficking, and provides information for historical studies. As more refined capabilities are still required, novel approaches are being sought, such as genetic testing by next-generation sequencing, mass spectrometry, chip arrays and pyrosequencing. Single nucleotide polymorphisms offer the potential to analyze severely compromised biological samples, to determine the facial phenotype of decomposed human remains and to predict the bioancestry of individuals, a new focus in analyzing this type of markers.     

Forensic uncertainty,fragile remains,and DNA as a panacea: an ethnographic observation of the challenges in twenty-first-century Disaster Victim Identification

This is an account of ethnographic research examining the specialist scientific processes known as ‘Disaster Victim Identification’ (DVI) in three settings: Québec, the United States, and the United Kingdom. In cases of multiple deaths, a series of actions accompanied by a plethora of tools are often invoked, housed at a disaster scene, forensic laboratories, a family assistance centre, and a mortuary. In this article, I examine a process dedicated to connecting the biological remains of the deceased with a confirmed validation of personhood. I describe a situation where responders/scientists will attempt multiple testing and re-testing of human remains, often pushing boundaries of available science. I argue that the search for certainty in identification lies at the heart of the activation of DVI processes, particularly when it is connected to DNA testing. Observing intimate forensic settings and the bricolage of the forensic anthropologist's labour has allowed me to track the production of the science of identity. I then reflect on the wider implications of these observations for affected communities and the responding scientists. Finally, I argue that there is complexity and ambivalence surrounding the increased use of technologies when applied to identification of victims.     

Cancerous tissues in forensic genetic analysis

Microsatellites or short tandem repeats (STRs) markers are important tools for mapping disease-causing genes by linkage, for performing investigations in forensic medicine, for population genetic studies and for studying genetic modifications in tumors. In forensic applications neoplastic tissues can be used as a source of genetic information for personal identification or paternity testing when no other specimen is available. Cancer tissues can show microsatellite instability (MSI) and loss of heterozygosity (LOH) also for the STRs used in the forensic field. In this study, we screened 56 sporadic gastrointestinal carcinomas in order to provide further data for the evaluation of the incidence of allelic alterations for 15 STR loci and the suitability of using cancerous tissues in forensic applications. Sixty-six percent of the cancerous tissues were found to possess allelic alterations of the microsatellites analyzed with a high incidence of MSI-L (microsatellite instability low) when compared to the corresponding normal tissue. The most frequently altered loci were D18S51, VWA, and FGA. From a forensic perspective, great care must be taken in evaluating the DNA typing results obtained from cancerous tissue samples.     

Ancient DNA: Using molecular biology to explore the past

Ancient DNA has been discovered in many types of preserved biological material, including bones, mummies, museum skins, insects in amber and plant fossils, and has become an important research tool in disciplines as diverse as archaeology, conservation biology and forensic science. In archaeology, ancient DNA can contribute both to the interpretation of individual sites and to the development of hypotheses about past populations. Site interpretation is aided by DNA-based sex typing of fragmentary human bones, and by the use of genetic techniques to assess the degree of kinship between the remains of different individuals. On a broader scale, population migrations can be traced by studying genetic markers in ancient DNA, as in recent studies of the colonisation of the Pacific islands, while ancient DNA in preserved plant remains can provide information on the development of agriculture.     

Identification of individuals by analysis of biallelic DNA markers, using PCR and solid-phase minisequencing.

We have developed a new method for forensic identification of individuals, in which a panel of biallelic DNA markers are amplified by the PCR, and the variable nucleotides are detected in the amplified DNA fragments by the solid-phase minisequencing method. A panel of 12 common polymorphic nucleotides located on different chromosomes with reported allele frequencies close to .5 were chosen for the test. The allele frequencies for most of the markers were found to be similar in the Finnish and other Caucasian populations. We also introduce a novel approach for rapid determination of the population frequencies of biallelic markers. By this approach we were able to determine the allele frequencies of the markers in the Finnish population, by quantitative analysis of three pooled DNA samples representing 3,000 individuals. The power of discrimination and exclusion of the solid-phase minisequencing typing test with 12 markers was similar to that of three VNTR markers that are routinely used in forensic analyses at our institute. The solid-phase minisequencing method was successfully applied to type paternity and forensic case samples. We also show that the quantitative nature of our method allows typing of mixed samples.     

DNA methylation profiling in cancer: from single nucleotides towards methylome

Genomic DNA methylation pattern (methylome) represents epigenetic program of a cell. It controls expression of genetic information. In tumor cells, significant alterations in DNA methylation take place, which can be identified as one of the earliest and most consistent features of tumorigenesis. Detailed survey of methylcytosines' distribution in genome is extremely important for understanding of real tumor etiology and early diagnostics. Progress in the field has been hampered by the unavailability of methods for large-scale determination of methylation patterns. Nowadays, variety of techniques is in development that allow for highly parallel regime of samples analysis (high-throughput analysis) or large loci DNA profiling (large-scale analysis). Aim of the work is to consider the main trends in the field of new methods development. The principles of the most frequently used approaches to DNA methylation studies are reviewed as well as their application and results. Most attention is paid to DNA microarrays as a technology of choice for epigenetic tumor analysis (oligonucleotide microarrays, BAC-arrays etc.). Alternative DNA sequencing based techniques are discussed, which can soon take on the leadership. Results of a large-scale analysis can be used for identification of new epigenetic markers and epigenetic classification of neoplasia.     

Basic research in evolution and ecology enhances forensics

In 2009, the National Research Council recommended that the forensic sciences strengthen their grounding in basic empirical research to mitigate against criticism and improve accuracy and reliability. For DNA-based identification, this goal was achieved under the guidance of the population genetics community. This effort resulted in DNA analysis becoming the 'gold standard' of the forensic sciences. Elsewhere, we proposed a framework for streamlining research in decomposition ecology, which promotes quantitative approaches to collecting and applying data to forensic investigations involving decomposing human remains. To extend the ecological aspects of this approach, this review focuses on forensic entomology, although the framework can be extended to other areas of decomposition.     

X-Chromosome STR markers data in a Cabo Verde immigrant population of Lisboa

Population genetic data of 12 X chromosomal short tandem repeats markers (DXS10074, DXS10079, DXS10101, DXS10103, DXS10134, DXS10135, DXS10146, DXS10148, DXS7132, DXS7423, DXS8378 and HPRTB) were analysed in 54 females and 95 males of an immigrant population from Cabo Verde living in Lisboa. The obtained results for forensic statistical parameters such as observed heterozigosity, polymorphism information content, power of discrimination and mean exclusion chance, based on single allele frequencies, reveal that this multiplex system is highly informative and can represent an important tool for genetic identification purposes in the immigrant population of Cabo Verde. Since the studied short tandem repeats genetic markers are distributed on four linkage groups, that can provide independent genotype information, we studied those groups as haploytes. The forensic efficiency parameters for the linked groups were all higher than 0.97, with linkage group I being the most polymorphic and linkage group III the less informative.     

DNA profiling

Although some concerns still remain in standard DNA profiling technology over the assumptions from population genetics used to calculate expected match frequencies, forensic scientists are preparing for the introduction of the next generation of DNA profiling techniques based on the polymerase chain reaction. These new techniques offer the prospect of dramatically increasing the speed and sensitivity of DNA profiling and have already been applied in some casework studies.     

Checking of individuality by DNA profiling

A review of methods of DNA analysis used in forensic medicine for identification, paternity testing, etc. is provided. Among other techniques, DNA fingerprinting using different probes and polymerase chain reaction-based techniques such as amplified sequence polymorphisms and minisatellite variant repeat mapping are thoroughly described and both theoretical and practical aspects are discussed.     

Familial identification: population structure and relationship distinguishability

With the expansion of offender/arrestee DNA profile databases, genetic forensic identification has become commonplace in the United States criminal justice system. Implementation of familial searching has been proposed to extend forensic identification to family members of individuals with profiles in offender/arrestee DNA databases. In familial searching, a partial genetic profile match between a database entrant and a crime scene sample is used to implicate genetic relatives of the database entrant as potential sources of the crime scene sample. In addition to concerns regarding civil liberties, familial searching poses unanswered statistical questions. In this study, we define confidence intervals on estimated likelihood ratios for familial identification. Using these confidence intervals, we consider familial searching in a structured population. We show that relatives and unrelated individuals from population samples with lower gene diversity over the loci considered are less distinguishable. We also consider cases where the most appropriate population sample for individuals considered is unknown. We find that as a less appropriate population sample, and thus allele frequency distribution, is assumed, relatives and unrelated individuals become more difficult to distinguish. In addition, we show that relationship distinguishability increases with the number of markers considered, but decreases for more distant genetic familial relationships. All of these results indicate that caution is warranted in the application of familial searching in structured populations, such as in the United States.     

Interpreting anonymous DNA samples from mass disasters--probabilistic forensic inference using genetic markers

MOTIVATION: The problem of identifying victims in a mass disaster using DNA fingerprints involves a scale of computation that requires efficient and accurate algorithms. In a typical scenario there are hundreds of samples taken from remains that must be matched to the pedigrees of the alleged victim's surviving relatives. Moreover the samples are often degraded due to heat and exposure. To develop a competent method for this type of forensic inference problem, the complicated quality issues of DNA typing need to be handled appropriately, the matches between every sample and every family must be considered, and the confidence of matches need to be provided. RESULTS: We present a unified probabilistic framework that efficiently clusters samples, conservatively eliminates implausible sample-pedigree pairings, and handles both degraded samples (missing values) and experimental errors in producing and/or reading a genotype. We present a method that confidently exclude forensically unambiguous sample-family matches from the large hypothesis space of candidate matches, based on posterior probabilistic inference. Due to the high confidentiality of disaster DNA data, simulation experiments are commonly performed and used here for validation. Our framework is shown to be robust to these errors at levels typical in real applications. Furthermore, the flexibility in the probabilistic models makes it possible to extend this framework to include other biological factors such as interdependent markers, mitochondrial sequences, and blood type. AVAILABILITY: The software and data sets are available from the authors upon request.     

Predicting genetic predisposition in humans: the promise of whole-genome markers

Although genome-wide association studies have identified markers that are associated with various human traits and diseases, our ability to predict such phenotypes remains limited. A perhaps overlooked explanation lies in the limitations of the genetic models and statistical techniques commonly used in association studies. We propose that alternative approaches, which are largely borrowed from animal breeding, provide potential for advances. We review selected methods and discuss the challenges and opportunities ahead.