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.

Wilms' tumor: a search for the critical lesion.

A Wilms' tumor from a 12-month-old boy showed epithelial and mainly rhabdomyoblastic differentiation. In addition, the kidney contained foci of nephroblastomatosis, a lesion predisposing to the development of nephric tumors. Flow cytometry indicated that the tumor DNA content was in the diploid range with an increased S-phase. Chromosome studies of the cultured tumor cells showed a dominant pattern of 49,XY, +8,9qh+, +12, +12,18q+, without obvious deletion of 11p. A few cells showed additional losses, deletions, or structural rearrangements superimposed on the basic pattern, but no normal metaphases were observed. The DNA from the tumor was probed for several loci on 11p because variations of 11p (deletion or translocation) have been reported in roughly one third of Wilms' tumors, and the critical gene in Wilms' has been localized to 11p13. In this case, 11p genes maintained heterozygosity or showed no detectable alteration in gene dosage when compared with peripheral-blood DNA. Therefore, despite histologic indication of an underlying constitutional defect, no genomic lesion of 11p was identified.

Preoperative aspiration cytology of breast tumors.

During a ten-year period, 1,942 aspirations of 1,906 mammary tumors in 1,874 patients were performed before excisional biopsy or mastectomy. The cytology findings were categorized as positive (1,107 cases), suspicious (152 cases), atypical (183 cases), benign (166 cases) and unsatisfactory (298 cases). All cytologically positive cases with follow-up were confirmed histologically or by clinical observation. Follow-up showed that 96% of the cases in the suspicious category, 86% of the cases in the atypical category, 51% of the cases in the benign category and 72% of the cases in the unsatisfactory category had malignant neoplasms. Aspiration cytology diagnosed 1,031 of 1,539 primary malignant mammary neoplasms (67%) and 19 of 28 neoplasms (68%) metastatic to the breast; if unsatisfactory cases are excluded, these figures become 1,031 of 1,365 cases (75%) and 19 of 25 (76%), respectively. If those cases reported as suspicious are included with the positive cases and those reported as atypical are included with the negative cases, aspiration cytology would have a sensitivity of 84% for the presence of carcinoma, a specificity of 97% for the absence of carcinoma, a predictive value of 99% for a positive diagnosis and a predictive value of 56% for a negative diagnosis; the diagnostic efficiency would be 86%. Our findings reaffirmed that the cytologic diagnosis of mammary carcinomas is reliable but that negative or inconclusive cytologic findings should not be regarded as a definitive diagnosis if there is clinical suspicion of a malignant neoplasm.

Abscisic acid and the regulation of synthesis of specific seed proteins and their messenger RNAs during culture of soybean embryos.

The ability of soybean (Glycine max (L.) Merr.) embryos cultured in vitro in the absence of abscisic acid (ABA) to germinate precociously increased as the embryos matured. Exogenous ABA prevented precocious germination at all stages of development, concentrations below 10(-5) M being partially effective. Growth (fresh weight increase) of mid-maturation embryos was dependent on ABA, and such embryos required ABA for continued synthesis of storage proteins during culture. Two complementary-DNA clones for different members of the family of 11S storage proteins, and one for the 7S storage proteins were used in Northern blot hybridizations to analyze the effects of ABA on the levels of the 11S and 7S mRNAs. In addition, filter hybridizations with in-vivo-labeled [(3)H]polyadenylated RNA to the cloned DNA for one of the 11S proteins were carried out to study transcription of 11S mRNA. Midmaturation embryos cultured with ABA continued to transcribe mRNAs for the storage proteins during the 21 d of culture studied, whereas in the absence of ABA these mRNAs disappeared from the cotyledons within about 5 d. The optimum concentration of ABA for the synthesis of storage-protein mRNAs was 10(-5) M. The effect of ABA on the concentration of 11S mRNAs was stage-dependent. Abscisic acid caused a decrease in the mRNA in embryos cultured at the cotyledon stage; it was necessary for high levels of the mRNA to be achieved in early- and mid-maturation embryos; and it did not reverse the decline in the mRNA levels in embryos cultured at the late-maturation stage.

Single Nucleotide Polymorphisms and Microarray Technology in Forensic Genetics - Development and Application to Mitochondrial DNA.

Variations in the genome, due to base substitutions, insertions, or deletions at single positions, are known as single nucleotide polymorphisms (SNPs). Approximately 85% of human variation is based on such polymorphisms. Therefore, there is an abundance of human SNPs that are available for forensic identity testing purposes. SNP analyses also may be suitable for some forensic identity cases, because they can be detected in smallsized amplicons, allowing for genetic analysis of substantially degraded DNA. While SNP analysis is unlikely to replace short tandem repeat loci typing for routine casework, SNPs may prove useful for certain circumstances, for example, typing mitochondrial DNA (mtDNA). Although sequencing mtDNA enables detection of all SNPs contained within the region of interest, it is currently not a practical approach for simultaneously typing SNPs that reside throughout the entire mtDNA genome. A variety of alternate methods to detect SNPs are available that may facilitate mtDNA analysis. All the methods include amplification, typically by the polymerase chain reaction, of the region containing the SNP of interest. Most assays are based on either hybridization of a probe to amplified product or primer extension chemistry, and multiplexing is possible. Some of these methodologies are: chips, SNaP shot™, Luminex 100™, SNPstream® UHT, and Pyrosequencing™. SNP analysis of mtDNA, both in the noncoding and coding regions, has been demonstrated using a number of these formats.

Chemiluminescent detection of DNA probes in forensic analysis.

The conditions for hybridization and detection of enzyme-labeled probes have been optimized in our laboratory for use with oligonucleotides coupled to alkaline phosphatase. We have examined several enzyme-linked probe which are complimentary to commonly used variable number of tandem repeats (VNTR) loci to determine the feasibility of using chemiluminescence for routine application in forensic DNA analysis. It was found that a chemiluminescent detection system employing an alkaline phosphatase activated dioxetane in the presence of chemiluminescent enhancers provides a high degree of sensitivity in hybridization protocols with a significant savings in overall filter processing time. The chemiluminescent system achieved equal or greater sensitivity that observed for 32P-labeled probes in much shorter development times. Furthermore, a new chemiluminescent substrate, Lumi-Phos Plus, has recently been investigated and found to further decrease the filter development time for forensic assays.

Analysis of the VNTR locus D1S80 by the PCR followed by high-resolution PAGE.

Allelic data for the D1S80 locus was obtained by using the PCR and subsequent analysis with a high-resolution, horizontal PAGE technique and silver staining. Compared with RFLP analysis of VNTR loci by Southern blotting, the approach described in this paper offers certain advantages: (1) discrete allele resolution, (2) minimal measurement error, (3) correct genotyping of single-band VNTR patterns, (4) a nonisotopic assay, (5) a permanent record of the electrophoretic separation, and (6) reduced assay time. In a sample of 99 unrelated Caucasians, the D1S80 locus demonstrated a heterozygosity of 80.8% with 37 phenotypes and 16 alleles. The distribution of genotypes is in agreement with expected values according to the Hardy-Weinberg equilibrium. Furthermore, the observed number of alleles and the level of heterozygosity, obtained through the protocol described here, were congruent with each other in accordance with the expectation of a mutation-drift equilibrium model for a single, homogeneous, random-mating population. Therefore, the analysis of D1S80 and similar VNTR loci by amplified fragment length polymorphism (AMP-FLP) may prove useful as models for population genetic issues for VNTR loci analyzed by RFLP typing via Southern blotting.

Genes expressed in the male gametophyte of flowering plants and their isolation.

Recombinant cDNA libraries to poly(A)RNA isolated from mature pollen of Zea mays and Tradescantia paludosa have been constructed. Northern blot analyses indicate that several of the clones are unique to pollen and are not expressed in vegetative tissues. The majority, however, are expressed both in pollen and vegetative tissues. Southern hybridizations show that the pollen specific sequences in corn are present in one or a very few copies in the genome. By using several of the clones as probes, it was found that there are at least two different groups of mRNAs with respect to their synthesis. The mRNAs of the first group represented by the pollen specific clones are synthesized after microspore mitosis and increase in concentration up to maturity. The second group, exemplified by actin mRNA, begins to accumulate soon after meiosis, reaches its maximum by late pollen interphase, and decreases thereafter. Although the actin mRNA and the pollen specific mRNAs studied show very different patterns of initiation of synthesis and accumulation during pollen development, the rates of decline of these mRNAs during the first 60 minutes of germination and pollen tube growth in Tradescantia are similar and reflect the previously observed declines in rates of protein synthesis during this period.