Validation of the InnoXtract™ DNA extraction method for bone, teeth, and rootless hair.

Forensic casework samples often include human hairs, teeth, and bones. Hairs with roots are routinely processed for DNA analysis, while rootless hairs are either not tested or processed using mitochondrial DNA. Bones and teeth are submitted for human remains identifications for missing persons and mass disaster cases.

Development and validation of a novel method "SpermX™" for high throughput differential extraction processing of sexual assault kits (SAKs) for DNA analysis.

The Sperm X method uses a nanotechnology derived polymer membrane that functions as a separation medium to effectively trap sperm cells while enabling efficient flow through of the digested epithelial cell DNA. This specialized membrane enabled development of a method that could significantly increase a forensic laboratory's ability to obtain male sperm fraction DNA profiles. The SpermX device provides a rapid, reproducible procedure that is easy to implement in a single-tube format as well as high-throughput truly automated hands-free workflows.

Successful nuclear DNA profiling of rootless hair shafts: a novel approach.

Historically, rootless hair shaft samples submitted to a forensic laboratory for DNA analysis are reserved for mitochondrial DNA (mtDNA) analysis due to the presence of highly degraded as well as insufficient amounts of nuclear DNA. Although mtDNA has been very successful in obtaining results from rootless hair, this system has its limitations, namely, it is a lineage marker that cannot differentiate between maternally related genotypes. Given the high incidence of hairs as forensic evidence, there is a need for the use of a nuclear DNA test system capable of producing reliable results for hair shaft forensic evidence.

Development and validation of a novel multiplexed DNA analysis system, InnoTyper 21.

We report here a novel multiplexed DNA analysis system consisting of 20 Alu markers and Amelogenin for analysis of highly degraded forensic biological samples. The key to the success of the system in obtaining results from degraded samples is the primer design yielding small amplicon size (60-125bp) for all 20 markers. The markers included in the InnoTyper 21 system are bi-allelic, having two possible allelic states (insertion or null) and thus termed INNULs.

Development and validation of InnoQuant™, a sensitive human DNA quantitation and degradation assessment method for forensic samples using high copy number mobile elements Alu and SVA.

There is a constant need in forensic casework laboratories for an improved way to increase the first-pass success rate of forensic samples. The recent advances in mini STR analysis, SNP, and Alu marker systems have now made it possible to analyze highly compromised samples, yet few tools are available that can simultaneously provide an assessment of quantity, inhibition, and degradation in a sample prior to genotyping. Currently there are several different approaches used for fluorescence-based quantification assays which provide a measure of quantity and inhibition.

INNULs: A novel design amplification strategy for retrotransposable elements for studying population variation.

Objectives: Retrotransposable elements (REs), consisting of long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs), are a group of markers that can be useful for human identity testing. Until now, however, due to the inherent size difference (up to 6 kb in some instances) associated with insertion and null alleles (or INNULs), the use of REs for facilitated population studies has not been sought or practical. The size of the insertion elements (from a few hundred to several thousand bp) has proven to limit their utility as a marker because of the inefficient amplicon yield with PCR.

Synthesis, molecular modeling and bio-evaluation of cycloalkyl fused 2-aminopyrimidines as antitubercular and antidiabetic agents.

An economical and efficient one step synthesis of a series of 8-(arylidene)-4-(aryl)-5,6,7,8-tetrahydro-quinazolin-2-ylamines and 9-(arylidene)-4-(aryl)-6,7,8,9-tetrahydro-5H-cycloheptapyrimidin-2-ylamines by the reaction of bis-benzylidene cycloalkanones and guanidine hydrochloride in presence of NaH has been developed. All the synthesized compounds were evaluated against Mycobacterium tuberculosis H(37)Rv strain and the α-glucosidase and glycogen phosphorylase enzymes. Few of the compounds have shown interesting in vitro activity with MIC up to 3.

Synthesis and bio-evaluation of alkylaminoaryl phenyl cyclopropyl methanones as antitubercular and antimalarial agents.

A series of 4-alkylaminoaryl phenyl cyclopropyl methanones (6a-6u and 8a-8c) were synthesized from 4-fluorochalcones (3a and 3b) by cyclopropanation of double bond followed by nucleophilic substitution of F with different amines. The compounds were screened for their antitubercular and antimalarial activities against Mycobacterium tuberculosis H37Rv and Plasmodium falciparum 3D7 strains in vitro respectively. Several compounds (6a, 6d-6h, 6p, 6q and 8a-8c) exhibited good in vitro antitubercular activities with MIC values 3.

Synthesis and optimization of antitubercular activities in a series of 4-(aryloxy)phenyl cyclopropyl methanols.

A series of [4-(aryloxy)phenyl]cyclopropyl methanones were synthesized by reaction of different benzyl alcohols with 4-chloro-4'-fluorobutyrophenone in DMF in the presence of NaH/TBAB. The methanones were further reduced to respective methanols. The antitubercular activity of these compounds was evaluated in vitro against Mycobacterium tuberculosis H37Rv.

Microparticles induce variable levels of activation in macrophages infected with Mycobacterium tuberculosis.

Activation of human macrophages infected with Mycobacterium tuberculosis was investigated following exposure to microparticles (MP) possessing high anti-tubercular efficacy in mice. A small set of innate responses (cytokine profiles, NO production, Annexin-V staining and caspase-8, caspase-9 and caspase-3 activities) of differentiated THP-1 cells or human monocyte-derived macrophages infected 1:10 in vitro were compared. Cytokines of THP-1 macrophages were comparable in trends, but not in magnitude, with five human genotypes studied.

Application of Huisgen (3+2) cycloaddition reaction: synthesis of 1-(2,3-dihydrobenzofuran-2-yl-methyl [1,2,3]-triazoles and their antitubercular evaluations.

1,4-Disubstituted-1,2,3-triazoles (3-27) have been synthesized by [3+2] cycloaddition of different 2-(azidomethyl)-dihydronaptho(benzo)furans (2a, 2b, 2c and 2d) with different alkynes. All the compounds were screened for antitubercular activity against Mycobacterium tuberculosis H37Rv. Compounds 2a, 7, 9, 12 and 14 exhibited antitubercular activities with MIC ranging from 12.

Solution-phase synthesis of a library of carbapeptide analogues based on glycosylamino acid scaffolds and their in silico screening and antimicrobial evaluation.

A well-organized and efficient approach toward the solution phase synthesis of a library of carbapeptide analogues based on glycosyl amino ester scaffold is described. The reported synthetic route involves a five step preparation of heptofuranuronamides 6a-h and octopyranuronamide 7e from glycosyl amino esters 1 and 7, respectively. Coupling of glycosyl amino esters 1 or 7 with three different N-Fmoc protected amino acids afford the N-Fmoc protected intermediates 2a-c and 7a.

A facile synthesis of alpha,alpha'-(EE)-bis(benzylidene)-cycloalkanones and their antitubercular evaluations.

An economical and facile synthesis of alpha,alpha'-(EE)-bis(benzylidene)-cycloalkanones was achieved by the reaction of cycloalkanones with different aromatic aldehydes using ethanolic KOH in good yields. Few of the selected compounds were reduced with NaBH(4) to the respective alpha,alpha'-(EE)-bis(benzylidene)-cycloalkanols. All these compounds and our earlier synthesized cyclohexyl phenyl methanols were evaluated for their antitubercular, antifungal and antibacterial activities.

Preparation and reactions of sugar azides with alkynes: synthesis of sugar triazoles as antitubercular agents.

5-azido-5-deoxy-xylo-, ribo-, and arabinofuranoses were prepared by the reaction of the respective 5-O-(methanesulfonyl) or p-toluenesulfonyl derivatives with NaN3 in DMF. The intermediate 5-azido-5-deoxy glycofuranoses on 1,3-cycloaddition with different alkynes in the presence of CuSO4 and sodium ascorbate gave the corresponding sugar triazoles in very good yields. The synthesized sugar triazoles were evaluated for their antitubercular activity against Mycobacterium tuberculosis H37Rv, where one of the compounds displayed mild antitubercular activity in vitro with MIC 12.

Human genomic DNA quantitation system, H-Quant: development and validation for use in forensic casework.

The human DNA quantification (H-Quant) system, developed for use in human identification, enables quantitation of human genomic DNA in biological samples. The assay is based on real-time amplification of AluYb8 insertions in hominoid primates. The relatively high copy number of subfamily-specific Alu repeats in the human genome enables quantification of very small amounts of human DNA.

Multiplex polymerase chain reaction for simultaneous quantitation of human nuclear, mitochondrial, and male Y-chromosome DNA: application in human identification.

Human forensic casework requires sensitive quantitation of human nuclear (nDNA), mitochondrial (mtDNA), and male Y-chromosome DNA from complex biomaterials. Although many such systems are commercially available, no system is capable of simultaneously quantifying all three targets in a single reaction. Most available methods either are not multiplex compatible or lack human specificity.

Y-chromosome STR system, Y-PLEX 12, for forensic casework: development and validation.

The Y-PLEX 12 system, developed for use in human identification, enables simultaneous amplification of eleven polymorphic short tandem repeat (STR) loci, namely DYS392, DYS390, DYS385 a/b, DYS393, DYS389I, DYS391, DYS389II, DYS 19, DYS439 and DYS438, residing on the Y chromosome and Amelogenin. Amelogenin provides results for gender identification and serves as internal control for PCR. The validation studies were performed according to the DNA Advisory Board's (DAB) Quality Assurance Standards.

Utility of the Y-STR typing systems Y-PLEX 6 and Y-PLEX 5 in forensic casework and 11 Y-STR haplotype database for three major population groups in the United States.

The Y-PLEX 6 and Y-PLEX 5 systems enable analysis for 11 Y-STR loci. We present here the utility of these systems in forensic casework. A total of 188 samples, including 127 evidence samples, were analyzed using either or both of the systems.

Quantitative PCR for DNA identification based on genome-specific interspersed repetitive elements.

We have designed and evaluated a series of class-specific (Aves), order-specific (Rodentia), and species-specific (equine, canine, feline, rat, hamster, guinea pig, and rabbit) polymerase chain reaction (PCR)-based assays for the identification and quantitation of DNA using amplification of genome-specific short and long interspersed elements. Using SYBR Green-based detection, the minimum effective quantitation levels of the assays ranged from 0.1 ng to 0.

Development and validation of the Y-PLEX 5, a Y-chromosome STR genotyping system, for forensic casework.

A genotyping system, Y-PLEX 5, has been developed for use in human identification. The Y-PLEX 5 enables simultaneous amplification of five polymorphic short tandem repeat (STR) loci residing on the Y-chromosome, which are DYS389I, DYS389II, DYS439, DYS438, and DYS392. As little as 0.

Quantitative intra-short interspersed element PCR for species-specific DNA identification.

We have designed and evaluated four assays based upon PCR amplification of short interspersed elements (SINEs) for species-specific detection and quantitation of bovine, porcine, chicken, and ruminant DNA. The need for these types of approaches has increased drastically in response to the bovine spongiform encephalopathy epidemic. Using SYBR Green-based detection, the minimum effective quantitation levels were 0.

Human DNA quantitation using Alu element-based polymerase chain reaction.

Human forensic casework requires sensitive quantitation of human nuclear DNA from complex sources. Widely used commercially available systems detect both nonhuman and human primate DNA, often require special equipment, and have a detection limit of approximately 0.1ng.