Screening for single nucleotide polymorphisms in highly degraded DNA by using the amplified fragment length polymorphism technique.

Short tandem repeat (STR) analysis is generally used for human identification of forensic samples; however, standard STR analysis sometimes fails to generate full profiles since DNA is frequently degraded by various environmental factors. Recently, single nucleotide polymorphism (SNP) analysis has attracted attention for human identification since the shorter amplicons are better suited for degraded samples. Though various SNP loci are used for analysis of degraded samples, it is unclear which ones are more appropriate. To identify SNPs that were resistant to degradation, we artificially degraded DNA obtained from the buccal swabs of six volunteers and the K562 cell line by heat treatment. Subsequently, the amplified fragment length polymorphism (AFLP) technique was used for SNP screening. We focused on the AFLP bands detected in both the heat-treated and untreated samples, and DNA extracted from these bands was directly sequenced. DNA degradation increased as the duration of heat treatment increased, and no STR profiles could be generated after 6h of heat treatment. When the AFLP band patterns were compared between 6h heat-treated and untreated samples, eight common bands were detected. The sequences of the DNA fragments of these common bands had higher adenine-thymine (A-T) content and included 17 SNPs. The SNPs detected in the heat-treated and untreated samples were considered to be resistant to degradation. Although there was a little information available in databases regarding the nine SNPs identified in this study, this study shows that some of these SNPs might be useful for human identification of extremely degraded DNA.

Investigation of single nucleotide polymorphism loci susceptible to degradation by ultraviolet light.

DNA in biological fluids is often degraded by environmental factors. Given that single nucleotide polymorphism (SNP) analyses require shorter amplicons than short tandem repeat (STR) analyses do, their use in human identification using degraded samples has recently attracted attention. Although various SNP loci are used to analyze degraded samples, it is unclear which ones are more appropriate. To characterize and identify SNP loci that are susceptible or resistant to degradation, we artificially degraded DNA, obtained from buccal swabs from 11 volunteers, by exposure to ultraviolet (UV) light for different durations (254 nm for 5, 15, 30, 60, or 120 min) and analyzed the resulting SNP loci. DNA degradation was assessed using gel electrophoresis, STR, and SNP profiling. DNA fragmentation occurred within 5 min of UV irradiation, and successful STR and SNP profiling decreased with increasing duration. However, 73% of SNP loci were still detected correctly in DNA samples irradiated for 120 min, a dose that rendered STR loci undetectable. The unsuccessful SNP typing and the base call failure of nucleotides neighboring the SNPs were traced to rs1031825, and we found that this SNP was susceptible to UV light. When comparing the detection efficiencies of STR and SNP loci, SNP typing was more successful than STR typing, making it effective when using degraded DNA. However, it is important to use rs1031825 with caution when interpreting SNP analyses of degraded DNA.

Characterization of cellular and extracellular DNA in saliva.

Although the presence of extracellular DNA in various body fluids was discovered long ago, only recently has it begun to attract attention for examining the genetic profiles of individuals in forensics studies. However, information on extracellular DNA is scarce. Among human body fluids, saliva is known to be rich in extracellular DNA. In this study, to analyze the possibility of identifying individuals and body fluids by using only extracellular DNA of saliva, we investigated the amount, size distribution, short tandem repeat (STR) profile, and methylation pattern of extracellular DNA from saliva and compared these with those of cellular DNA. The amount and size distribution of extracellular DNA was different from that of cellular DNA. However, their respective STR profiles were the same. The methylation patterns of the BCAS4 gene were different among donors, but no significant difference was observed between cellular and extracellular DNA. The results of our study suggest that identification of individuals and body fluids from saliva may be possible without the need for cells.

A simple ABO genotyping by PCR using sequence-specific primers with mismatched nucleotides.

In forensics, the specific ABO blood group is often determined by analyzing the ABO gene. Among various methods used, PCR employing sequence-specific primers (PCR-SSP) is simpler than other methods for ABO typing. When performing the PCR-SSP, the pseudo-positive signals often lead to errors in ABO typing. We introduced mismatched nucleotides at the second and the third positions from the 3'-end of the primers for the PCR-SSP method and examined whether reliable typing could be achieved by suppressing pseudo-positive signals. Genomic DNA was extracted from nail clippings of 27 volunteers, and the ABO gene was examined with PCR-SSP employing primers with and without mismatched nucleotides. The ABO blood group of the nail clippings was also analyzed serologically, and these results were compared with those obtained using PCR-SSP. When mismatched primers were employed for amplification, the results of the ABO typing matched with those obtained by the serological method. When primers without mismatched nucleotides were used for PCR-SSP, pseudo-positive signals were observed. Thus our method may be used for achieving more reliable ABO typing.

Changes in the brain after intracerebral implantation of a lead pellet in the rat.

In gunshot wounds to the head, the bullet containing neurotoxic lead may remain in the brain after trauma, and brain damage is therefore anticipated. We developed an animal model incorporating a lead ball implanted in the brain, or a glass ball as a control, and analyzed histological and biochemical changes in the brain for 28 days after surgery. The concentration of lead in the brain increased with time after implantation of the lead ball, while lead was not detected in brains implanted with a glass ball. A number of changes were noted following implantation of the lead ball. The number of neutrophils increased significantly at 7-28 days, while the number of macrophages also increased significantly with time. The number of neurons was significantly decreased by 28 days, and apoptotic cells were identified at 14-28 days. Expression of genes encoding N-methyl-d-aspartate receptors, which are related to cognitive function and help regulate apoptosis, was significantly suppressed at 7-21 days after implantation. Expression of metallothionein protein increased significantly with time. Metallothionein detoxifies lead and its overexpression is a known method of protection against lead neurotoxicity. These findings indicate that intracerebral lead induces neuronal degeneration that is attributable in part to the inflammation and apoptosis caused by leached lead. Although the severity of the brain damage primarily depends on the destructive effect of the moving bullet, the results of this study indicate that lead leaching from the residual bullet also damages the brain.

Selection of a biotin protein ligase by phage display using a combination of in vitro selection and in vivo enzymatic activity.

We report the development of a phage display-based method for the direct selection of enzymatic activity by a combination of selection in vitro and an assay of enzymatic activity in vivo. We describe the selection of a biotin protein ligase (BPL) that specifically recognizes neurotensin as a substrate, as an example of the utility of our method. We constructed an enzyme library with a diversity of 8.8 x 10(6) by insertion of four randomized regions into a BPL backbone and used it for the selection of variant BPLs. We obtained an active BPL, which was identical to wild-type BPL, using the combination of selection in vitro and an assay of enzymatic activity in vivo. Our results indicate that our method is suitable for the selection of enzymes with specific functions of interest.

Selection of an active enzyme by phage display on the basis of the enzyme's catalytic activity in vivo.

We have developed a novel phage display method based on catalytic activity for the in vivo selection of an enzyme. To confirm the validity of our method and to demonstrate its potential utility, we used biotin protein ligase (BPL) from Escherichia coli as a model enzyme. We were able to demonstrate the potential value of our method by selective enrichment for the birA gene, which encodes BPL, in a mixed library. The presented method for in vivo selection should allow selection of various enzymes that catalyze modification of peptides or proteins, such as protein ligase, acetylase, kinase, phosphatase, ubiquitinase, and protease (including caspase). The method should be useful in efforts to analyze mechanisms of signal transduction, to find unidentified enzymes encoded by cDNA libraries, and to exploit artificial enzymes.

Reversed-phase HPLC determination of chlorophyll a' and phylloquinone in Photosystem I of oxygenic photosynthetic organisms. Universal existence of one chlorophyll a' molecule in Photosystem I.

Chlorophyll (Chl) a', the C132-epimer of Chl a, is a constituent of the primary electron donor (P700) of Photosystem (PS) I of a thermophilic cyanobacterium Synechococcus (Thermosynechococcus) elongatus, as was recently demonstrated by X-ray crystallography. To determine whether PS I of oxygenic photosynthetic organisms universally contains one molecule of Chl a', pigment compositions of thylakoid membranes and PS I complexes isolated from the cyanobacteria T. elongatus and Synechocystis sp. PCC 6803, the green alga Chlamydomonas reinhardtii, and the green plant spinach, were examined by simultaneous detection of phylloquinone (the secondary electron acceptor of PS I) and Chl a' by reversed-phase HPLC. The results were compared with the Chl a/P700 ratio determined spectrophotometrically. The Chl a'/PS I ratios of thylakoid membranes and PS I were about 1 for all the organisms examined, and one Chl a' molecule was found in PS I even after most of the peripheral subunits were removed. Chl a' showed a characteristic extraction behaviour significantly different from the bulk Chl a in acetone/methanol extraction upon varying the mixing ratio. These findings confirm that a single Chl a' molecule in P700 is the universal feature of PS I of the Chl a-based oxygenic photosynthetic organisms.