Reena Roy

Protein synthesis in rabbit reticulocytes XIX: EIF-2 promotes dissociation of Met-tRNAf-EIF-1-GTP complex and Met-tRNAf binding to 40S ribosomes.

Abstract The peptide chain initiation factor, EIF-2 has been partially purified from the 0.5 M KCl ribosomal wash. The molecular weight of EIF-2 is approximately 450,000. The purified EIF-2 preparation promotes the dissociation of the ternary complex, Met-tRNA f ·EIF-1·GTP in the presence of Mg ++ and is also required along with EIF-1 for AUG-directed Met-tRNA f binding to 40S ribosomes.

AmpliType® PM and HLA DQα Typing from Pap Smear, Semen Smear, and Postcoital Slides

Deoxyribonucleic acid (DNA) samples extracted from stained pap smear, semen smear and postcoital slides were amplified by the polymerase chain reaction (PCR) and typed for the AmpliType® PM and HLA DQα alleles. HLA DQ α and PM types consistent with blood controls from the donors were obtained. Stained cells fixed to slides can provide a valuable source of material for determining a genetic profile of the sample donor, particularly in sexual assault cases and possibly in missing person cases involving women.

Autosomal STR Variation in a Basque Population: Vizcaya Province

ABSTRACT We have characterized 68 unrelated Basque individuals from Vizcaya, Spain, for 13 tetrameric short tandem repeat (STR) loci: CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX, and VWA. Interpopulational analyses were also performed for 21 European and North African population data sets for nine of the STRs typed in the Basque sample. Heterozygosity values for the Vizcayan Basques were found to be high, ranging from 0.662 to 0.882, and none of the STR loci significantly deviated from Hardy-Weinberg equilibrium. Based on the comparative population data set, the average GST score is 0.7%, indicating a low degree of genetic differentiation. However, neighbor-joining trees and multidimensional-scaling plots of DA genetic distances indicate that the Vizcayan Basques are an outlier relative to both neighboring Iberians and North African populations.

An evaluation of direct PCR amplification

Aim To generate complete DNA profiles from blood and saliva samples deposited on FTA® and non-FTA® paper substrates following a direct amplification protocol. Methods Saliva samples from living donors and blood samples from deceased individuals were deposited on ten different FTA® and non-FTA® substrates. These ten paper substrates containing body fluids were kept at room temperature for varying lengths of time ranging from one day to approximately one year. For all assays in this research, 1.2 mm punches were collected from each substrate containing one type of body fluid and amplified with reagents provided in the nine commercial polymerase chain reaction (PCR) amplification kits. The substrates were not subjected to purification reagent or extraction buffer prior to amplification. Results Success rates were calculated for all nine amplification kits and all ten substrates based on their ability to yield complete DNA profiles following a direct amplification protocol. Six out of the nine amplification kits, and four out of the ten paper substrates had the highest success rates overall. Conclusion The data show that it is possible to generate complete DNA profiles following a direct amplification protocol using both standard (non-direct) and direct PCR amplification kits. The generation of complete DNA profiles appears to depend more on the success of the amplification kit rather than the than the FTA®- or non-FTA®-based substrates.

Multiplex amplification of STR loci with gender alleles using infrared fluorescence detection.

The analysis of short tandem repeat (STR) polymorphisms has proven extremely useful for gene mapping, paternity testing, and forensic analysis. Several commercial products are currently available for performing amplification and analysis of STRs. We have adapted Promega Geneprint Systems for use with a high sensitivity infrared (IR) fluorescent automated DNA sequencer. IR-labeled amplification products are generated by including a small quantity of IR-labeled dATP in the reaction. Several Geneprint STR loci can be multiplexed together with the amelogenin sex identification locus in a single amplification reaction. We have successfully amplified up to five Geneprint STR loci together with the amelogenin locus thus improving the throughput of analysis. Purified genomic DNA as well as simulated forensic samples have been utilized for these multiplex amplifications.

Protein synthesis in rabbit reticulocytes XXXI: Purification of Co-eIF-2C and studies of its roles in peptide chain initiation.

Abstract Co-eIF-2C activity has been purified from high salt wash of reticulocyte ribosomes. The purified preparation is free from detectable levels of eIF-2, Co-eIF-2A, Co-eIF-2B and RF activities. The final step in the purification process involves use of a phosphocellulose chromatography and elution of Co-eIF-2C activity from the column with a buffer containing 2 M urea. This step results in complete removal of the contaminating Co-eIF-2B activity from the purified Co-eIF-2C preparation. Upon polyacrylamide gel electrophoresis, the final Co-eIF-2C preparation shows a single protein band under non-denaturing conditions. In the presence of SDS, the gel picture shows five prominent polypeptide bands (approximate mol. wt: 100,000; 67,000; 53,000; 45,000; and 40,000) and several faint bands. Purified Co-eIF-2C preparation strongly stimulates Met-tRNA f ·40S·AUG complex formation in the presence of eIF-2 and such stimulation is almost completely inhibited by HRI plus ATP. This study thus delineates the minimum factor requirements, namely, eIF-2 and Co-eIF-2C for formation of a stable Met-tRNA f ·40S·AUG complex.

Protein synthesis in rabbit reticulocytes XXII: A heat stable dialyzable factor (EIF-1∗) modulates Met-tRNAf binding to EIF-1☆

Abstract The peptide chain initiation factor EIF-1 forms a ternary complex, Met-tRNA f ·EIF-1·GTP in the absence of Mg ++ and the preformed complex is stable to Mg ++ . However, with homogeneous preparations of EIF-1, addition of Mg ++ during the initial formation of the ternary complex strongly inhibits the complex formation. A heat stable dialyzable factor (EIF-1 ∗ ) which mostly remains associated with the high molecular weight protein complex, EIF-2 (TDF) during purification of the peptide chain initiation factors, has been purified using a phenol extraction procedure. EIF-1 ∗ restores the Met-tRNA f binding activity of EIF-1 in the presence of 1 mM Mg ++ ; in the presence of EIF-1 ∗ , Met-tRNA f binding by EIF-1 shows a sharp Mg ++ optimum around 1 mM. EIF-1 ∗ is heat stable, alkali stable, dialyzable and pronase sensitive. The same EIF-1 ∗ preparation also strongly inhibits Met-tRNA f binding to EIF-1 in the absence of Mg ++ and stimulates protein synthesis in a mRNA-dependent rabbit reticulocyte lysate system.

Evaluation of Direct PCR Amplification Using Various Swabs and Washing Reagents

DNA profiles were generated via direct amplification from blood and saliva samples deposited on various types of swab substrates. Each of the six non‐FTA substrates used in this research was punched with a Harris 1.2 mm puncher. After 0.1 μL of blood or 0.5 μL saliva, samples were deposited on each of these punches, samples were pretreated with one of four buffers and washing reagents. Amplification was performed using direct and nondirect autosomal and Y‐STR kits. Autosomal and Y‐STR profiles were successfully generated from most of these substrates when pretreated with buffer or washing reagents. Concordant profiles were obtained within and between the six substrates, the six amplification kits, and all four reagents. The direct amplification of substrates which do not contain lysing agent would be beneficial to the forensic community as the procedure can be used on evidence samples commonly found at crime scenes.

Detection of insertion/deletion polymorphisms from challenged samples using the Investigator DIPplex® kit.

This research focuses on detection of bi-allelic insertion/deletion polymorphisms (InDels) from challenged samples using the Investigator DIPplex® Kit from Qiagen. The study included analyzing body fluids from humans, as well as pristine and degraded samples. For the purpose of assessing species specificity, samples from various animals were included. At first, an analytical threshold (AT) for the detection of alleles was established based on an assessment of the noise in the system. Then, InDel profiles were obtained from samples exposed to detrimental environmental conditions, washed bloodstains, lipsticks, ChapStick®, ancient Croatian bone samples, and every day products such as toothbrushes and dental floss. Concordant profiles were obtained from different body fluids of the same donor. InDel profiles were also generated successfully when body fluids were deposited on substrates and directly amplified without pre-treatment with buffer or washing reagents. InDels can provide additional information when only partial STR profiles are generated from challenged samples.

Comparison of Quantifiler® Trio and InnoQuant™ human DNA quantification kits for detection of DNA degradation in developed and aged fingerprints

The development techniques employed to visualize fingerprints collected from crime scenes as well as post-development ageing may result in the degradation of the DNA present in low quantities in such evidence samples. Amplification of the DNA samples with short tandem repeat (STR) amplification kits may result in partial DNA profiles. A comparative study of two commercially available quantification kits, Quantifiler(®) Trio and InnoQuant™, was performed on latent fingerprint samples that were either (i) developed using one of three different techniques and then aged in ambient conditions or (ii) undeveloped and then aged in ambient conditions. The three fingerprint development techniques used were: cyanoacrylate fuming, dusting with black powder, and the columnar-thin-film (CTF) technique. In order to determine the differences between the expected quantities and actual quantities of DNA, manually degraded samples generated by controlled exposure of DNA standards to ultraviolet radiation were also analyzed. A total of 144 fingerprint and 42 manually degraded DNA samples were processed in this study. The results indicate that the InnoQuant™ kit is capable of producing higher degradation ratios compared to the Quantifiler(®) Trio kit. This was an expected result since the degradation ratio is a relative value specific for a kit based on the length and extent of amplification of the two amplicons that vary from one kit to the other. Additionally, samples with lower concentrations of DNA yielded non-linear relationships of degradation ratio with the duration of aging, whereas samples with higher concentrations of DNA yielded quasi-linear relationships. None of the three development techniques produced a noticeably different degradation pattern when compared to undeveloped fingerprints, and therefore do not impede downstream DNA analysis.