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The goal of this study was to develop a simple method to improve DNA recovery from challenging bone samples. To this end, an optimized procedure was developed that combined the demineralization and DNA extraction into a single step, followed by DNA purification using an automated silica-coated paramagnetic bead procedure. This method replaced a previous silica-membrane-based procedure, which was able to recover sufficient DNA to obtain full autosomal and Y chromosome STR profiles from greater than 90% of the samples, including samples greater than 20 years old. The development process began with the evaluation of buffer and demineralization systems to determine the best reagent combination. During the developmental process, we observed that the addition of EDTA and DTT affected silica-based DNA purification methods by raising the pH of the digest buffer. The protocols with buffer ATL, PK, EDTA, and DTT followed by lowering the pH with sodium acetate just before purification resulted in the best yields. The method reduced the extraction volume from 10 to 1.5 ml and used commercially available reagents already being utilized in forensic DNA casework. Because of the simplicity and small volume needed for the procedure, many steps where contamination could be introduced have been eliminated or minimized. This study demonstrated a new method of recovering DNA from bone samples capable of extracting trace quantities of DNA, removing potential inhibitors, and minimizing the potential for exogenous DNA contamination.

The correlation of growth hormone (GH) mRNA abundance and expression of specific transcription factors was studied in pituitaries of panhypopituitary (Ames df/df and Snell dwJ/dwJ dwarf), isolated GH-deficient (lit/lit), and GH-overproducing (growth hormone-releasing hormone [GHRH] transgenic) mice compared with normal littermates. A fluorescence-based reverse transcriptase polymerase chain reaction assay was developed for seven target mRNAs: GH, prolactin (PRL), pro-opiomelanocortin (POMC), alpha-subunit of the glycoprotein hormones (alphaSU), Pit-1, Prop-1, and Zn-16. Amplification parameters for each of these primer pairs were determined in order to calculate initial mRNA transcript number. The reproducibility of the assay was found to be +/-10% for either Pit-1 or Zn-16 mRNAs measured in characterized murine GHFT1-5 somatotroph precursor cells. The cell extracts also showed an increased abundance of both Zn-16 and Pit-1 mRNAs when compared with whole pituitary extracts. Measurement of copy number in normal pituitaries showed that for every 10(6) GH or PRL mRNAs, there were 3 x 10(5) POMC, 4 x 10(4) alphaSU, 2 x 10(3) Pit-1, and only 70 Zn-16 or Prop-1 transcripts. Transcript abundance in GH-altered mice as a percentage of copy number per normal gland showed that POMC was significantly reduced in dwJ/dwJ (p0.01) and df/df (p0.05) mice. AlphaSU mRNA was reduced in df/df (p0.05), dwJ/dwJ (p0.05), and lit/lit (p0.05) mice, but not in GHRH-excess mice. PRL mRNA was not detected in dwarf mice, reduced to 52% of normal in lit/lit (p0.05), and unchanged in GHRH-excess animals. GH mRNA was not detected in dwarf mice, reduced to 1.3% in lit/lit (p0.005), and increased to 242% in GHRH-excess mice (p0.05). Pit-1 mRNA was not detected in dwarf mice, was 2.9% of normal in lit/lit (p0.005) mice, and increased to 200% in GHRH-excess mice (p0.05). Prop-1 was not present in dwarf mice, was decreased to 1.4% in lit/lit (p0.01), and increased to 223% in GHRH-excess mice (p0.05). Zn-16 abundance in df/df mice was significantly reduced (p0.05) to 4.8% of normals, to 6.3% of normals in dwJ/dwJ (p0.005), to 6.1% of normals in lit/lit (p0.005) mice, and significantly elevated in GHRH-excess mice to 197% (p0.05). Altered pituitary mRNA abundance was found for several products not previously measured, or thought not to be affected by these mutations. Correlation of GH mRNA abundance with transcription factor copy number showed a significant correlation for Pit-1, Prop-1, and Zn-16. These quantitative analyses provide the first in vivo evidence that Zn-16 mRNA abundance correlates with GH expression.

Electrokinetic injection (EI) is the primary method used in forensic laboratories to load amplified PCR product in capillary electrophoresis for short tandem repeat (STR) fragment separation. Because all samples subjected to capillary electrophoresis use internal lane standard (ILS), this study investigated the consequence of varying the volume of ILS and its effects on allele peak heights and number of alleles detected. Results demonstrated that when the volume of ILS is reduced, the average peak height and number of alleles increased, thereby increasing the sensitivity of the detection method. Sizing anomalies were observed; however, they did not adversely affect accuracy and precision. The method developed in this study offers a simple and universal procedure to increase the alleles detected in forensic STR analysis. Reducing the volume of ILS to achieve greater sensitivity is applicable to all STR amplification kits and capillary electrophoresis instruments currently used in forensic DNA analysis.

A Y-chromosome multiplex polymerase chain reaction (PCR) amplification kit, known as Y-PLEX 6, has been developed for use in human identification. The Y-PLEX 6 kit enables simultaneous amplification of six polymorphic short tandem repeat (STR) loci located on the non-recombinant region of the human Y-chromosome. These loci are: DYS393, DYS19, DYS38911, DYS390, DYS391, and DYS385. Our studies show that as little as 0.2 ng of template DNA can be used for analysis. The specificity of the amplification reaction enabled analysis of male DNA in a male:female DNA mixture at a ratio of 1:125. Among the six Y-STR loci, the maximum mean stutter percentage was 11.9 for allele at DYS38911 locus. Attempts at amplification of DNA from various animal sources revealed that the Y-PLEX 6 primers are human specific. Details of the development of the kit, generation and description of the allelic ladders, and validation of the multiplex PCR are presented. In addition, Y-STR allele and haplotype frequencies in three populations have been investigated. The data indicate that results obtained using the Y-PLEX 6 kit are robust, sensitive, and reliable and can be used in human forensic and male lineage identification cases.

The forensic community continues to seek improvements in DNA typing methods on aspects such as sensitivity and efficacy. Reducing the volume of the AmpFlSTR Profiler Plus reagents offered greater sensitivity and improved the chance of obtaining useful results for samples with very low quantities of DNA and multiple source samples. On the downside, amplifications initiated with less than 0.4 ng of DNA exhibited a twofold increase in the standard deviation of peak ratios. This research suggested a twofold approach to analyzing samples. For samples with greater than 0.25 ng of DNA, a 25 microL reaction is appropriate. Samples that did not demonstrate quantifiable results, or that have less than 0.25 ng, can be amplified by drying the sample directly in the PCR tube and amplifying in a 5 microL reaction. The analyst can expect at least limited results with as little as 0.03 ng of DNA in the 5 microL reaction.

Rat Zn-15 is a transcription factor activating GH gene expression by synergistic interactions with Pit-1, named for 15 DNA-binding zinc fingers, including fingers IX, X, and XI that are responsible for GH promoter binding. In this study, a mouse cDNA for Zn-15 was characterized. The predicted 2192-amino acid mouse protein is 89% identical to rat (r) Zn-15 overall, and is 97% similar in the C-terminal domain necessary for binding the GH promoter. However, the mouse cDNA encodes 16 zinc fingers, and sequences of rZn-15 pituitary cDNAs were the same as the mouse (m) Zn-16; the rat sequence in GenBank has a one nucleotide offset of a 17-bp segment in the finger V region. The mouse and corrected rat sequences contain four tandemly repeated fingers in the N-terminus, each separated by seven amino acids, typical of zinc finger proteins of the transcription factor IIIA-type. Analysis of mZn-16 expression by RT-PCR showed that the mRNA is produced at similar levels in normal and GH-deficient Ames dwarf (Prop-1 ) mouse pituitaries at postnatal day 1. Mouse Zn-16 mRNA also was detected by ribonuclease protection assay in the pre-somatotrophic mouse cell line GHFT1-5. The Zn-16 protein is bipartite in that the N-terminal half displays tandem spacing typical of most zinc finger proteins, while the C-terminal portion contains long linkers between fingers that cooperatively bind to a DNA response element. Expression in early postnatal pituitary and in pre-somatotrophic cells suggests that Zn-16 could play a role in pituitary development prior to somatotroph differentiation.

Publisher Summary This chapter provides an overview of the study to investigate the use of RT-PCR to detect mRNAs relevant to elucidating the patterns of gene expression in the developing Ames dwarf pituitary. The use of RT-PCR for GH mRNA provides a rapid and reliable means of determining dwarfism at ages before body-size differences become apparent. The chapter presents a method that provides classification of dwarfs vs normals that is 100% correct for animals that could be identified by size. GH mRNA is not present in the Ames dwarf pituitary within three days of the initiation of transcription in the normal mouse pituitary. The Ames mutation appears to be involved in the blockage of GH transcription, implicating the involvement of Pit-1 in the effects of the mutation. The assays of Pit-1 confirmed the absence of detectable mRNA in dwarfs at 30 days of age or older. A study discussed in the chapter shows that Pit-1 is not detectable in dwarfs even at seven days of age.

The forensic community continues to seek improvements in DNA typing methods on aspects such as sensitivity and efficacy. Reducing the volume of the AmpFlSTR Profiler Plus reagents offered greater sensitivity and improved the chance of obtaining useful results for samples with very low quantities of DNA and multiple source samples. On the downside, amplifications initiated with less than 0.4 ng of DNA exhibited a twofold increase in the standard deviation of peak ratios. This research suggested a twofold approach to analyzing samples. For samples with greater than 0.25 ng of DNA, a 25 microL reaction is appropriate. Samples that did not demonstrate quantifiable results, or that have less than 0.25 ng, can be amplified by drying the sample directly in the PCR tube and amplifying in a 5 microL reaction. The analyst can expect at least limited results with as little as 0.03 ng of DNA in the 5 microL reaction.