Browsing by Subject "extraction"
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Item Comparison of DNA Extraction Methods From Bone to be Used With the DNA IQ System on the Maxwell 16 for Human Identification(2008-08-01) Lopez, Kristen; John Planz; Arthur Eisenberg; Joseph WarrenLopez, Kristen M., Comparison of DNA Extraction Methods From Bone to be Used with the DNA IQ System on the Maxwell 16 for Human Identification . Masters of Science (Graduate School of Biomedical Sciences), August, 2008, 52 pp., 11 tables, 15 figures, bibliography, 24 titles. Extraction and purification of DNA from human bones is essential for correctly identifying the remains through DNA analysis. Current DNA extraction methods include a demineralization step, which extracts calcium and phosphate from the bone matrix, inactivation of DNAses, and the removal of Polymerase Chain Reaction (PCR) inhibitors. These methods often use harsh chemicals and may allow for residual DNA to be discarded in various wash steps. To assess the effectiveness of DNA extraction from bone samples, two extraction protocols were compared. The first method included a bone demineralization pretreatment solution of Sodium N-Laurylsarcosinate, 0.5 M EDTA, and Proteinase K (20 mg/ml). The second included a pretreatment using a Bone Incubation Buffer by Promega Corporation, with an addition of Proteinase K (18mg/ml). Various incubation times were included to assess the extraction at different time intervals. All extracted samples were purified with the DNA IQ Reference Sample Kit on the automated Maxwell 16 Instrument (Promega Corp.). Full and partial profiles were obtained from samples extracted with the Bone Incubation pretreatment, regardless of incubation time. Profiles were not observed with the standard demineralization pretreatment when amplified at 28 cycles, with partial profiles present in a few samples when amplified at 32 cycles.Item Comparison of Four Differential DNA Extraction Methods for Casework Analysis of Sexual Assault Kit Swabs(2016-05-01) Brignac, Francine J.; Joseph E. WarrenSexual assault kits make up 40-50% of a typical Forensic Laboratory caseload. The traditional method to process these samples is time-consuming and requires the use of hazardous chemicals such as Phenol:Chloroform:Isoamyl Alcohol (PCIA). This study compares another manual differential extraction method and two automated methods to the traditional standard differential extraction. Results indicate that as sperm sample concentration decreases, automated methods produce superior results both in DNA quantity obtained and in quality of STR profiles produced. Automated methods reduce hands-on time, facilitate higher through-put of samples, and reduce analyst contact with hazardous chemicals such as PCIA, making it an excellent choice for labs.Item The Comparison of Four DNA Extraction Techniques From Blood for Forensic Application(2003-08-01) Otto, Jennifer Blake; John Planz; Joseph Warren; Arthur EisenbergThe results of the phenolphthalein sensitivity study indicate that dilutions of blood down to a 1:1,000 level can be detected with the phenolphthalein presumptive test. Even though this value is not as sensitive as reported by other studies (17), a 1:1,000 level of detection is adequate for day to day use for forensic casework blood presumptive testing. The 1:1,000 dilution level samples were able to amplify and genotype complete or almost complete profiles, so any evidentiary items that give a positive phenolphthalein result should provide an analyst with a good genetic profile. Results from the Quantiblot quantification procedure for the four extraction methods show that the organic extraction with PLG yields the most human DNA out of the four techniques at the 1:10, 1:100, and 1:1,000 blood dilution levels. The organic extraction with PLG tubes also provided the best genetic profiles at the 1:100 and 1:1,000 levels. These results support the use of the organic extraction technique with the addition of the Phase Lock Gel tubes. This method will provide a forensic analyst with the highest yield of human DNA from low copy number blood samples. The organic method provides an analyst with a clean and complete profile at dilution levels lower than the other techniques tested in this project. Although any of the four DNA extraction techniques would provide a high DNA yield and complete genetic profile for known samples with ample DNA, the organic method provides the highest yield of DNA at low levels where sample/evidence consumption may be an issue. When comparing the costs of the four extraction methods per reaction, Chelex 100 is by far the most affordable. One 50g bottle of Chelex 100 resin can be used for approximately 5000 extractions at ~$0.02 per sample. The organic method with PLG tubes is the next affordable, with a price of ~$0.80 per sample. The QIAGEN and DNA IQ kits are more expensive and both cost ~$1.70 per sample. Based on this cost analysis, it may be cost effective to use the Chelex 100 method for known blood samples or evidentiary samples with a large stain present, and the organic method with PLG tubes for any evidentiary blood samples that are small and in limited quantity.