Browsing by Subject "genetic profile"
Now showing 1 - 2 of 2
- Results Per Page
- Sort Options
Item Construction of a Cost Effective Nested-PCR Reaction for Use with the Applied Biosystems AmpFLSTR Identifiler Kit(2005-12-01) Mikeska, Margo M.; John Planz; Joseph Warren; Arthur EisenbergHuman STR analysis has greatly increased the ability to perform identity testing for many different situations. These situations include, but are not limited to, the identification of individuals involved in violent crimes, establishing paternity, and identification of unknown human remains. The most common type of DNA information currently used for identity testing is the short tandem repeat, or STR. STR testing utilizes the number of repeating units in the DNA to assign an allele. Alleles from several different loci are used to establish a genetic profile. Currently, the United States used a standard of 13 different DNA loci to establish identity. These 13 loci can be typed by using a number of different multiplex kits such as the Applied Biosystems Profiler Plus, Cofiler, and Identifiler Kits [1,2]. The 13 loci were selected based on a number of parameters. Each locus was required to be polymorphyic, and a tetranucleotide repeat. The loci also could not display any linkage between each other and extensive population studies had to be conducted to both verify the absence of linkage and to establish allelic frequencies [1]. The goal of this research was the construction of a more cost effective method of utilizing the Applied Biosystems Identifiler Kit. Across the country there is a large backlog of samples that need to be processed in order to obtain a genetic profile. If these samples could be tested using a more cost effective method, more funding could be directed to other endeavors. Paternity testing, as well as some research endeavors could be conducted at a fraction of the cost, leaving resources for other projects or additional staff. Although it would be inadvisable to use this technique on forensic samples, the implications on paternity and research samples would be positive. This research attempted to design a nested PCR reaction and subsequently dilute the Applied Biosystems Primers in order to reduce the cost. The first step was to design new primers for the first round of PCR, followed by testing of those primers. The new primers then required optimization so that they all worked effectively together. After optimization was accomplished, the Identifiler primers were diluted until loci began dropping out of the genetic profile.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.