Browsing by Subject "direct amplification"
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Item DEVELOPMENT OF A FULL MITOCHONDRIAL GENOME SPECIFIC TARGET-ENRICHED LIBRARY FOR NEXT GENERATION SEQUENCING APPLICATIONS(2013-04-12) Oatts, SarahPurpose: The purpose of this study is to develop a target-enriched full mitochondrial genome library for a set of database samples from Easter Island to be used for downstream next generation sequencing applications. The samples did not amplify with a previously-developed polymerase chain reaction (PCR) primer multiplex that was expected to amplify overlapping mitochondrial DNA (mtDNA) fragments of approximately 1,500 -3,000 base pairs (bp) in length. Possible explanations of this unsuccessful amplification are either the sample quality is low or the FTATM cards on which the samples are stored prevents amplification of very large fragments of DNA. It is hypothesized that successful amplification of the samples can be achieved using a multiplex of PCR primers with a smaller target amplicon size. Methods: Twenty-four primer pairs developed by Rieder et al. were obtained that amplify the entire mitochondrial genome in overlapping fragments approximately 900 bp in length. The primers were tested in single-plex reactions first to ensure each primer pair was amplifying as expected. PCR results were observed and analyzed using the Agilent 2100 Bioanalyzer, an instrument that uses electrophoresis to separate, size and quantify fragments of DNA in a sample. Once all the fragments covering the entire mtDNA were amplified in single PCR reactions, 2 multiplex reactions each with 12 primer pairs were developed so that only 2 reactions were needed to generate the whole mtDNA amplicon pool instead of 24. An amplicon ladder for each of the two multiplexes was created by compiling the amplicons from the single-plex reactions. These ladders were used for comparison with the multiplex reaction results to ensure all fragments successfully amplified and were at balanced concentrations in the multiplex PCR reactions. Results: Electropherograms displaying the size and concentration of the DNA fragments amplified in each PCR reaction were generated by the Agilent 2100 Bioanalyzer. The electropherograms of the multiplex reactions closely resembled the electropherograms of the amplicon ladders. All of the expected amplicons were present in the multiplexed reactions and at relatively balanced concentrations. Conclusions: Successful whole mtDNA amplification was accomplished by using PCR primers with smaller target amplicons. These newly developed amplicon pools can now be used to create next generation sequencing libraries. The multiplex primer reactions developed can be used for similarly challenged samples in the future.Item Developmental and Internal Validation of a Mitochondrial DNA Direct Amplification Kit for Forensic Reference Samples(2015-05-01) Alicea-Centeno, Alessandra; Arthur J. Eisenberg; Rhonda Roby; Dixie L. PetersEvaluation of the control region of the mitochondrial genome is a common practice for forensic casework and research purposes. Since no kit is currently commercially available for the amplification of mitochondrial DNA (mtDNA), its sequencing procedure is time-consuming and laborious. Six steps are generally followed: DNA extraction, quantification and normalization, amplification of two regions (hypervariable regions 1 and 2), cycle sequencing, capillary electrophoresis and data analysis. This project evaluated a mtDNA direct amplification kit by performing developmental and internal validations. The studies performed included sensitivity, stability, reproducibility, case- type samples, mixtures and accuracy. The mtDNA direct amplification kit successfully amplified reference samples used in each study without the need of extraction and quantification steps. In addition, mtDNA profiles were obtained from the sequenced amplification products. Using the validated direct amplification procedure in the laboratory will improve workflow, decrease operational cost and reduce the possibility of error by minimizing sample handling.Item Increasing the Efficiency of Mitochondrial DNA processing of Reference Samples(2013-05-01) Morgan, Katherine N.; Rhonda RobyMitochondrial DNA (mtDNA) analysis in forensic testing is especially useful for human skeletal remains and hair samples. When a profile is generated for unidentified human remains or hair samples, comparison to a reference sample is critical to the case. The following steps are involved in the processing of family reference samples for mtDNA: DNA extraction, HV1 and HV2 amplification, cycle sequencing, electrophoresis, and analysis. Some procedures require DNA quantification and normalization. Analysis of mtDNA is expensive and time-consuming. A direct lysis and amplification method was previously shown to eliminate the need for DNA extraction, quantification, and normalization of reference samples. This study was performed to further develop, optimize, and validate its use for future implementation in routine casework of reference samples. The results have shown that quality mtDNA sequencing data can be obtained using a direct amplification method from blood and buccal samples from a variety of collection devices.