Author: Bolanle Ariyo
Publisher:
ISBN:
Category :
Languages : en
Pages : 92

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Author:
Publisher:
ISBN:
Category : Dissertations, Academic
Languages : en
Pages :

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Author: Sarah Copeland
Publisher:
ISBN: 9781339543420
Category :
Languages : en
Pages :

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Book Description
Mitochondrial DNA (mtDNA) analysis is a useful method for analyzing samples with limited or degraded DNA due to the high copy number per cell. The most common approach uses Sanger sequencing and capillary electrophoresis to analyze the two hypervariable regions (HVI and HVII) within the non-coding portion of the mitochondrial genome. However, this approach does not allow for mixture analysis, misses potentially discriminating information in the coding region, and fails if the mtDNA is too badly degraded. Next-generation sequencing (NGS) is a high throughput, massively parallel technique that allows for clonal amplification of single DNA molecules making it ideal for analyzing mixtures and heteroplasmy in mtDNA. Also, the analysis of single molecules is a more sensitive approach which can be used for analysis of limited DNA or potentially degraded samples.This study had two aims. The first was to use the Roche 454 GS Jr to sequence the hypervariable regions of mtDNA from five comingled bone samples originating from the 20th century AD, five bone samples from the 15th century AD, and one bone sample from the 3rd century AD. The second aim was to use probe capture and the Illumina MiSeq NGS platform to sequence the whole mitochondrial genome of artificially degraded K562 DNA, naturally degraded DNA from a modern bone sample (~50 years old), and extremely degraded DNA from a 15th century AD bone and a 3rd century AD bone. A sub-aim was to compare a selection of small fragment removal methods following adaptor ligation to minimize sample loss at this step of library preparation. Prior linear array and degradation qPCR results indicated that comingled remains from the 20th century AD were highly degraded. HVI & HVII sequencing using the Roche 454 was attempted because, in spite of also using long amplicons, it is a more sensitive assay. 29 comingled remains samples from the 20th century AD were amplified for 454 analysis, but only three showed successful results when viewed by gel. Those samples were sequenced by 454 along with two samples that appeared to fail amplification from this same group. All five of the comingled remains were successfully sequenced except for the HVII region of one sample, however, all were mixtures. The five 15th century AD bone samples and 3rd century AD bone sample were highly degraded. Two of the 15th century AD samples failed sequencing entirely and another failed HVII. The 3rd century AD sample also failed HVII sequencing. There were mixtures among these samples as well, which points to the need for optimized methods of sample cleaning to remove more of the exogenous DNA present on bone samples. A probe capture technique followed by whole genome sequencing on the Illumina Miseq was used to analyze one highly degraded bone sample from each of the 15th and 3rd centuries AD along with a naturally degraded modern bone purchased from The Bone Room in Berkeley, CA, and artificially degraded commercial K562 DNA (human chronic myelogenous leukemia cell line). Probe capture doesn't utilize PCR amplification, therefore no specific sites are required to be intact for the assay to be successful, potentially making it a viable alternative for samples that are too degraded for PCR-based HVI and HVII analysis. The protocol calls for a small fragment removal step after adaptor ligation that could potentially remove precious sample along with the unligated adaptor, so a variety of small fragment removal methods were tested to limit the sample loss at this step of library preparation. All of the artificially degraded samples had 100% coverage regardless of the small fragment removal method and had an average percent aligned reads of 95.59% indicating high specificity for the probe capture. Three of the four naturally degraded modern bone samples from The Bone Room had 100% coverage whereas the last sample only had a 23 bp unsequenced portion in an area with low coverage for the other samples as well. The average percent aligned reads was 49.28% indicating a capture of a substantial amount of exogenous DNA by the probe capture technique. The highly degraded ancient bone samples failed sequencing after appearing to have successfully completed library preparation, once again potentially indicating a substantial amount of exogenous DNA. These two NGS assays were able to successfully sequence degraded bone samples. However, before they can be successful at sequencing highly degraded samples, a method of cleaning bones to remove exogenous DNA and a method of purifying extracted DNA to remove inhibitors must be optimized.

Author: Cassandra Danelle Calloway
Publisher:
ISBN:
Category :
Languages : en
Pages : 554

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Author: Alessandra Alicea-Centeno
Publisher:
ISBN:
Category :
Languages : en
Pages : 42

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Book Description
Evaluation 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.

Author: Cassandra Taylor
Publisher:
ISBN: 9781369616446
Category :
Languages : en
Pages :

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Book Description
Forensic DNA samples are often highly degraded, making them unsuitable for traditional methods of DNA analysis, such as STR analysis, because target sequences and primer binding sites are not intact. Mitochondrial DNA (mtDNA) is useful for analyzing degraded DNA because of its high copy number; the high copy number makes successful typing more likely. Traditional methods of mtDNA analysis, including Sanger sequencing, are commonly used to only target only the HVI/HVII regions of the mitochondrial genome. These analysis methods miss discriminating information outside of the HV1/HVII, limiting the discriminatory power of mitochondrial DNA analysis. Probe capture is a novel technique that uses mtDNA sequence-specific probes to enrich and capture the entire mitochondrial genome. Analyzing the entire mitochondrial genome allows detection of discriminating information outside of the HVI/HVII regions and increases the discriminatory power of mitochondrial DNA analysis. Next Generation Sequencing, a massively parallel, clonal, and high-throughput technique, is an excellent tool for analyzing the entire mitochondrial genome. The purpose of this project was to test the efficiency of Dr. Calloway’s optimized probe capture assay on degraded DNA by analyzing the entire mitochondrial genome of highly degraded bone samples. Throughout the course of this project, three sets of bones samples, dating to 100, 2000, and 4000 years old respectively, were tested. Seven highly degraded bone samples recovered from a comingled tomb in Rijeka, Croatia and dating to approximately 100 years old were successfully sequenced with coverage of the mitochondrial genome ranging from approximately 52 to 98%. Haplogroups were determined for six of these samples based on the sequenced variants present in the entire mitochondrial genome. Additionally, six prehistoric bone samples dating to approximately 2000 years ago were sequenced with coverage of the mitochondrial genome ranging from 26 to 100%. The last set of samples, dating to 4000 years old, was recovered from a necropolis on the island of Kor?ula, Croatia. These six samples were successfully sequenced with coverage of the mitochondrial genome ranging from 26 to 100%.

Author: Dhavendra Kumar
Publisher: Academic Press
ISBN: 0127999221
Category : Science
Languages : en
Pages : 360

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Book Description
Medical and Health Genomics provides concise and evidence-based technical and practical information on the applied and translational aspects of genome sciences and the technologies related to non-clinical medicine and public health. Coverage is based on evolving paradigms of genomic medicine—in particular, the relation to public and population health genomics now being rapidly incorporated in health management and administration, with further implications for clinical population and disease management. Provides extensive coverage of the emergent field of health genomics and its huge relevance to healthcare management Presents user-friendly language accompanied by explanatory diagrams, figures, and many references for further study Covers the applied, but non-clinical, sciences across disease discovery, genetic analysis, genetic screening, and prevention and management Details the impact of clinical genomics across a diverse array of public and community health issues, and within a variety of global healthcare systems

Author: Pankaj Shrivastava
Publisher: Springer Nature
ISBN: 9811566550
Category : Science
Languages : en
Pages : 673

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Book Description
The book explores the fundamental principles, advances in forensic techniques, and its application on forensic DNA analysis. The book is divided into three modules; the first module provides the historical prospect of forensic DNA typing and introduces fundamentals of forensic DNA typing, methodology, and technical advancements, application of STRs, and DNA databases for forensic DNA profile analysis. Module 2 examines the problems and challenges encountered in extracting DNA and generating DNA profiles. It provides information on the methods and the best practices for DNA isolation from forensic biological samples and human remains like ancient DNA, DNA typing of skeletal remains and disaster victim identification, the importance of DNA typing in human trafficking, and various problems associated with capillary electrophoresis. Module 3 emphasizes various technologies that are based on SNPs, STRs namely Y-STR, X-STR, mitochondrial DNA profiling in forensic science. Module 4 explores the application of non-human forensic DNA typing of domestic animals, wildlife forensics, plant DNA fingerprinting, and microbial forensics. The last module discusses new areas and alternative methods in forensic DNA typing, including Next-Generation Sequencing, and its utility in forensic science, oral microbes, and forensic DNA phenotyping. Given its scope, the book is a useful resource in the field of DNA fingerprinting for scientists, forensic experts, and students at the postgraduate level.

Author: Colin Aitken
Publisher: John Wiley & Sons
ISBN: 047001122X
Category : Mathematics
Languages : en
Pages : 540

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Book Description
The first edition of Statistics and the Evaluation of Evidence for Forensic Scientists established itself as a highly regarded authority on this area. Fully revised and updated, the second edition provides significant new material on areas of current interest including: Glass Interpretation Fibres Interpretation Bayes’ Nets The title presents comprehensive coverage of the statistical evaluation of forensic evidence. It is written with the assumption of a modest mathematical background and is illustrated throughout with up-to-date examples from a forensic science background. The clarity of exposition makes this book ideal for all forensic scientists, lawyers and other professionals in related fields interested in the quantitative assessment and evaluation of evidence. 'There can be no doubt that the appreciation of some evidence in a court of law has been greatly enhanced by the sound use of statistical ideas and one can be confident that the next decade will see further developments, during which time this book will admirably serve those who have cause to use statistics in forensic science.' D.V. Lindley

Author: Manjunath.R
Publisher: Manjunath.R
ISBN:
Category : Antiques & Collectibles
Languages : en
Pages : 2658

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Book Description
This book takes readers back and forth through time and makes the past accessible to all families, students and the general reader and is an unprecedented collection of a list of events in chronological order and a wealth of informative knowledge about the rise and fall of empires, major scientific breakthroughs, groundbreaking inventions, and monumental moments about everything that has ever happened.