Dental Forensic Kit (DFK®) and Quick Extract™ FFPE DNA extraction kit, a new workflow for obtaining dental DNA for human genetic identity.

BACKGROUND: Most of the protocols described for obtaining DNA from dental tissues are methods that involve major laboratory equipment and many hours of work. They are also methodologies that almost destroy the tooth. AIM: Our aim was to develop an innovative workflow for optimizing dental DNA extraction from teeth. Our methodological proposal is a new workflow for obtaining dental DNA for human genetic identity using Dental Forensic Kit (DFK®) and Formalin-Fixed Paraffin-Embedded (FFPE™) DNA extraction kit. METHODS: Two different dental samples groups were assayed with DFK® and FFPE™. The first group corresponded to extracted teeth from living donors and the second group was considered in real conditions with challenging teeth from corpses. Genomic Dental DNA was amplified and genotyped with platforms Identifiler Plus™, Power Plex 21™ and Global Filer ™ kits. RESULTS: Our workflow was useful in obtaining dental DNA and partial and complete genetic profiles, from teeth of both study groups. DFK® kit worked in a conservative treatment of teeth generating dental tissues (pulp and cement) and FFPE™ for DNA extraction was a very cheap, quick and easy method for obtaining genomic dental DNA. CONCLUSIONS: The innovative method and the workflow proposed herein allows obtaining robust and reliable genetic profiles, from dental tissues. DFK® kit works optimizing the treatment of dental tissues and FFPE™ demonstrates a new use in forensic genetics.

Optimizing DNA recovery and forensic typing of degraded blood and dental remains using a specialized extraction method, comprehensive qPCR sample characterization, and massively parallel sequencing.

Human dental remains encountered in criminal casework evidence, missing person cases, or mass disaster tragedies provide a valuable sample source for DNA typing when suitable soft tissue is unavailable. Using traditional methods, teeth samples can be challenging to process, resulting in low-quantity and/or quality nuclear DNA and insufficient profiles for comparisons. This study examines the performance of a three-part nuclear DNA analysis workflow for teeth samples based on (1) improved dental tissue recovery using the Dental Forensic Kit (DFKMR) (Universidad de los Andes) and DNA extraction with QuickExtract™ FFPE DNA Extraction Kit (Lucigen®), (2) quantification with InnoQuant® HY (InnoGenomics Technologies) for sensitive assessment of total human and male DNA quantity/quality, and (3) massively parallel sequencing for simultaneous genotyping of 231 short tandem repeat (STR) and single-nucleotide polymorphism (SNP) markers with the ForenSeq® DNA Signature Prep Kit (Verogen, Inc.). Initial evaluation of artificially degraded blood samples (n = 10) achieved highly sensitive and informative quantification results with InnoQuant® HY, enabling successful first pass genotyping with the MiSeq FGx® System. Twenty-three STR alleles (out of 85) and 70 identity informative SNP loci (out of 94) were recovered from two pg total long target DNA input (0.86 ng total short target input) and an InnoQuant degradation index (DI) of 460 (severely degraded). The three-part workflow was subsequently applied to teeth samples (dental pulp, root cement tissues; n = 13) with postmortem intervals (PMI) of the teeth ranging from 8 days to approximately 6 months. Informative SNP and STR DNA profiles were obtained, to include 78 STR alleles and 85 identity informative SNP loci typed (of 94 total SNP targets) in a 1 month, four-day PMI root cement sample with one pg total long target DNA input and a DI of 76. These data indicate successful performance of the proposed workflow from degraded DNA from teeth samples.

Histological transformations of the dental pulp as possible indicator of post mortem interval: a pilot study.

BACKGROUND: The correct estimation of the post mortem interval (PMI) can be crucial on the success of a forensic investigation. Diverse methods have been used to estimate PMI, considering physical changes that occur after death, such as mortis algor, livor mortis, among others. Degradation after death of dental pulp is a complex process that has not yet been studied thoroughly. It has been described that pulp RNA degradation could be an indicator of PMI, however this study is limited to 6 days. The tooth is the hardest organ of the human body, and within is confined dental pulp. The pulp morphology is defined as a lax conjunctive tissue with great sensory innervation, abundant microcirculation and great presence of groups of cell types. AIM: The aim of this study is to describe the potential use of pulp post mortem alterations to estimate PMI, using a new methodology that will allow obtainment of pulp tissue to be used for histomorphological analysis. The current study will identify potential histological indicators in dental pulp tissue to estimate PMI in time intervals of 24h, 1 month, 3 months and 6 months. MATERIALS AND METHOD: This study used 26 teeth from individuals with known PMI of 24h, 1 month, 3 months or 6 months. All samples were manipulated with the new methodology (Carrasco, P. and Inostroza C. inventors; Universidad de los Andes, assignee. Forensic identification, post mortem interval estimation and cause of death determination by recovery of dental tissue. United State patent US 61/826,558 23.05.2013) to extract pulp tissue without the destruction of the tooth. The dental pulp tissues obtained were fixed in formalin for the subsequent generation of histological sections, stained with Hematoxylin Eosin and Masson's Trichrome. All sections were observed under an optical microscope using magnifications of 10× and 40×. RESULTS: The microscopic analysis of the samples showed a progressive transformation of the cellular components and fibers of dental pulp along PMI. These results allowed creating a chart of qualitative and quantitative parameters to be used on the estimation on PMI based on microscopic degradation of dental pulp. CONCLUSIONS: The histological transformations of dental pulp as a function of time can be used as PMI indicators.