Preservation and Direct PCR of DNA from Fresh and Decomposing Human Tissue

While I am away this week, I decided to pick an easy topic to write about: my own. I started independent research working with my advisor in 2013 while I was working towards my masters degree in forensic science. Mass disasters are  events because they typically result in mass casualties in less than ideal situations, where loss of facilities and/or electricity can happen. Then we are posed with the question about what do we do with the bodies. As remains are left to decompose, DNA is degrading because it is exposed to heat, humidity, and/or UV damage. So we wanted to see if there was a way to easily collect and preserve DNA samples in heat and humidity for later disaster victim identification (DVI). We collected decomposing skin and muscle from three cadavers at different stages of decomposition to evaluate room temperature tissue preservatives over a span of three months of storage. Besides preserving the DNA in tissues at room temperature, we wanted to see if these preservatives would lyse the cells and release nuclear DNA into solution, which would make DNA analysis faster by removing the tissue lysis step that takes hours to several days. We had success obtaining DNA from the tissue and liquid preservative for up to three months storage from 4 out of the 5 preservatives; however, the quantity and quality of DNA varied. The manuscript just got accepted this summer. My second project, although my first published manuscript, was a collaboration project with a group of scientists in Australia. We evaluated these same 4 preservatives that facilitated leaching of DNA from tissues for direct polymerase chain amplification (PCR). Because the DNA is suspended in solution, an aliquot of sample can be added directly to amplification reagents and amplified, thereby skipping extraction and quantitation all together. This type of analysis is commonly performed on DNA reference samples due to its efficiency and cost effectiveness. My next project is combining these two projects and testing even more methods to collect, preserve, and analyze DNA in decomposing tissue samples in hopes of making recommendations on the best, cheapest, fastest way to process DNA for DVI purposes or crime scene field-work.


References:

1) Sorensen A, Rahman E, Canela C, Gangitano D, Hughes-Stamm S. Preservation and rapid purification of DNA from decomposing human tissue samples. FSI:Genetics 25 (2016) 182-190.

2) Sorensen A, Berry C, Bruce D, Gahan M E, Hughes-Stamm S, McNevin D. Direct-to-PCR tissue preservation for DNA profiling.

Marijuana Short Tandem Repeats (STRs)

My friend in the forensic science PhD program recently got published for her work on developing a Marijuana STR multiplex system. Marijuana is a prevalent drug related to crime, especially in this area so close to the Mexican border. In 2014, marijuana became legalized in some states for medical and recreational use. Therefore, it has become a challenge to track and prevent legal marijuana from entering states where it is illegal. The goal of this project was to aid in individualization of seized marijuana samples and to link drug cases together. They modified and optimized a 13-loci multiplex (meaning 13 loci can be amplified and sequenced in one reaction) according to guidelines and standards required in forensic science. They also designed an allelic ladder for sequencing and developed and validated a real-time quantitative PCR method for nucelar DNA specific to Marijuana samples. In the end, they were able to generate full profiles from 127 samples part of 11 U.S. Customs and Border Protection seizures and develop a reference Marijuana population database with allele frequencies.  Future work includes looking into tetranucleotide markers opposed to the dinucleotide markers and massive parallel sequencing.

Reference:

 Houston R, Birck M, Hughes-Stamm S, Gangitano D. Evaluation of a 13-loci STR multiplex system for Cannabis sativa genetic identification. Int J Legal Med (2016) 130:635-647.

Pharmacogenetics

Besides forensic DNA, another field that interests me is pharmacogenetics, which is the study of how an individual's genetic make-up affects his/her drug metabolism and response. This field began in the 1950's and can be useful for personalized medicine for diseases and addiction treatment, as well as death investigations to name a few. Since cancers and diseases can arise from many factors besides genetic ones, the field involves the study of how the environment plays a role with genetics to cause human diseases using biomarkers to determine more effective drug treatment than traditionally done by trial and error. With whole genome sequencing becoming more commonly used, it seems like the world of tomorrow will be like the movie GATTACA. If you haven't seen it, it makes for a pretty good watch.

One article I found interesting is titled, "Pharmacogenetics of alcohol, nicotine and drug addiction treatments" by Jessica E Sturgess et al (2011). Alcohol and nicotine dependence as well as cocaine and heroin addition, although less common, result in negative health impacts and premature deaths causing a major socioeconomic issue. The article goes in depth to provide a review of pharmacogenetic studies, treatments, and genes associated with addiction. For example, naltrexone is a common treatment for alcohol dependence and a few studies found that participants taking this and had the G allele on the OPRM1 gene had reduced rates of relapse compared to those with the genotype AA.

Another interesting read is titled, "Personalizing medicine with clinical pharmacogenetics" by Stuart A Scott (2011). In 1977, a hepatic cytochrome P450 oxidase polymorphism (CYP2D6 gene) was discovered. This enzyme is in involved in the metabolism of  approximately 25% of commonly used drugs. Some individuals can inherit this "poor metabolism trait." Genotypes have been associated with phenotypes such as ultrarapid, extensive, intermediate, and poor. You can start to see how pharmacogenetic information might play a role for personalized medicine and addiction treatment.

References:

1) Jessica E Sturgess et al. Pharmacogenetics of alcohol, nicotine and drug addiction treatments. Addicition Biology. 2011 16:357-376.

2) Stuart A Scott. Personalizing medicine with clinical pharmacogenetics. Genet Med. 2011 Dec; 13 (12):987-995.

Human DNA from Mosquitos?

So let me tell you all a little bit about me. I am a PhD Candidate at Sam Houston State University in the forensic science program. My concentration is in Forensic DNA and my research topic is human identification (HID) following mass disasters. However, I figured I should probably branch off to new topics, but first I thought I would start with an interesting read I found a little while ago titled, “Identification of person and quantification of human DNA recovered from mosquitoes (Culicidae)” by Garan Curic, et al. The article is a short communication about obtaining human DNA from the gut of mosquitoes. The study apparently stemmed from a case of a dead body found on the beach and the only evidence linking the victim to the suspect was a mosquito found at the suspect’s house that contained the blood of the victim. The use of human DNA from mosquitoes may sound weird. However, forensic science is all about the probative value of evidence. Any little piece of evidence is important if it can help tell the story of a crime committed, especially if it’s the only probative evidence found. I thought it would be a good article for those of you interested in forensic DNA because it touches on common DNA analyses for HID and a common problem that we face, such as DNA degradation.

Common DNA analysis in crime labs consists of (1) evidence collection and sampling, (2) DNA purification, (3) quantification, (4) amplification, and (5) short tandem repeat (STR) typing. The main criteria for successful STR typing is single source DNA of sufficient quantity and quality. Extracting human DNA from the gut of a mosquito could be an issue. We work with nanograms and picograms of DNA, and although that seems very small, degraded or low amounts of DNA makes STR typing more difficult. According to the article, the very little amount of human DNA in the gut of a mosquito is digested by digestive enzymes and bacterial activity. However, they were able to obtain full profiles up to 32-48 hours after feeding. Any time longer than that, they were only able to obtain partial DNA profiles. They were able to obtain more reportable alleles after reanalysis of these samples by increasing the number of cycles during amplification and using a mini-STR kit, which are common techniques used to overcome DNA degradation. I found it odd that they discussed DNA profiling before quantitation but anyway, the quantity of human DNA decreased as the digestion period increased. Low amounts of human DNA were reported (<1pg/uL in some cases). However, successful STR typing usually requires 500pg/uL to 1ng/uL of DNA. The solution is concentrating the purified DNA to obtain more DNA per microliter.

In conclusion, human DNA obtained from the gut of mosquitoes is suitable for STR typing more than three days after the feeding. I thought that was pretty interesting. It reminds me of another article that was able to obtain DNA from the worm in tequila. Look it up if you’re interested. Here’s a link to a brief description about it: http://www.scienceagogo.com/news/20100110033959data_trunc_sys.shtml. By the way, that’s sorta the basis of my research with Dr. Sheree Hughes-Stamm.