Here at Promega we receive some interesting requests…
Take the case of Virginia Riddle Pearson, elephant scientist. Three years ago we received an email from Pearson requesting a donation of GoTaq G2 Taq polymerase to take with her to Africa for her field work on elephant herpesvirus. Working out of her portable field lab (a tent) in South Africa and Botswana, she needed a polymerase she could count on to perform reliably after being transported for several days (on her lap) at room temperature. Through the joint effort of her regional sales representative in New Jersey/Pennsylvania (Pearson’s lab was based out of Princeton University at the time) and our Genomics product marketing team, she received the G2 Taq she needed to take to Africa. There she was able to conduct her experiments, leading to productive results and the opportunity to continue pursuing her work. Continue reading “Of Elephant Research and Wildlife Crime – Molecular Tools that Matter”
Forensic lab validations can be intimidating, so Promega Technical Services Support and Validation teams shared these tips for making the process go more smoothly.
Prepare Your Lab. Make sure all of your all of your instrumentation (CEs, thermal cyclers, 7500s, centrifuges) and tools (pipettes, heat blocks) requiring calibration or maintenance are up to date.
Start with Fresh Reagents. Ensure you have all required reagents and that they are fresh before beginning your validation. This not only includes the chemistry being validated, but any preprocessing reagents or secondary reagents like, polymer, buffers, TE-4 or H2O.
Develop a Plan. Before beginning a validation, take the time to create plate maps, calculate required reagent volumes, etc. This up-front planning may take some time initially, but will greatly improve your efficiency during testing.
Create an Agenda. After a plan is developed, work through that plan and determine how and when samples will be created and run. Creating an agenda will hold you to a schedule for getting the testing done.
Determine the Number of Samples Needed to Complete Your Validation. Look at your plan and see where samples can be used more than once. The more a sample can be used, the less manipulation done to the sample and the more efficient you become.
Select the Proper Samples for Your Validation. Samples should include those you know you’ll obtain results with be similar to the ones you’ll most likely be using, and your test samples should contain plenty of heterozygotes. When you are establishing important analysis parameters, like thresholds, poor sample choice may cause more problems and require troubleshooting after the chemistry is brought on-line.
Perform a Fresh Quantitation of Your Samples. This will ensure the correct dilutions are prepared. Extracts that have been sitting for a long time may have evaporated or contain condensation, resulting in a different concentration than when first quantitated.
Stay Organized. Keep the data generated in well-organized folders. Validations can contain a lot of samples, and keeping those data organized will help during the interpretation and report writing phase.
Determine the Questions to Be Answered. While writing the report, determine the questions each study requires to be answered. Determining what specifically is required for each study will prevent you from calculating unnecessary data. Do you need to calculate allele sizes of your reproducibility study samples when you showed precision with your ladder samples?
Have fun! Remember, validations are not scary when approached in a methodical and logical fashion. You have been chosen to thoroughly test something that everyone in your laboratory will soon be using. Take pride in that responsibility and enjoy it.
Need more information about validation of DNA-typing products in the forensic laboratory? Check out the validation resources on the Promega web site for more information for the steps required to adopt a new product in your laboratory and the recommended steps that can help make your validation efforts less burdensome.
That is the prevailing question I’m asked when someone learns of my occupation as Deputy Sheriff Criminalist for the Contra Costa County (CA) Office of the Sheriff. Alas, my life is not quite so glamorous. It actually often entails entering formulas into an excel spreadsheet while being placed on hold as I order some pipette tips.
But, why does it have to be that way?
I have attended my fair share of professional conferences and workshops and written numerous journal articles. As a forensic scientist I do believe in the importance of sharing data, new techniques, and new methodologies with my colleagues. Yet what I think is not highlighted enough is the one element that differentiates our field from any other scientific field—our involvement with the criminal justice system. Every case we work on involves a mystery, a crime, a victim(s), and a suspect(s). And while scientists in other fields typically only speak to other scientists, in my world, forensic scientists usually interact with a person in a black robe who has the power to strongly influence the outcome of a case. These wildly frustrating, invigorating, and challenging cases are the most interesting things about our field, and yet we hardly share our stories.
Today’s blog is written by guest blogger, Douglas R. Storts, PhD, head of Research, Nucleic Acid Technologies, Promega Corporation.
Massively parallel sequencing (MPS), also called next generation sequencing (NGS), has the potential to alleviate some of the biggest challenges facing forensic laboratories, namely degraded DNA and samples containing DNA from multiple contributors. Unlike capillary electrophoresis, MPS genotyping methods do not require fluorescently-labeled oligonucleotides to distinguish amplification products of similar size. Furthermore, it is not necessary to design primers within a color channel to generate amplicons of different sizes to avoid allele overlap. Consequently, all the amplicons can be of a similar, small size (typically <275 base pairs). The small size of the amplicons is particularly advantageous when working with degraded DNA. Because the alleles are distinguished by the number of repeats and the DNA sequence, additional information can be derived from a sample. This can be especially important when genotyping mixtures. As previously demonstrated (1), this sequence variation can help distinguish stutter “peaks” from minor contributor alleles.
Because there is no reliance upon size and fluorescent label, significantly greater multiplexing is possible with MPS approaches. In addition to autosomal short tandem repeats (STRs), we can also sequence Y-STRs, single nucleotide polymorphisms (SNPs), and the mitochondrial DNA control region. The advantage to this approach is the forensic analyst does not need a priori knowledge whether a sample would benefit most from the different methods of genotyping.
Every year, hundreds of forensic analysts attend the International Symposium on Human Identification to learn about new advances in the field of DNA typing. The talks and posters are informative, and it is a great opportunity to network with DNA analysts from around the world. As the editor of Profiles in DNA, I am fortunate to attend the meeting to gather feedback about the publication and to get ideas for future content.
Sitting on my kitchen counter, atop a pile of junk mail I have yet to throw away, is the current issue (May 2009) of the National Geographic. Emblazoned in red letters are the words “Ice Baby: Secrets of a Frozen Mammoth” under the picture of an incredibly well preserved wooly mammoth baby. The feature article tells the story of “Baby Lyuba” a baby mammoth discovered in 2007 on the Yamal Peninsula in Siberia. A companion article focuses on the possibility of bringing extinct animals—like the mammoth— back to life.
This article heralds the sequencing of a large portion of the nuclear genome of the mammoth by a team from Pennsylvania State University led by Webb Miller and Stephan C. Schuster (1). A happy coincidence because I was working on a blog about this same subject. The idea of sequencing the genome of an animal that last walked this earth tens of thousands of years ago captures my imagination. Continue reading “Sequencing Ancient DNA: A Mammoth on the Tip of the Iceberg?”
“We are on our way to Machu Picchu”. These were the words to which I awoke on a warm July morning in 1984 as I struggled to make sense of where I was and regain my memory of the previous week. My family and I had come to Peru on a mission to learn more about the Incas, a civilization that I knew little about. We had spent two days in the coast-hugging capital city of Lima, seen much of the colonial architecture and even experienced the full force of a midnight earthquake (which because of sheer exhaustion I had slept right through). We had taken a plane down the coast in the hope of visiting the smaller city of Arequipa, but cloud cover had forced us to fly several hundred miles further south to a town called Tacna and take an overnight taxi ride to Arequipa through the Peruvian desert. Continue reading “Machu Picchu, Lost Civilizations, and the Resolving Power of DNA Analysis”