Simplifying Next Generation Sequencing Workflow with QuantiFluor® ds-DNA System

DNA SequenceNext-generation sequencing (NGS), also known as high-throughput parallel sequencing, is the all-encompassing term used to describe a number of different modern sequencing technologies. These include Illumina (Solexa) sequencing, Roche 454 sequencing, Ion torrent: Proton / PGM sequencing and SOLiD sequencing to name a few [1].

With the advent of these technologies sequencing DNA and RNA has become much more facile and affordable in comparison to the previously used Sanger sequencing. For these reasons NGS has been the game-changer in the field of modern genomics and molecular biology.

A common starting point for template preparation for NGS platforms is random fragmentation of target DNA and addition of platform-specific adapter sequences to flanking ends. Protocols typically use sonication to shear input DNA, coupled with several rounds of enzymatic modification to produce a sequencer-ready product [2].

Accurate quantification of DNA preparations is essential to ensure high-quality reads and efficient generation of data. Too much DNA can lead to issues such as mixed signals, un-resolvable data and lower number of single reads. Too little DNA, on the other hand, might result in insufficient sequencing coverage, reduced read depth or empty runs, all of which would incur higher costs. The quality of DNA can also vary depending on the source or extraction method applied and further reinforces the need for appropriate management of the input material. Continue reading

DNA Amplification Using Body Heat, No Instrument Required

Cartoon by Ed Himelblau

Cartoon by Ed Himelblau Copyright Ed Himelblau.

When I was in the lab, there was more than a few times I held tubes in my hand (maybe even under my arm) to make them thaw faster, especially reaction buffers. However, I never considered whether this could be a strategy for actual incubation although humans run at about 37°C and many restriction enzyme reactions proceed most efficiently at 37°C. But research published in PLOS ONE by Crannell, Rohrman and Richards-Kortum took this idea and decided to experiment with the possibility of eliminating an instrument-based DNA amplification. Continue reading

Optimizing PCR: One Scientist’s Not So Fond Memories

primer_tubesThe first time I performed PCR was in 1992. I was finishing my Bachelors in Genetics and had an independent study project in a population genetics laboratory. My task was to try using a new technique, RAPD PCR, to distinguish clonal populations of the sea anemone, Metridium senile. These creatures can reproduce both sexually and asexually, which can make population genetics studies challenging. My professor was looking for a relatively simple method to identify individuals who were genetically identical (i.e., potential clones).

PCR was still in its infancy. No one in my lab had ever tried it before, and the department had one thermal cycler, which was located in a building across the street. We had a paper describing RAPD PCR for population work, so we ordered primers and Taq DNA polymerase and set about grinding up bits of frozen sea anemone to isolate the DNA. [The grinding process had to be done using a mortar and pestle seated in a bath of liquid nitrogen because the tissue had to remain frozen. If it thawed it became a disgusting mass of goo that was useless—but that is a topic for a different blog.] Since I had never done any of the procedures before, my professor and I assembled the first set of reactions together. When we ran our results on a gel, we had all sorts of bands—just what he was hoping to see. Unfortunately, we realized that we had added 10X more Taq DNA polymerase than we should have used. I repeated the amplification with the correct amount of Taq polymerase, and I saw nothing. Continue reading

Insights into the Function of P7C3 Compounds in Neuroprotection

The multiple Lombardi trophies won by Pittsburgh Steelers.  Image used under Wikimedia Creative Commons, and attributed to daveynin.

The multiple Lombardi trophies won by Pittsburgh Steelers. Image used under Wikimedia Creative Commons, and attributed to daveynin.

It is fall and the season for American football. For this football fan, watching the game is a bit less enjoyable than it used to be, as more and more information is available about the serious and permanent brain injuries suffered by football players.

In the introduction to a recent paper in the journal Cell, “P7C3 Neuroprotective Chemicals Function by Activating the Rate-Limiting Enzyme in NAD Salvage”, not a word about American football is mentioned.

However, the paper begins, “No substantive therapeutics are available for the treatment of almost any form of disease entailing nerve death” (1). The authors list a range of neurodegenerative disorders such as Huntington’s, Alzheimers and Parkinson’s diseases, as well as ALS  or Lou Gherig’s disease. They also note that there are currently no effective treatments for trauma to the brain or peripheral nervous system.

The authors note that a chemical treatment that could interfere with nerve cell death would have a “transformative impact in modern medicine”. Continue reading

Optimizing Tryptic Digestions for Phosphoproteomics Analysis

11296971-DC-CR-KinaseProtein phosphorylation is the most widespread type of post-translational modification. It affects every basic cellular process, including metabolism, growth, division, differentiation, motility, organelle trafficking, membrane transport, muscle contraction, immunity, learning and memory (1,2). Protein kinases catalyse the transfer of the phosphate from ATP to specific amino acids in proteins. In eukaryotes, these are usually Ser, Thr and Tyr residues. Due to the development of specific phosphopeptide enrichment techniques and highly sensitive MS instruments, phosphoproteomics has enabled researchers to gain a comprehensive view on the dynamics of protein phosphorylation and phosphorylation based signaling networks.

Due to its high cleavage specificity, trypsin is the commonly used proteolytic enzyme in MS-based proteomics, cleaving peptides carboxyterminal of the amino acids lysine and arginine. However, various factors such as the tertiary structure of a protein, adjacent basic amino acids or negatively charged residues close to cleavage sites as well as PTMs are known to impair proteolysis.

To gain closer insights into the impact of phosphorylation on tryptic digestion, a recent publication(3) systematically characterized the digestion efficiency of model peptide sequences that are known to be prone to incomplete digestion.

The results indicated that increasing trypsin concentrations up to a trypsin to peptide ratio of 1:10 led to a significant gain (1) in the overall number of phosphorylation sites (up to 9%) and in the intensities of individual phosphopeptides, thereby improving the sensitivity of phosphopeptide quantification.

The effect of organic solvents (ACN, acetonitrile and TFE trifuorethanol was also evaluated). Positive results were noted with TFE when determining the digestion of individual peptides. However TFE interfered with TiO2 phosphopeptide enrichment and therefore was not recommended for use with complex samples.

  1. Engholm-Keller, K and Larsen, M.R. (2013) Technologies and challenges in large scale phosphoroproteomics. Proteomics 13, 910–31.
  2. Beausoleil, S. A. et al. (2010) Tissue-specific atlas of mouse protein phosphorylation and expression. Cell 143, 1174–89.
  3. Dickhut, C. et al. (2014) Impact of Digestion Conditions on phosphoproteomics. J. Proteome Res. 13, 2761–70.

Telepathy and Mind Control: From Science Fiction Movies to Reality?

BrainInterfaceThere is something very futuristic, and perhaps scary, about the idea of nonverbally transferring one person’s thoughts to another person, especially for the purpose of controlling or influencing a person’s actions and behaviors. Maybe that’s why telepathy and mind control are favorite topics of many science fiction movies. However, there are times when direct, nonverbal transfer of thoughts would be advantageous, for example when communicating complex concepts or feelings that are difficult to convey. Direct transfer also would circumvent the need to translate information from one language to another. For these reasons, scientists are currently developing technologies to allow such thought transfers. A recent PLOS ONE article describes a simple brain-to-brain interface in humans and shows how this interface can be used to capture a thought generated by one person and communicate that information directly to the brain of a second person and elicit a physical response (1).

Continue reading

6 + 1 Ways Dual-Reporter Assays Can Save Your Data

Dual-Reporter-AssayTransient transfection is often used to perform reporter assays.  We have advocated using a dual-reporter system for decades to normalize the data obtained and gain a clearer understanding of your results.  The experimental reporter should vary with treatment and the control reporter should vary little with treatment. The control reporter thus serves as a marker to help you understand the relative activity of your experimental reporter. Here are seven ways in which dual-reporter assays help you avoid misinterpreting results.

Simply comparing the ratio of the experimental to the control reporter can resolve differences in:

  1. Number of Cells/Well: When manually pipeting cells into a 96-well plate, there is always a chance of having variable numbers of cells in each well. This variation is cell number will affect the experimental and control reporters equally, so the ratio of experimental:control reporter activity will eliminate false interpretation of the experimental data–whether it affects an entire row or column on the plate or individual wells.
  2. Transfection Efficiency: The variations in transfection efficiency will equally affect both the experimental and control reporters so the ratio will normalize the data.
  3. Continue reading

Tips and Tricks for Successful Nucleic Acid Preparation from FFPE Samples: Webinar Preview

FFPE_molecular_analysis_workflowFormalin-fixed, paraffin-embedded (FFPE) tissue samples are extremely common sample types. In this form, tissue is easy to store for extremely long periods of time and useful for immunohistochemical studies. Additionally FFPE samples are fairly inexpensive to produce. However the formalin fixation procedure, which was developed long before the advent of molecular biology, results in chemical crosslinking of nucleic acid and protein molecules inside the cells. This crosslinking presents a challenge for isolating intact, high-quality nucleic acid DNA; so getting at the wealth of molecular information within an FFPE sample can be difficult.

In the upcoming webinar “Successfully Overcoming the Challenges of Working with FFPE Samples”, Dr. Trista Schagat of Promega Corporation discusses some of the key considerations for anyone who is attempting to isolate nucleic acid from FFPE samples. Continue reading

Biotechnology Youth Apprenticeship Program Fosters Young Scientists

Student working in laboratory.

Photo credit: BTC Institute.

Ellyn Lepinski is an intern at Promega who started her biotechnology career path five years ago as a high school junior taking a course from the BTC Institute (www.btci.org) as part of the Biotechnology Youth Apprenticeship Program.

Ellyn credits the program with helping her achieve her goals:

“Over the course of two years in which I was a Youth Apprentice, I obtained numerous skills, both inside and outside of the lab. I gained valuable scientific experience, including techniques like gel electrophoresis, nucleic acid purification, PCR, SDS-PAGE, Western blotting, cell culture and more.

On a personal level, I became very close with other students in the class and with our instructors, Barbara Bielec and Chad Zimprich. Everyone involved was always very approachable and willing to help with both laboratory tasks and in terms of giving advice for the future.

Through the program, I was placed in Dr. Que Lan’s entomology lab at UW-Madison, beginning in 2009. While there, I worked on a project involving sterol carrier protein-2, a protein involved in cholesterol uptake in mosquitoes.Notably, I am still working in Dr. Lan’s lab, however my research focus has shifted to bacterial fermentation. In between working in Dr. Lan’s lab, I also worked at the Forest Products Laboratory (USDA).

Additionally, this past June, I began an internship at Promega in the Scientific Applications department. Here I work to develop new applications for existing projects. This November marks five years of laboratory research for me, which would not have been possible without the Youth Apprenticeship Program and everyone involved. In addition to the specific labs that I have had the opportunity to work in, my experience in the Youth Apprenticeship Program has allowed me to emerge as a leader in my college lab courses. The program has clearly made a phenomenal impact on my life and is something I am very grateful for.”

Photo credit: BTC Institute.

Photo credit: BTC Institute.

Since 1993, the BTC Institute in partnership with the Dane County School Consortium has helped make such opportunities possible to nearly 300 students from public schools throughout Dane County. The program includes a paid apprenticeship in an industry or UW-Madison research lab and specialized instruction. In addition to being paid for their work, students receive high school credit for their participation in the worksite and the specialized biotechnology course held at the BTC Institute.

One aspect of the program that makes it so effective and unique is the amount of time that students spend working. Youth apprentices who start as juniors in the program must work 900 paid work hours to earn the Science, Technology, Engineering and Math (STEM) Skill Standards Certificate from the State of Wisconsin, youth apprentices who start work as seniors must earn 450 work hours. Students have had employment at a variety of companies and UW-Madison research labs, a few examples that have hired multiple apprentices include Genus PIC (ABS), MOFA Global, Promega and laboratories in the UW-Madison Departments of Bacteriology, Biochemistry, Entomology, Genetics, Horticulture, Plant Pathology and Surgery. Many of the students, like Ellyn, continue to be employed by their worksite long after they graduate from high school—proof of how effective this program is in helping to create the next generation STEM workforce.

Each year the BTC Institute hosts a Youth Apprenticeship Program preview night for all of the Dane County youth apprenticeship options: biotechnology, automotive technician, health services, and many more (www.dcsc.org). This year the preview nights will be held February 24 and 25 starting at 5:00pm. Students in grades 10 and 11 who are interested in learning more about the program are encouraged to attend one of the evening sessions with a parent.

Two-Sentence Scary Lab Stories

jackolantern_editYour bloggers at Promega Connections like Halloween. In the past we have reviewed our top scary blogs and provided lists of things to do with pumpkins and suggestions for what to do when you have too much leftover Halloween candy. This year we are jumping on the 2-sentence horror story bandwagon with a twist: the 2-sentence scary/funny lab story. Here are a few of our creations. If you have one of your own, leave it in the comments.

saveI had just finished writing the final chapter of my dissertation, when a pop up box appeared :“Are you sure you want to exit without saving changes”. Then the screen went blank. Continue reading