What's in a miniprep? A "short" history of a taken for granted technique

If you’re a scientist chances are the miniprep was one of the first techniques you learned. I still remember opening my first blue box as an undergraduate and have done countless preps since. For non-scientists, a miniprep is a way to separate plasmid DNA from chromosomal DNA. Plasmids are DNA encoded on an enclosed circle that replicate inside bacteria independently from the much bigger, linear chromosomal DNA. Bacteria transfer these plasmids to each other, which give those bacteria special properties such as antibacterial resistance. Scientists have hijacked this system to put whatever DNA we want on this plasmid and put it inside bacteria which will quickly make more of that DNA, After purifying this foreign DNA away from bacterial junk, we can then do whatever it is we evil scientists do with foreign DNA…perhaps genetically modify some tomatoes just to make them less flavorful?  

The miniprep’s ease lies in its step-by-step instructions and pre-made buffers. The name doesn’t hurt either, sounding rather diminutive and much less threatening than a phenol/chloroform extraction. Many an older faculty has complained that science has become so automated young scientists don't even understand the basis for the miniprep technique. So for a description of how a miniprep kit works, see the end of the article, as "it's proprietary" probably won't work as a response on your GBO. 

But where did the miniprep technique come from? Probably not many people have wondered this and I hadn’t either until this week for some reason. Perhaps I’m starting to crack under the pressure of the new postdoc, but I decided to put some time into investigating, which, with the Internet, is not that difficult.

The basis of the miniprep kit, the alkaline lysis, was published by Birnboim and Doly in 1979 (Birboim and Doly 1979). In 1988, Dr. Birnboim recounted how he developed the technique in Citation Classics (Birnboim 1988), a feature in Current Contents that ran from 1977 to 1993 and sought to show the human side involved in putting out some of the most widely-cited papers. During a sabbatical in Paris, H. Chaim Birnboim decided he needed a fast and reproducible way to purify plasmid DNA. He was not sufficiently fluent in French and was therefore a little isolated in the lab, for which he attributes his ability to focus on solving this problem rather than working on another lab project. What Dr. Birnboim was actually interested in was identifying and studying a new class repetitive sequences in higher eukaryotes. The sequence (a polypyrimidine tract) was resistant to acid treatment so the idea was to examine clones from a mouse DNA library. He would screen for those regions with long pyrimidine tracts using acid treatment that would break other DNA sequences into short fragments. The DNA was labeled with ³²P, a radioactive isotope of phosphate, before acid treatment and the slow-moving piece of DNA containing the repetitive sequence would be identified with autoradiography. This screen required analysis of many clones, and hence, a quick way to purify the DNA. At the time of the article he had not received any awards, but said that it was “personally gratifying to have developed a procedure that has survived for nearly a decade.” 

Of course this is only the first part of the miniprep technique, the second being the spin column, which relies on the ability of nucleic acids to bind a silica membrane under certain conditions, such as high salt. Dr. Birnboim published the use of silica-glass powder to bind and purify nucleic acids in 1982 (Marko et al. 1982) and the technique was improved upon by other scientists using different chaotropic agents (Boom et al. 1990). By the mid 1980s, Qiagen began selling kits for purification of plasmids and in the early 1990s companies began patenting the technology with Promega filing a patent in 1995 for “Nucleic acid purification on silica gel and glass mixtures” (US Patent number: 5658548) and Qiagen filing a patent in 1994 for “A method for the purification and separation of a nucleic acid mixture by chromatography” (Patent number 6383393). And so the miniprep battle began. But that’s for the company reps to worry about.

I’ve always found minipreps to be pretty relaxing because of their foolproofness (aside from a mistaken miniprep attempt after Happy Hour). Despite their ease, there is enough room to make you feel as though you have somehow improved on the design. For example, “psst, if you heat the water before elution, it will increase your yield” or “hey, pass the elution over the column again, my friend.”

I am terrible with numbers. I can’t remember my anniversary and regularly invert the birth year and day for my husband. I also recently wrote 7/14/20 on an eppendorf tube. BUT if I get hit in the head and someone wants to assess my mental faculties you can ask me the protocol for a miniprep: 250 microliters P1, 250 microliters P2, 350 N2….and don’t forget to heat the water.

How a miniprep works: The first buffer (P1) is simply to resuspend the cells and digest RNA. The P2 lysis buffer contains a detergent to lyse the cell membrane and a high pH that denatures the DNA. This is because the hydroxide ions pull hydrogen ions off the DNA molecule, disrupting the hydrogen bond network that holds the DNA strands together. Addition of the neutralization buffer (N3) lowers the pH and allows the circular DNA to go back to being double-stranded while the large, bulky chromosomal DNA cannot and forms a precipitate. High-speed centrifugation pellets the chromosomal DNA away from the soluble plasmid DNA. The supernatant containing plasmid DNA is then added to a silica membrane under high salt conditions, which allow double-stranded, but not single-stranded RNA and DNA to bind. A chaotropic agent, such as guanidium HCl disrupts the hydration shell around DNA and allows positively charged ions to form a salt bridge between the phosphate backbone and negatively-charged silica membrane. The membrane is washed and then DNA is eluted with a low ionic strength buffer, such as water. And there you have it, more than you wanted to know about minipreps.

References:

Birnboim, H.C. and Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7(6), 1513-23.

Birnboim, H.C. (Nov 7 1988). Citation Classic – A rapid alkaline extraction procedure for screening recombinant plasmid DNA. CC/Life Sci. 45, 12-1

Boom, R., Sol, C.J., Salimans, M.M., Jansen, C.L., Wertheim-van Dillen, P.M., van der Noordaa, J. (1990). Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 3, 495-503.

Marko, M.A., Chipperfield, R., Birnboim, H.C. (1982). A procedure for the large-scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder. Anal Biochem. 2, 382-7.

Very good read and insight into a scientist's life and passion

Saw this article from one of my favorite news organizations: BBC News Magazine. It is about the scientist who sought to identify the causative agent of Ebola:

The virus detective who discovered Ebola in 1976

The summer of scientific missteps

What is going on with scientists this summer? Misplaced smallpox strains, contamination at the CDC? It took me over a week to decide what I really thought about all this before I could write about it. Is the media blowing things out of proportion, only increasing the public’s mistrust in science? How did this happen and is it part of a bigger problem?

First came news from the director of the Centers for Disease Control and Prevention (CDC) in Atlanta, GA, that two laboratories, working with highly pathogenic organisms would be shut down and all shipment of materials from BSL-3 and -4 level labs (high biosafety levels) stopped pending review. In one case, a low-pathogenic influenza virus, H9N2, was contaminated with the highly pathogenic H5N1 (of “bird flu” fame) and sent to the Department of Agriculture who discovered the mistake. In a separate incident the bacteria responsible for anthrax disease had been improperly inactivated before transferring it to a lower biosafety level lab. Apparently, approved sterilization techniques were not followed, nor was their a standard protocol in the lab for inactivating and transferring the anthrax bacteria to other labs.

Then in early July, 6 vials of the variola virus (responsible for smallpox) were found in a storage freezer (a freezer probably very much resembling a deep freezer) at the National Institute of Health (NIH) in Bethesda, MD. The samples were prepared and stored in 1954. Previously the only remaining stocks of virus (which was eradicated in 1981) were thought to be held at the CDC and at the State Research Center of Virology and Biotechnology in Siberia.

So how could this happen?
Upon hearing the news, I shook my head, but was not surprised. Scientists are quite fallible, especially when it comes to lab organization and thinking they are immune to lab safety practices. The fast pace required to publish and not perish can often lead to sloppiness: sloppiness in record keeping and in properly training and overseeing safety measures. I have experienced both of these first hand. It is not hard to imagine at all how 6 vials, even vials of deadly and potential agents to be used for bioterrorism, went unnoticed amidst the ~40,000 vials that fit inside a standard laboratory -80˚C freezer. Also, there is a lot of turnover in science: graduate students change every 4.5 years while postdoctoral fellows come and go as often as every year (though three is more likely). This combined with the high variability in record keeping practices often leads to loss of years of work, and in this case, a potentially deadly mistake. Because of time constraints for advisors, there is usually little oversight into the record keeping practices of the researchers actually making the samples. It is usually not until that researcher leaves that there is a mad scramble to make sure everything is in “order.” The best-case scenario is that samples will have been entered into a database. But in too many cases, there is no database and samples are poorly labeled (and anecdotally, written in another language), labels are worn off, and of course, encrusted in ice. Unless someone is immediately taking over that researcher’s work, these “orphaned” boxes of material are often shoved to the back of the freezer, apparently in some cases, waiting to cause a scandal half a century later.

Though there have been multiple calls from the World Health Organization for scientists to go through their inventories and find remaining vials of smallpox throughout the decades since it was eradicated, there was little impetus and no oversight for scientists to actually go through the arduous (and finger-numbing) procedure. Really, the virus poses little public health threat left alone at the bottom of a deep freezer. Also, on a positive note, the researcher who identified the virus notified his superior and the viral stocks were transfer to the CDC for analysis and destruction. One can imagine a worse case scenario where an unknowing researcher somehow disposed the vials in a receptacle that was not subsequently autoclaved (exposing the virus to pressurized steam that kills viruses and bacteria). However low, there was a chance for these viral stocks to make it into the environment alive.

Many scientists I talked to were frustrated by these glaring oversights because they worried it would put smallpox virus (and other “extinct” disease) research at risk. There has been an ongoing debate by countries around the world about not so much as whether to destroy the virus, but when. Why keep the virus around? Is there really a chance for smallpox to return or be used by terrorists? Well, in 2002, scientists were able to synthesize poliovirus de novo using mail-order DNA segments that were assembled using the genome sequence of the virus as a blueprint and molecular biology techniques known to any graduate student (Cello et al, 2002). There was immediate uproar from both the science community and general public and the potential creation of the smallpox virus specifically cited as a major concern. And so scientists argue, we need to keep a few smallpox stocks around just in case.

It seems like a silly response to the NIH incident to destroy the remaining known smallpox stocks when the real danger, it seems, is from the stocks we don’t know about, not from those in Russia or at the CDC…oh wait. I can understand why the general public mistrusts scientists. In 2003, the NIH accidentally sent anthrax to a Children’s Hospital in California for Pete’s sake! And the most recent investigations into the CDC discovered anthrax specimens in unlocked refrigerators in a hallway where many workers pass through. And again, record keeping was found to be inadequate. If we want to argue to keep around deadly pathogens, “just in case”, we need to get it together people.

Of course, this is easy for me to say from my budding yeast field. The worst my biological agent will do is give you a hang over (not true at all, but I couldn’t resist). And of course, I always follow proper safety precautions, never eating or drinking in lab and always wear long pants and closed shoes. But although I started off thinking the media was potentially blowing things out of proportion, I found myself in the end, angry at scientists but not really having any answers. Clearly more oversight into protocols is necessary and I believe Dr. Thomas Friedman, the director at the CDC, will do it. But as long as time is of such limited supply and demands are so high on supervisors, sloppiness will continue to contribute to incidents like these. But that's just my two cents, what do you all think?

References:
Cello J, Paul AV, Wimmer E (2002) Chemical synthesis of poliovirus cDNA: generation of infectious virus in the absence of natural template. Science 297: 1016–1018.

Transitioning from a life of woe to a life of wow: from malaria to yeast

When I started the blog not very long ago, the intention was to post an article once a week. But last week came and went without posting anything. I can’t even really blame this on my Birthday and having my family in town as this all happened after the self-imposed deadline of Thursday. I have some articles in the works but find all the legwork of diving into the primary literature unappealing at this moment. So instead I will discuss my personal experience with transitioning from biochemistry to yeast genetics. After years struggling with the malaria parasite Plasmodium falciparum in my PhD; cloning from a 70% AT-rich genome, trying to express, purify, and crystallize proteins that are generally larger and more disordered than their human homologues in E. coli, and trying to do western blots with not very good antibodies, I thought I would do myself a favor and focus on the model eukaryotic organism, Saccharomyces cerevisiae. I would still study the biological processes I was interested in, autophagy and protein trafficking, but in a system with some proper tools. Plus yeast smells a heck of a lot better than bacteria!

So how are things going nearly 3 months in?

One thing that has taken getting used to is the amount of planning that is required when working with yeast. I was very used to deciding to purify a protein from bacteria and two days later, having the materials to do so. Once the proteins were purified and stably frozen away, experiments could be conducted as soon as I planed them. But with yeast, more time is required to first grow on a plate from a glycerol stock, then transform a plasmid and wait three more days for colonies, then start a small overnight culture, and the next day: microscopy. A week can easily go by where I don’t really have anything tangible to show. This is thankfully changing, but requires a lot of multi-tasking and planning ahead so that while strains are growing for one project, I am doing experiments for another. Right now, my method of juggling this has evolved into each day reviewing the previous days “to-do” list and writing a new one for that day. I can only imagine there are or will soon be, lab apps that will send text reminders for different experiments.

I am also learning that yeast are tricky little guys. They readily undergo homologous recombination, which is great for manipulating the genome, but they will do anything to stay alive and seem to shuffle things around as need be so that I am left wondering where the heck in the genome did my GFP tag go? I cannot complain too much about this, as I have deleted a gene in less than a month, compared to the oft-predicted one-year time frame for P. falciparum. I was hoping the microscopy would be easier with yeast, but really they are not much bigger than the asexual forms of Plasmodium (5-10 µm vs 1-2 µM). I still find myself looking longingly at the images of human cells or tissues appearing on the microscope computer screen next to me. But at least tagging yeast genes is easy, allowing for live microscopy. However, tagging a gene can be deleterious for that protein’s function and I have struggled for weeks trying to C-terminally tag several proteins in a complex without success. So I am about to begin the laborious task (this is relative, of course) of N-terminal tagging several genes to hopefully get around this problem. Which is to say that science is never easy.

So how are things going? Generally pretty good. I am learning a lot about genetics and working with yeast. Maybe in 3 more months I will actually have some worthwhile results…or be working for a brewery.