Wednesday, 17 August 2016 13:44

Zika A, G, C, U

Written by 
Published in Zika Diaries
Audrey Warren preparing DNA samples to be sent out for sequence analysis. Audrey Warren preparing DNA samples to be sent out for sequence analysis.

Part of our work on Zika virus is to determine the genome sequences of multiple virus isolates, and learn how the differences affect infection. That’s our technician Audrey in the photo preparing samples to be sent out for sequence analysis. Not very long ago, if you wanted to determine the nucleotide sequence of a DNA, you had to do the whole process by yourself. It took me over one year to obtain the 7,442-nucleotide sequence of poliovirus RNA! The same task could be done today in an hour.

When I arrived in David Baltimore’s laboratory at MIT in 1979, it was my project to make a DNA copy of the RNA genome of poliovirus, and then determine its nucleotide sequence. The method I used for DNA sequence analysis was called the Maxam-Gilbert technique, after its two creators, Allan Maxam and Walter Gilbert. To sequence the DNA copy of the poliovirus genome that I had made, I used restriction enzymes to cut the DNA into ~500 base pair pieces, which I then purified by agarose gel electrophoresis. I took each DNA fragment and enzymatically attached an isotopically labeled molecule of phosphate to each end. Then I cut the "hot" DNAs in half with another restriction enzyme and purified the halves by gel electrophoresis. Finally the DNAs were treated with four different chemicals, and the resulting products were separated on a very thin and long polyacrylamide gel.

Then I repeated the whole process using different enzymes to cut the DNA, to make sure that we sequenced across each restriction enzyme cleavage site.

It took me one year to determine the 7,442-base sequence of poliovirus RNA by this technique.

Audrey is creating DNA copies of the RNA of many different Zika virus isolates from all over the world. She determines the nucleotide sequence of each DNA copy to make sure that no errors have been introduced. To determine the genome sequences, she amplifies short pieces of the Zika virus RNA genome by polymerase chain reaction, and then sends off the DNA to a local company (there is a dropbox in our building for leaving samples—the company picks them up once a day). The sequences are determined by next-generation sequencing—a very fast, high-throughput process that has revolutionized molecular biology. The next day we get the results on a website.

I do not regret for a moment having spent a year on a process that can today be done so much faster. I’m just happy that we have the technology now, and it can be used to work on so many amazing projects—such as studying the genome sequences of all isolates of Zika virus and their biological implications. 

Fast, high-throughput nucleotide sequencinganother great example of how technology pushes science forward.

Last modified on Tuesday, 23 August 2016 15:56
Vincent Racaniello

Vincent Racaniello, Ph.D. is Professor of Microbiology at Columbia University Medical Center. As principal investigator of his laboratory, he oversees the research that is carried out by Ph.D. students and postdoctoral fellows. He also teaches virology to graduate students, as well as medical, dental, and nursing students.

Vincent entered the world of social media in 2004 with virology blog, followed by This Week in Virology. Videocasts of lectures from his undergraduate virology course are on iTunes University and virology blog. You can find him on WikipediaTwitter, Facebook, and Instagram. His goal is to be Earth’s virology professor. In recognition of his contribution to microbiology education, he was awarded the Peter Wildy Prize for Microbiology Education by the Society for General Microbiology. His Wildy Lecture provides an overview of how he uses social media for science communication.