When the 

 material is cooled, 

 the primers reform 

 bonds on the 

 conserved regions 

 broken during the 

 heating process. 

 Enzymes then begin 

 building DNA on 

 the new strand, 

 which fills in the 

 rest of the gene 

 around the already 

 matched conserved 

 regions. These areas 

 will be either 

 semiconserved 

 (with a degree of 

 similarity in all 

 organisms) or 

 variable (unique for 

 each organism). The 

 end result is a copy 

 of the original 

 DNA. 



This process is 

 repeated 30 to 50 

 times, each time 

 doubling the 

 number of strands, 

 to generate millions 

 of copies. Rublee 

 then looks at the 

 sequences of these 

 copies and com- 

 pares them to 

 sequences of other 

 organisms similar 

 to Pfiesteria to 

 determine whether 

 it is truly unique. 



If it is unique, Rublee can design 

 a probe complementary to it, a piece 

 of synthesized DNA called an 

 oligonucleotide that will seek out its 

 complement on DNA extracted from 

 waters or sediments where the suspect, 

 Pfiesteria, may reside. If Pfiesteria is 

 present, the oligonucleotide will bind 

 to that target. If not, it won't. 



The mystery isn't solved yet. 

 Next, the scientists testing for the 

 dinoflagellate must confirm that the 

 binding has actually taken place. 



Loading an electrophoresis gel 

 to check the results of polymerase chain reaction 



Hitting the Target 



One method to confirm binding is 

 to use the probe as a primer in the 

 polymerase chain reaction, which 

 would yield many copies of the target 

 if it is present. But certain chemical 

 conditions have to be met for this 

 procedure to work, and all the un- 

 knowns about Pfiesteria make that a 

 chancy course to take. 



A second method is blotting, 

 chemically hooking another molecule 

 to the probe so that it is larger and has 



a marker attached. 

 Rublee has blotted 

 with a radioisotope 

 but says that this 

 method requires too 

 many precautions. 



"We want to use 

 high tech to develop 

 a low-tech method," 

 he says. 



So Rublee is 

 focusing on fluores- 

 cent in situ hybrid- 

 ization (FISH), the 

 detection strategy he 

 calls "the coolest." 

 Rather than binding 

 to material extracted 

 from the cell, the 

 probe — equipped 

 with a fluorescent 

 marker — enters the 

 cell and binds with 

 the target material 

 inside. The cell can 

 be examined under a 

 microscope after 

 excess probe material 

 has been washed 

 away. If it glows, 

 binding has occurred. 



FISH requires 

 minimal equipment 

 and a few hours of 

 time from sampling 

 to results. With a 

 power source and 

 few chemicals on 

 hand, it could be 

 used to test waters 

 on site. 



But using this method on Pfiesteria 

 poses a few thorny problems that Rublee 

 is working to solve. 



First, after consuming algae, 

 Pfiesteria retains some of the natural 

 fluorescence from the plant's chloro- 

 plasts. Also, the cell walls of the 

 dinoflagellate itself fluoresce. These 

 natural sources of fluorescence could 

 interfere with the probe's effectiveness 

 by masking the specific fluorescence 

 from the probe. Rublee equates this to 



Continued 



COASTWATCH 7 



