Wednesday, March 29, 2017
University of Hawaii at Manoa
University of Hawaii System
College of Tropical Agriculture and Human Resources

1. Understanding the polymerase chain reaction (PCR).

2. PCR testing of field soil for Ralstonia solanacearum. To extract DNA from soil, a phosphate buffer solution is added to the soil sample. The sample with buffer is placed in a shaker for 5 minutes. After shaking the sample is allowed to sit for an additional 5 minutes, when the particulates will settle. Microbes remain suspended in the supernatant from which a sample is extracted. The supernatant sample is added to an SMSA enrichment broth, which contains antibiotics that select for growth of Ralstonia solanacearum by suppressing other organisms.The tube is shaken for 5 days. Aliquots are taken every 24hrs over a 5 day period, and tested for the presence of Ralstonia solanacearum. By allowing the samples to enrich over a period of 5 days, Ralstonia solanacearum is allowed to multiply overtime, which allows for greater sensitivity in PCR testing. PCR reactions are then made up and placed in a thermocycler. When the program is completed the PCR reactions are removed. 

PCR is a technique used for rapidly synthesizing large quantities of a specific DNA segment. The procedure involves separating the DNA into its two complementary strands, using DNA polymerase to synthesize two-stranded DNA from each single strand, and repeating the process until a massive number of identical DNA particles have been synthesized. PCR reactions are loaded into an agarose gel and electric current is applied. Because DNA is negatively charged, when electric current is applied, it will migrate towards the positive pole. Larger molecules move more slowly through the gel while the smaller molecules move faster. When DNA molecules have had enough time to migrate through agarose gel the gel is then transferred to a transilluminator to be visualized. Stains used to visualize DNA molecules are only visible under UV light. DNA molecules, depending on their size have a specific length measured in base pairs or (bp) which are visible on the gel. In the video is an image of a gel loaded with samples that were collected at different times of enrichment. The samples 2 & 5 show a band at 165 bp, indicating that amplification of the R. solanacearum occurred within these samples and thus the presence of the pathogen in the soil was confirmed.

Video: PCR testing (polymerase chain reaction)
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