FRET

G. Ronald Jenkins & Luke A. Shokere, Deana Namuth & Leah Sandall Author

03/15/2019 Added

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FRET is an acronym for Fluorescence Resonance Energy Transfer. It is a DNA detection tool.

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[00:00:00.160]This video clip shows an overview of setting up a real time PCR experiment
[00:00:05.476]using a FRET probe format.
[00:00:10.000]FRET stands for Fluorescence Resonance Energy transfer.
[00:00:16.280]Luke is working in a sterile environment within a laminar flow hood and wearing
[00:00:21.046]gloves to reduce the possibility of contaminating his samples.
[00:00:25.600]Previously, in other steps, DNA from his seed samples was extracted
[00:00:29.923]and quantified to a concentration of 50 nanograms per microliter.
[00:00:34.920]Now shown here, Luke is making serial dilutions of 10%,
[00:00:40.555]1%, .1% and .01% for his reference DNA.
[00:00:45.480]These will be used to produce a standard curve,
[00:00:48.173]which is needed for measuring the amount of GMO present in unknown seed samples.
[00:00:54.960]Next, he adds a buffer containing nucleotides
[00:00:58.268]and salt to Taq DNA polymerase at a 10X concentrate.
[00:01:02.800]Taq DNA polymerase is a heat stable enzyme that is a necessary component of
[00:01:08.153]the PCR process.
[00:01:10.120]It makes copies of DNA based upon a specific gene sequence,
[00:01:14.208]in this case identifying the presence of a GMO.
[00:01:19.040]Luke labels the tube to indicate that the buffer and enzyme are now mixed.
[00:01:24.560]Now he must prepare his master mix.
[00:01:30.600]The master mix contains aliquots from the 10X Taq DNA polymerase, nucleotide buffer,
[00:01:37.453]water, magnesium primers, and FRET probes.
[00:01:41.520]The primers and FRET probes he is adding are specific to the GMO he is testing for.
[00:01:58.080]Throughout the entire experiment, Luke must adjust the pipetter before
[00:02:02.698]adding a volume of each reaction component.
[00:02:06.640]He is also using fresh sterile tips at each step to prevent contamination.
[00:02:13.760]Here he is aliquoting the master mix into the individual capillary tubes.
[00:02:19.840]Notice that the capillary tubes are set in a metal cold block.
[00:02:24.120]This cold block is stored in a refrigerator prior to use.
[00:02:28.640]It keeps the reaction components at stable cool temperatures
[00:02:32.040]to prevent primer denaturation and minimize the reactivity of Taq DNA
[00:02:37.423]polymerase. 100 nanograms of DNA sample is added to
[00:02:41.422]their respective capillary tubes.
[00:02:44.840]Both positive and negative control reactions must be included during the
[00:02:49.080]experiment.
[00:02:50.720]Finally, he caps each tube.
[00:02:59.160]Keeping the samples in the cold block, Luke next takes them to the centrifuge.
[00:03:03.840]Notice that each capillary tube stays inside a cold metal canister.
[00:03:08.080]During the centrifugation step.
[00:03:11.320]He quickly spins down the solutions to the bottom of each capillary tube with
[00:03:15.640]minimal centrifugal force.
[00:03:21.160]The samples are carefully removed from the centrifuge and placed back into the
[00:03:25.794]cold block.
[00:03:31.480]This is one of the small capillary tubes he is using.
[00:03:34.920]It is made of clear borosilicate glass to allow highly efficient heat transfer to
[00:03:40.084]the samples.
[00:03:42.520]Now the tubes are ready to be put inside the light cycler.
[00:03:46.680]This type of Thermo cycler heats and cools the tubes efficiently at a rate of
[00:03:51.678]20° per second.
[00:03:53.400]This enables the PCR process to proceed quickly.
[00:03:57.200]The Light Cycler detects fluorescence using an LED fluorimeter,
[00:04:00.875]which measures fluorescence from individual samples.
[00:04:04.520]In essence, this instrument measures the amount of
[00:04:07.630]product present upon completion of each cycle in the reaction process.
[00:04:13.560]Finally, Luke utilizes a software program that
[00:04:17.004]allows him to set up reaction conditions and detect for a GMO in a real time
[00:04:22.647]format.
[00:04:24.080]After a short time, the reactions are complete.
[00:04:27.400]Luke can now analyze the data and determine the percent of GMO in his
[00:04:31.635]samples.

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FRET

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