The figure shown is an autoradiograph of a Southern hybridization experiment don
ID: 133394 • Letter: T
Question
The figure shown is an autoradiograph of a Southern hybridization experiment done by class. The sizes and mobilities of the molecular size markers are shown on the figure. The dark line at the top of the figure is the position of the wells. ("**Hint: The two fainter bands on the audiograph for the samples digested with EceRl are the result of incomplete digestion. That means that there are one or more EceRI restriction sites on those fragments that were not cleaved by the restriction enzyme U - Undigested yeast genomic DNA E-EcaRI digested genomic DNA H-Hind? digested genomic DNA U E H 23.1 kb 9.4 kb 6.6kb 4.4 kb 2.3kb 2.0kb 1. The autoradiograph clearly shows that the probe hybridizes to the genomic DNA that was not digested with a restriction enzyme (Lane U). Is this consistent with your expected results? If not, what is the most reasonable interpretation of the data? The autoradiograph clearly shows hybridization to two Hindll fragments. Is this consistent with your expected results? If not, what is the most reasonable interpretation of the data? 2. The autoradiograph also clearly shows hybridization to a single EcRI fragment? Is this consistent with your expected results? If not, what is the most reasonable interpretation of the data? 3.Explanation / Answer
Please find the answers below:
Answer 1: A hybridization assay is designed for combination of a probe and a target in the total pool of DNA. This can be possible only if the DNA contains a complementary site for the probe sequence. According to the information, the probe binds to uncut DNA and gives a band in the gel. This is ideally not possible since un-cut DNA would not present any potentially binding sites for the probe. This result suggests that the DNA is damaged at some point thus exposing its sequences for the probe.
Answer 2: Yes. According to the information, there are two potential sites for EcoRI enzyme and one for HindIII enzyme. Hence, a single digest with HindIII will present two bands of DNA, each on either side of the break.
Answer 3: No. According to the informaiton, the DNA contains two potential sites for EcoRI enzyme. Hence, at least 4 potential bands must be present in the gel. The presence of a single bands shows that the digestion sites are too close to each other in the DNA so that the resolution of the resulting bands is not prefectly visible as separate.