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The revolution brought about by molecular biology depended heavily on nucleic acid hybridization procedures. These techniques are used extensively in the research laboratory for detecting specific nucleotide sequences in DNA and RNA and are increasingly being applied in medicine for diagnosing disease. Modern Biology Inc. is proud to introduce these methods into your teaching laboratory. In this laboratory program, students use hybridization procedures to investigate key topics in contemporary biology including the evolution of the vertebrate genome, the complexity of the viral chromosome, and the analysis of a specific nucleotide sequence in mammalian DNA. The package comes complete with all of the biochemicals that you will need although a water bath incubator that will maintain a temperature of 60-65°C is required for these experiments. The procedures used in these exercises are safe for your students because the four hybridization probes provided with the chemical package were prepared using a nonradioactive labeling system. 401. Evolution of the Vertebrate Genome The history of evolution is recorded in the genomes of present-day organisms and enlightened guesses of evolutionary events can be made by comparing DNA sequences in different species. Evidence drawn from this approach has led to the construction of family trees of organisms that agree remarkably well with those obtained from more traditional procedures. In fact, on a number of occasions, comparative DNA sequence analysis has been used to clarify and expand on phylogenetic relationships that were derived from classical studies. For example, although biologists have long disagreed about the taxonomic placement of the giant panda, recent studies using DNA hybridization techniques strongly suggest that this animal is more closely related to bears than to raccoons. These important concepts are illustrated in this exercise where students use a dot-blot hybridization procedure to compare DNA sequences in salmon, turkey, chicken and cow. In the analysis, single strands of chicken or cow DNA that have been previously linked to biotin are mixed with single strands of DNA from a second species. The two types of DNA are allowed to form double-stranded hybrid molecules and the extent of hybridization is measured using a simple color-producing enzyme reaction. This exercise requires approximately two 2-3 hour laboratory periods and electrophoresis equipment is not required. 402. Application of the Southern Blot Procedure In l975, Edward M. Southern at the University of Edinburgh, developed a powerful technique for DNA analysis which has become known as Southern blotting. Here your students use the Southern blotting procedure to identify the major control region for transcription and replication in the lambda phage genome. Following the step-by-step procedures in their manuals, students digest lambda DNA with a restriction endonuclease, electrophorese the DNA, and then transfer the separated fragments on the gel to a nylon membrane. The DNA fragment containing the control region is then identified by hybridization analysis using a biotinylated probe for the control region and the enzyme-color-producing assay. This exercise provides a wealth of practical information on one of the most powerful methods in molecular biology and illustrates an important strategy for mapping simple and complex genomes. This experiment requires about three 2-3 hour laboratory periods and typical results are shown below. 403. Detecting a Specific Sequence in the Mammalian Genome Southern hybridization analysis can now be used for tracing defective genes in human DNA including those involved in Huntington’s disease, Duchenne Muscular Dystrophy, and Cystic Fibrosis. This application is becoming increasingly important for diagnosing these diseases in members of afflicted families and it seems likely that the method will be used to detect additional abnormal and even normal human genetic traits in the near future. In this exercise students perform Southern hybridization analysis in order to characterize a specific sequence in mammalian DNA. Students digest cow, sheep, and chicken DNA with EcoRI, electrophorese the DNA, and transfer the separated fragments to a nylon membrane. The DNA fragments containing the cow satellite is then detected by hybridization using a biotin-labeled probe made from a plasmid that contains the cow satellite sequence. This exercise requires about three 2-3 hour laboratory periods. Agarose Gel Southern Blot
Detection of the Control Region in Lambda Phage DNA Left: Restriction nuclease digests of lambda DNA were electrophoresed on an agarose gel and the gel was stained. Right: The separated DNA fragments on the gel were transferred to a nylon membrane and the DNA containing the control region was identified by hybridization analysis.
Price List - Standard Laboratory Program 4
Individual Experiments Each of the individual experiments is supplied with the chemicals and laboratory guides needed for 16 students working in pairs. (Please click here for additional details.) If you chose one or more of the experiments below, you should also order Electrophoresis Package 3/4. Electrophoresis Package 3/4 provides sufficient agarose, gel stain and electrophoresis buffers for up to 6 of the individual experiments in this series. Note that Electrophoresis Package 3/4 is also suitable for the experiments in Standard Programs 3 and 10.
The following Experiments include Chemicals and Instruction Guides.
IND-16. Identifying Viral DNA by Rapid Southern Blotting A limitation to the Southern blotting procedure for the teaching laboratory is that it requires multiple laboratory sessions. The procedures have been streamlined for this experiment so that they can be carried out in a single 3-hour laboratory period. In the exercise, students electrophorese three unknown DNA samples on an agarose gel. One of the samples (instructor keyed) contains biotinylated DNA from the bacterial virus lambda. Following electrophoresis, the DNA in the gel is transferred to a nylon membrane during a 15-minute blotting step and the viral DNA on the blot is then detected by using an enzyme-color-producing reaction. The experiment was designed for 8 groups of students and sufficient materials are provided so that four blots can be prepared and analyzed. The exercise requires Electrophoresis Package 3/4 and a water bath maintained at 37° C.
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