Notes from the September 3, 1998, lecture:

Basic Methods

Some background

From 1993 to 1997, this material was presented to students in BIR571, "Regulatory Mechanisms of Eukaryotic Cells." At that time, there was only one lecture and the emphasis was on molecular techniques for analysis of nucleic acids and proteins.

As a result of RPCI's ongoing modernization of its educational program, the methods lecture from BIR571 has been incorporated into the new core course, RPN530, "Oncology for Scientists," and has been expanded to include discussion of methods relevant to the genetics of cancer. In addition, the time allotted for presentation of this material has been expanded to two lectures.

Thus I intend to provide you, the students in RPN530, with the same material I've provided in the recent past to students in BIR571 (updated as necessary) plus additional material on genetic techniques. In addition, I will describe in greater detail some recently introduced, powerful molecular and genetic techniques that have not yet made their way into methods manuals. My goal is to provide you not only with the information necessary to understand the rest of the material in this course but also with tools that will help you in the laboratory research you'll be doing as Masters and Ph.D. degree students.

Why bother to learn about molecular and genetic techniques?

How much do you need to know?

This is an excellent question. There's far too much known about molecular and genetic methods for any one individual to learn even in several lifetimes. Fortunately, what you need to know for this course is limited.

Some important tips

Some of the techniques you need to understand

The following information is an updated version of the material on molecular methods that I've taught in the past. In the future, I'll add sections on genetic methods, as needed. The additional information available by clicking in the list below is in abbreviated lecture note format. For more complete information, see the sources listed above or ask me.

Perhaps the best way for you to find out how much you need to know is to start by taking the self-help exams on each of these subjects. If you can easily answer all of the questions on any of these exams, with full understanding of why your answer is the correct answer, then you don't need any further study in the area covered by the exam. If you do need further study, then the lecture notes (accessible from the list immediately below) covering the area of the exam are a good place to start. I also recommend further reading in textbooks and methods manuals, and please feel free to ask me for additional help.

I. Hybridization/annealing--used to detect specific DNA or RNA sequences
Click here to learn more about hybridization/annealing

II. Gel electrophoresis of nucleic acids--separation of molecules of interest from "junk"
Click here to learn more about gel electrophoresis

III. Restriction enzymes--permit the cutting of DNA molecules at precise sites
Click here to learn more about restriction enzymes

IV. Other enzymes commonly used in molecular biology
Click here to learn more about other enzymes

V. Cloning vectors
Click here to learn more about cloning vectors

VI. DNA cloning--permits the amplification of DNA segments of interest
Click here to learn more about cloning

VII. cDNA cloning--permits the amplification of DNA representations of RNA molecules of interest
Click here to learn more about cDNA cloning

VIII. DNA sequencing--what scientists 20 years ago could only dream of
Click here to learn more about sequencing

Click here to learn more about PCR

In addition to the methods listed above, various modifications and accessory techniques are also important for this course. These include methods for purification of nucleic acids and proteins; culture and maintenance of bacteria; Southern, Northern and Western blotting, and in vitro mutagenesis. For more information on these topics, see the sources listed above or ask me.

An example of the use of these techniques to solve a particular experimental problem

Imagine that you wish to clone and characterize the human homolog of a yeast DNA ligase. You have available the complete amino acid and nucleotide sequences of this particular DNA ligase from two yeasts, Saccharomyces cerevisiae  and Schizosaccharomyces pombe. You also have antibodies raised against the S. cerevisiae DNA ligase, and you know that these antibodies cross-react with the S. pombe  enzyme. How would you proceed?

Usually there are many possible ways to proceed, and this case is no exception. However, given the materials available, the procedure described below seems reasonable. The links are to notes (see previous section) that provide more detail about particular methods.

  1. First, use the S. cerevisiae and S. pombe amino acid sequences to screen the available protein, EST, cDNA and DNA databases to determine if all or a portion of the human gene has already been cloned and sequenced by a different research group. If that approach is not successful, then proceed to the next steps.
  2. Prepare a human cDNA library in an expression vector, such as lambda gt11, that permits easy screening of expressed proteins with antibodies as well as permitting screening with hybridization probes. Note that, if lambda gt11 is used, then the cDNAs to be cloned must have Eco RI linkers at their ends, so that they can be cloned into the Eco RI site of lambda gt11.
  3. Two strategies are available for detecting the correct cDNA clone:

    1. PCR against the cDNA library (or against a total cDNA prep) employing degenerate primers derived from conserved (in S. pombe and S. cerevisiae) portions of the amino acid sequence may permit specific amplification of a segment of the correct cDNA. That segment could subsequently be used as a hybridization probe to distinguish the correct clone from the rest of the clones in the library. Note that gel electrophoresis would be needed to detect a positive signal during the PCR step.
    2. Antibody screening of plaques produced by members of the lambda gt11 cDNA library. Note that this would involve a variation of Western blotting.
  4. The positive clone would be amplified and sequenced to determine the amino acid sequence of the protein.
  5. The positive clone would be used as a hybridization probe to screen a human genomic DNA library. This would allow the genomic sequences flanking and between the coding sequences (i.e. introns plus 3' and 5' flanking sequences) to be obtained. These should include the sequences important for regulating expression of the DNA ligase gene.
  6. Site-directed mutagenesis (see your textbook) could then be used to create mutations at desired locations in the coding sequence. The mutant cDNAs could then be transcribed and translated in vitro (kits are available for this purpose). The resulting polypeptide chains could be assayed for DNA ligase activity employing an assay in which the ligation of the ends of a linear DNA molecule to produce a circular molecule is followed by gel electrophoresis. In this way, the importance for function of each amino acid in the protein could be tested.

Note that, in this example, a great deal of biologically interesting information is potentially obtainable using the molecular biological methods that we instructors consider important for this course. These methods are equally important in real-life laboratory work!

Return to the Molecular and Genetic Methods Home Page

Go to the page for the lecture of September 8, 1998