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?

• Most biological research these days depends on these techniques
• You will need to use most or all of them in your own research
• The rest of the course, RPN530, will present results obtained using these techniques. Thus you need to understand these techniques in order to understand the rest of the course.

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.

• For purposes of this course, you should have a good understanding of the following:
  1. The 9 different areas covered by self-help exams. To learn this material:
     • Take each test
     • If you find that you can't understand the test questions, then try to use the resources listed below to develop the necessary understanding
     • If you're still lost, contact me for help
     • If sufficient students need help with a particular subject, I'll arrange for some remedial classes
     • You are responsible for determining how much studying you need to do.
     • Don't wait until the last minute before the exam to discover that you need help. At that point, I won't be able to help you.
  2. The material in my lectures, all of which will be posted on the Web
  3. The material in Chapter 3 of the textbook
• Understanding the biochemistry of proteins and nucleic acids, at least at the depth of a one-year biochemistry course, is essential background for understanding molecular methods, and understanding the basic principles of genetic recombination and of haploid and diploid genetics is essential background for appreciating genetic methods. If you don't have this background, you should read the appropriate textbooks.
• You should attempt to acquire an excellent understanding of the principles of each technique. I can't possibly cover this in two lectures. I've attempted to provide some help with additional notes posted on the Web, and you should get additional help from the sources below.
• Here are some useful information sources about molecular and genetic techniques.
  • Your textbook. Chapter 3, "Methods of Genetic Analysis," (pp 26-47) provides a good overview of modern molecular and genetic methods. In particular, it provides a good description of several techniques (especially genetic techniques such as gene mapping) that I do not cover in my lectures or in my materials on the Web.
  • Technique manuals. These are your best sources for information about techniques. They provide detailed protocols for a variety of manipulations as well as help with the principles of the techniques. NOTE: sometimes the authors of these manuals make mistakes, both in their protocol descriptions and in their presentation of theory. Even I make mistakes (you are likely to find some in these notes). Therefore, think for yourselves! Note: you can find most of these methods manuals in the RPCI Medical Library.
    • Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989.
    • Current Protocols in Molecular Biology, ed. by F. M. Ausubel et al., John Wiley & Sons, Inc., 1987-present.
    • Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988.
    • Birren, Green, Klapholz, Myers and Roskams, Genome Analysis: A Laboratory Manual Series, Cold Spring Harbor Laboratory Press:
      • Volume 1: Analyzing DNA,1997
      • Volume 2: Detecting Genes, 1998
      • Volume 3, Cloning Systems, due November, 1998
      • Volume 4, Mapping Genomes, due December, 1998
    • Winfrey, Rott and Wortman, Unraveling DNA: Molecular Biology for the Laboratory, Prentice-Hall, 1997
    • Others
  • BioTechniques, Analyt. Biochem., METHODS: a Companion to Methods in Enzymology, and other methods journals. Note that a free subscription to BioTechniques can be obtained by filling out one of the reply cards that can be found in any issue of BioTechniques or by accessing the BioTechniques web site.
  • World Wide Web sources
    • This set of resources, which can be accessed through the URL:
      http://mcbio.med.buffalo.edu/RPN530/Molecular_Genetic_Methods.html
    • The BioNet newsgroups. Current and past postings can easily be read at many Internet sites using a WWW browser, Gopher or news-reading software.
      • Currently, the best ways to read and post messages to these newsgroups are by using an Internet browser and making a WWW connection directly to one of two sites:
        1. the BioNet server at the following URL: http://www.bio.net/. At this site you can access any of the BioNet newsgroups. At each newsgroup, you can read current and past messages, and you can post your own messages. I recommend that you read the messages for the current week or current month to get an idea of the type of message that is posted. You will see that the messages consist mainly of specific questions about particular aspects of particular techniques, plus answers to those questions from multiple individuals. It is interesting to read the many answers that come in. Frequently, the recommendations are quite different from each other. It is evident that some of the answers are probably incorrect. Once again, it is necessary to think for yourself. Despite the fact that some of the answers that come in are incorrect, most of the information is valid, and all of it is well-intentioned. Note that one can see many questions posted that do not seem to be answered. That's because in many cases the people who have the answers (who are simply other molecular biologists reading the newsgroup) answer by sending an e-mail message directly to the person who posted the question rather than posting the answer to the whole newsgroup. In other words, a much higher proportion of questions is usually answered than one would infer from looking just at the posted messages.
        2. The DejaNews server, which allows you to access not only the BioNet news groups but also all other Usenet messages. To increase focus, I recommend clicking on "Biology" on the first page (under "Science" in the "Channels" section). Then you'll have the option of searching only newsgroups devoted to biology.
      • Both DejaNews and the BioNet server permit one to search previous messages for specific topics or keywords of interest. DejaNews offers more powerful seach methods than does the BioNet server, but you must take time to read the instructions (see Help above and to the right of the search box) in order to take advantage of these powerful methods. Despite searching all of Usenet, the DejaNews server seems as rapid or more rapid than the BioNet server. In both cases, if the message that you find is part of a larger discussion (called a "thread"), links are provided for you to read all the other messages in the thread if you wish.
      • The BioNet newsgroups that are likely to be of most interest to you include the following:
        • Bionet.molbio.methds-reagnts. This is the most active of the BioNet news groups, and therefore perhaps the most useful. I and the members of my laboratory have found this newsgroup to be extremely helpful, especially when we have posted our own specific questions there.
        • Bionet.immunology. This newsgroup can provide answers to your questions regarding immunological methods.
        • Bionet.molbio.genome-program. Here is where your questions regarding the human genome program and human gene mapping can be answered.
    • Some additional internet sources (including sites with methods descriptions and protocols and sites for genome analysis) are listed at the end of the Molecular and Genetic Methods home page.
  • Catalogs and Web sites of equipment and reagent vendors
  • The Materials and Methods sections of research papers
  • Friends and colleagues
• Use multiple information sources to get a complete picture. Single sources frequently omit important considerations. However, multiple sources frequently propagate errors, so rely also on your own experience.
• Working in your own laboratory with these techniques will provide you with command of the details of the techniques relevant to your own needs and will aid you in appreciating the principles and details of these techniques as they are used by others. IT IS IMPORTANT TO GET AS MUCH HANDS-ON EXPERIENCE WITH THESE TECHNIQUES AS POSSIBLE.
• All of these techniques are evolving. None is optimal yet. Try experimenting to improve the techniques you need for your own work. Stay informed; keep in touch with the literature so as to become aware as soon as possible of improvements introduced by others.

Some important tips

• In all biochemical reactions, alterations in pH, temperature and ionic composition can lead to dramatic effects. Be careful in adjusting the pH of your buffers. If a reaction does not work as intended, check the pH and ionic composition. Similarly, be careful to control temperatures properly. A microfuge tube with its base in ice and its top in the air is not at 0°, or even at 4°; it is at a higher temperature.
• Work rapidly and carefully. Plan all the steps of your experiment ahead of time, and check them off as you perform them. Don't take breaks for meals, rest, etc., except at points where you know that your materials won't be harmed by waiting.
• Keep accurate notes of your experiments. Record all variables that might affect the outcome. Write your notes so carefully that someone completely unfamiliar with the experiment (possibly you, two years later) could understand what was going on.

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

IX. PCR
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!

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