Notes from the September 3, 1998, lecture:
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:
- 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.
- The material in my lectures, all of which will be posted
on the Web
- 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
- Here are some useful information sources about molecular and genetic
- 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
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
- World Wide Web sources
- This set of resources, which can be accessed through the URL:
- The BioNet newsgroups. Current and past postings can easily
be read at many Internet sites using a WWW browser, Gopher or news-reading
- 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:
- 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
- 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
- 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:
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
This newsgroup can provide answers to your questions regarding immunological
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
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
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
III. Restriction enzymes--permit the cutting of DNA molecules at precise
Click here to learn more about restriction
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
Click here to learn more about sequencing
Click here to learn more about PCR
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
An example of the use of these techniques to solve a particular experimental
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.
- 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.
- 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.
- Two strategies are available for detecting the correct cDNA clone:
- 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
- Antibody screening of plaques produced by members of the lambda gt11
cDNA library. Note that this would involve a variation of Western blotting.
- The positive clone would be amplified and sequenced to determine the amino acid sequence of
- 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.
- 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
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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