Note: the information on this page is old. I last taught this class in 1998. In 2003 I updated key contact information and some key links, but the scientific content remains vintage 1998. You are welcome to use the information on this page, but you should be aware that some of it may be outdated.
I am indebted to Prof. Thomas M. Terry at the University of Connecticut, whose article in the August, 1995, issue of ASM News on "Teaching Microbiology with the World Wide Web" inspired me to put this material on molecular methods onto the Web. ASM News is the newsletter of the American Society for Microbiology. I'm also indebted to Richard Roy and Tom Plunkett at the University of Connecticut who wrote software for creating self-correcting interactive exams, kindly permitted me to use the software, and provided initial help with its use.
Ideally, all students taking RPN530 should previously have been thoroughly trained in the necessary molecular biological methods. The instructors realize, however, that this may not always be true. This section of the course is designed to help those students who are unfamiliar with one or more of the methods needed for the course.
To avoid taking too much class time with material that may not be necessary for all students, only two lectures are devoted to presentation of information about molecular biological methods.
Supplementary material is also accessible from this World Wide Web page:
As you study the above material, you are very likely to have questions. I would be happy to answer them. You can get in touch with me using any of the methods listed at the beginning of this page, or you can simply send me an e-mail message now, directly from your WWW browser. When you send me an e-mail message, be sure to specify whether it is a private message (that you do not want put on our bulletin board) or a public one. If a public message is OK with you, then I will post your question, along with my answer, in the next section of this document.
In order to sequence DNA, you must first know a portion of the nucleotide sequence in order to create a primer, correct? How do you make that primer?
When one wants to obtain the nucleotide sequence of a stretch of DNA that is totally unknown, one subclones the stretch of DNA into a cloning site of a known vector. Thus DNA with known sequence (the vector) is placed adjacent to the DNA with unknown sequence. One can then use an oligonucleotide complementary to a portion of the vector sequence to prime DNA sequencing reactions that extend into the unknown sequence.
I did not understand the example you gave about the sequencing of that Yeast ligase. Could you perhaps diagram or illustrate a flow chart or illustration of some methods that you would utilize to isolate and identify DNA, RNA, protein. In other words can you help me understand the situation where you have protein X and you want to isolate gene X, or you have gene X and want to get protein X. How does that interrelate to DNA and RNA?
OK, let's see if this helps. If you have gene X and want to get protein X, the thing to do is determine the nucleotide sequence of gene X. From the nucleotide sequence you can (by the standard genetic code) deduce the amino acid sequence of protein X.
If you have protein X and want to get gene X, you first need to determine the amino acid sequence of protein X. Amino acid sequencing is not one of the topics covered in these lectures, but it's a fairly straightforward procedure, especially when you have an Edman degradation machine or a mass spectrometer. After you've obtained the amino acid sequence, then you attempt to create the nucleotide sequence of the gene. This is difficult, because the genetic code is ambiguous when reading backwards (several different triplets code for each single amino acid). Nevertheless, investigators find that if they program their oligonucleotide synthesizers to incorporate two, three or four different nucleotides at ambigous positions in the nucleotide sequence, they can create a mixture of oligonucleotides that contains a sufficiently close match to the correct gene so that the mixture can be used to detect the correct gene by hybridization.
Concerning question #8 in the PCR exam: Why is the given statement true?
The strategy to be employed when cloning PCR products depends in the following way on whether or not the polymerase used in PCR has a proofreading exonuclease. If the polymerase does NOT have a proofreading exonuclease, then many of the PCR product molecules will contain one or more incorrect nucleotides. Under these conditions, it is essential to determine the nucleotide sequence of each cloned PCR product to make sure that the clone contains the same sequence as the starting DNA. If it does not, then other cloned molecules from the same PCR reaction need to be tested until a correct clone is found. If a polymerase with a proofreading exonuclease is used for PCR, it is much more likely that each cloned PCR product will have the correct sequence, so sequencing each PCR product may not be essential. Even a polymerase with a proofreading exonuclease can make mistakes, however, so a careful scientist might argue that it's important to determine the nucleotide sequence of the PCR product even when the polymerase has a proofreading exonuclease.
In the Cloning Methods Exam question# 3: Why not oligonucleotide probe, because would not it be too small to probe a relatively larger human gene?
If an oligonucleotide probe has been thoroughly tested and found to detect just a single sequence in genomic DNA, it can be used to screen a genomic DNA library. In contrast, an antibody probe can never be used to screen a genomic DNA library. That's because antibodies detect proteins, not nucleic acids. One can use antibodies to detect particular cDNAs in cDNA libraries in the special case when the cDNA library has been cloned in an expression vector, and the expression vector is "induced," forcing each host cell to express whatever protein is encoded by the cDNA it contains.
Can you explain what the question is seeking for in cDNA Cloning Exam #2?
Any primer for nucleic acid synthesis requires a 3'-OH end, because new
nucleotides are added on to 3'-OH ends. Thus the 3' end of the newly synthesized
strand can loop back (forming a hairpin) and prime second strand synthesis,
but the 5' end cannot do this. Is this clear? Let me know if you need more
help with this problem.
Thanks to Richard Roy and Tom Plunkett at the University of Connecticut for the software that drives these exams. They have kindly provided it for our use here at RPCI.
Additional thanks are due to Prof. Tom Terry at the University of Connecticut, whose recent article inspired me. In addition, in the interests of saving time, I have used his instructions for interactive self-help exams with only minor modifications.
The following exams are intended to help you determine whether or not you really understand the methods you need to know for this course. The exams are in multiple choice and/or short answer format, because those formats are easiest for a computer to handle. The methods question(s) on the exam may or may not employ these formats! In previous years I have chosen to rely primarily on essay questions. You can see examples of exams from previous years in the next section.
There are currently 9 separate exams, each designed to cover one of the methods we (instructors) consider most important for this course. Additional exams on other methods are likely to appear in the future. Each of these exams is designed to stimulate you into recognizing aspects of these molecular methods that you are uncertain about. It is your responsibility to learn enough about each of these methods so that you understand each of the questions asked on these exams and why the answer I have listed as correct is, indeed, the correct answer. The numerous books on molecular methods are your best source of information. In addition, I invite you to come to my office, call, FAX, or e-mail. I will try to help you get onto the right track if you are having problems. But I can't work miracles. If you wait until the day before the exam to seek my help, there is probably not much I can do for you.
Note: the answers that I have suggested to the questions on these
exams are not necessarily entirely correct. I, too, can make mistakes. In
September, 1998, a first-year student in RPN530 pointed out to me that my
answer to question 7 on the PCR exam was incorrect, and he explained why.
I was delighted to learn from him about my error, and I revised the exam
so that the correct answer is now displayed. I hope that the rest of you
will also discover errors and will let me know about them.
When you take the exam, click the circle (radio button) corresponding to your choice for each answer (multiple choice questions), or click in and then type a short answer into the dialog box (short answer questions). You may answer as many or as few questions as you wish. When you are done, click the "Submit" button at the bottom of the exam. This will cause your exam to be "graded". You can then review your exam to see whether each answer choice was correct or incorrect, and view your percentage correct at the end of the exam.
Short answer questions will not be graded, but your instructor's answer will be shown next to yours, so you can see what is expected and judge how well you did.
These results are for your use only. No scores are kept. You can, and should, retake the exam several times, continuing to work on questions you got wrong, until you can answer all questions successfully. Be sure to think about why you answered incorrectly, and then try to make an intelligent choice among the remaining options before answering the exam again.
You will not gain as much benefit from printing out exams and studying them as you will from taking, and retaking them, electronically. I will not post answer keys--the only way for you to find out how you did is to take the exam electronically, then have your answers scored, and continue working on the questions that you get wrong. The purpose of these exams is for you to identify areas that require more study and to practice your exam-taking skills, not to try to "collect" questions. Although these particular questions will probably not be on the actual exam you take, your work in learning how to answer these questions will be very valuable to you.
If you have suggestions for additional exam areas,
or additional types of questions within the exam areas listed below, that
would be helpful to you, please let
me know.
The following questions are ones that I have asked in the past few years. Please note that for 1993-1997, these lectures were given in BIR571 (Cellular Regulatory Mechanisms), and their emphasis was on molecular methods. During those years, I did not cover genetic methods.
After you read each question, try to write down your answer. After you have an answer, then click below each question to see the type of answer to that question which I found acceptable.
From 1998:
1. (7 points) List 5 independent sources that can be used to learn about
molecular and genetic techniques.
Answer to question 1 from 1998
2. (7 points) This question involves compass directions. What is a Southern blot? A Northern blot? A Western blot? Describe using no more than a single sentence for each type of blot.
Answer to question 2 from 1998
3. (11 points) You have discovered a human single-nucleotide polymorphism that is useful in predicting susceptibility to toenail cancer. Individuals homozygous for the sequence ACGGTATCCTAGA are 5 times more likely to develop toenail cancer than individuals homozygous for the alternative sequence ACGGTAGCCTAGA. Heterozygotes display the same susceptibility as individuals homozygous for the G allele. To help screen for toenail cancer susceptibility, you have developed a "genotyping array", an array of 24 oligonucleotides on a glass chip with the pattern shown in the leftmost third of this diagram (the 24 squares over the label "G/G Homozygote"):
The upper 12 squares in this pattern contain a 4L tiled array of oligonucleotides based on the G allele, while the lower 12 squares contain a 4L tiled array based on the T allele. Please place an X in each square where strong hybridization to this genotyping array is expected for individuals with each of the 3 indicated genotypes: G/G homozygote, G/T heterozygote and T/T homozygote.
Answer to question 3 from 1998
From 1997:
1. Imagine that you have succeeded in obtaining both genomic and cDNA clones
of the human gene encoding psychic ability, PSI, and that you have developed
a simple in vitro assay for PSI protein function.
Answer to question 1 from 1997
2. During electrophoresis in an SDS-containing polyacrylamide gel, toward which electrode (positive or negative) do the following substances migrate?
Answer to question 2 from 1997
From 1996:
1. (10 points) The following questions are designed to test how well you
understand the principles of gel electrophoresis--a technique that is essential
for modern molecular biology. Please answer the first three questions with
a single word, and please answer the last question in just a few sentences.
Answer to question 1 from 1996
From 1995:
1. (10 points) Jennifer had a collection of 10 different cloned DNA fragments,
all in M13 phage vectors. She had lost track of which clone was which, but
she knew that one of them was a DNA fragment containing the IMPORTANT gene.
It had become important for her to determine which of her M13 clones contained
the IMPORTANT gene. Since she also had a copy of the IMPORTANT gene cloned
in a plasmid vector, between promoters for T3 and T7 RNA polymerases (with
each promoter oriented to permit transcription of the DNA fragment containing
the gene, but from opposite directions), she decided to use this plasmid
clone as a probe to detect the corresponding DNA fragment in her collection
of 10 M13 clones.
Jennifer took the following experimental approach. She prepared single-stranded DNA from phage representing each of her M13 clones, then subjected the DNA to agarose gel electrophoresis, using one lane for each clone. After blotting the gel to a membrane, she hybridized the membrane with a radioactive RNA probe created by transcribing her plasmid clone with T7 RNA polymerase in the presence of radioactive rNTPs.
After a long autoradiographic exposure of her membrane, Jennifer was surprised to find that none of the gel lanes displayed a radioactive band. She was very disappointed. In hopes of finding an explanation, she described what she had done to her friend Judy. Judy immediately suggested that Jennifer make one experimental modification. When Jennifer did what Judy suggested, she detected a strongly radioactive band in just one of her gel lanes, thereby identifying the IMPORTANT M13 clone.
What do you think was the modification that Judy suggested to Jennifer?
Answer to question 1 from 1995
From 1994:
1. (10 points) Imagine that you are working in the laboratory and that you
obtain unexpectedly complicated results. With a sinking feeling, you realize
that the only way to understand these results is to do a new experiment,
an experiment that will require cloning into a triple-stranded DNA vector
followed by application of the ligase chain reaction and concluding with
genetic transformation of Paramecia. All of these techniques are
totally unfamiliar to you. List 4 independent sources from which you could
obtain help with these unfamiliar techniques.
Answer to question 1 from 1994
2. (23 points) You have succeeded in identifying a gene encoding a protein
required to enjoy the taste of peanut butter (also known as Peanut Butter
Taste, or PBT). From a cosmid library, you have obtained a clone of a large
DNA fragment containing the gene, and now you wish to subclone the gene
into a plasmid vector to facilitate nucleotide sequence determination. By
restriction mapping, you have identified two unique restriction sites outside
of and close to the left end of the gene, EcoRI and PstI,
and two additional sites outside of and close to the right end of the gene,
HindIII and XbaI. You wish to clone the gene into the "multiple
cloning site" of your plasmid vector. This multiple cloning site contains
recognition sites for three restriction enzymes: BamHI, SalI,
and SmaI. The recognition and cleavage sites for these enzymes are
(only the top strand is shown; ' indicates the cleavage position): EcoRI=G'AATTC;
PstI=CTGCA'G; HindIII=A'AGCTT; XbaI=T 'CTAGA; BamHI=G'GATCC;
SalI=G'TCGAC; and SmaI=CCC'GGG.
Suggest a strategy for subcloning the PBT gene into the plasmid vector. Provide names for all enzymes required. If you need to modify the ends produced by one or more restriction enzymes, specify which enzyme(s) would be needed for the modification(s).
Answer to question 2 from 1994
From 1993:
1. (10 points) List 4 sources from which you could obtain help if you were
planning an experiment and needed assistance with a molecular biological
technique (the kind of technique discussed in class).
Answer to question 1 from 1993
2. (15 points) You have succeeded in identifying a gene encoding a protein
required for mental telepathy (also known as extra-sensory perception or
ESP). From a cosmid library, you have obtained a clone of a large DNA fragment
containing the gene, and now you wish to subclone the gene into a plasmid
vector to facilitate nucleotide sequence determination. By restriction mapping,
you have identified two unique restriction sites outside of and close to
the left end of the gene, BclI and EcoRV, and two additional
sites outside of and close to the right end of the gene, SacI and
SalI. You wish to clone the gene into the "multiple cloning
site" of your plasmid vector. This "multiple cloning site"
contains recognition sites for three restriction enzymes: XhoI, EcoRV,
and BamHI, in that order. The recognition and cleavage sites for
these enzymes are (only the top strand is shown; ' indicates the cleavage
position): BclI=T 'GATCA; EcoRV=GAT 'ATC; SacI=GAGCT
'C; SalI=G'TCGAC; BamHI=G'GATCC; XhoI=C'TCGAG.
Suggest a strategy for subcloning the ESP gene into the plasmid vector.
Provide names for all enzymes required. If you need to modify the ends produced
by one or more restriction enzymes, specify which enzyme(s) would be needed
for the modification(s).
Answer to question 2 from 1993
There are (August, 1998) numerous information sources relevant to molecular and genetic methods available on the Internet, and the number is growing extremely rapidly. Below I have listed just a few of the many sites with relevant information. Other sites can be found using the links available at the sites I have listed here.