BITC2441 Molecular Biology Techniques
Name __________________
Molecular Diagnostics by Buckingham & Flaws
Guided Lecture Notes
Chapter 1. Fundamentals of Nucleic Acid Biochemistry: An Overview
After reading the whole chapter, answer the following questions into your lecture notebook. Leave spaces for additional information
that might come up during lecture. This preview of the lecture and will allow you to minimize the time needed to enter lecture notes into
your notebook during the lecture. With your notes outlined ahead of time, you will be able to ask questions during lecture over
information that you need help with.
After answering all the questions, sign the question sheet and circle the questions that you want to have emphasized during lecture,
either because you are unsure of your answer or because you need clarification. Hand in this sheet with your circled questions at the
beginning of class. You will not turn in your lecture notebook; you will be given a completion grade for your work.
1. DNA analysis has proved to be valuable in the research and the clinical laboratory. What are two fundamental techniques that are
unique to DNA analysis that makes it such an interesting target molecule?
2. Describe the structure of a nucleotide: what are the three basic components that all nucleotides share?
a. Which nucleoside bases belong in the category of purines, and which are pyrimidines?
b. Why are sugar carbons numbered with a “prime”?
3. Describe the structure of the DNA double helix: what bonds link the nucleotides in a strand, and what interactions hold the two
strands together?
a. What are the base pairs that are found in the double helix?
b. By convention, what is the polarity of a DNA sequence as it appears left to right in print?
c. Why is the double helix referred to as being “antiparallel”?
d. What is a “complementary” sequence, and why will it hybridize with its complement?
4. What are some natural base modifications that are found in DNA, and how do they come about?
a. What are some consequences of these modifications in the cell?
5. PROKARYOTES: Describe the enzymatic steps of DNA replication and the changes that the double helix undergoes during this
process.
a. What are the enzymes named that participate in DNA replication in the replisome?
b. In what direction is the template strand read, and in what direction is the daughter strand synthesized?
c. Explain why Okazaki fragments are necessary during DNA replication.
d. Which daughter strand is the leading strand, and which is the lagging strand?
6. DNA polymerases play important roles in amplification, sequencing, and cloning of DNA in the molecular lab. What are some
catalytic properties of DNA polymerases that find special applications in the molecular lab?
a. Explain why DNA polymerase III has so little activity in vitro, while it is the main polymerizing enzyme during bacterial
replication.
b. Why might a pyrophosphatase activity be an advantage for in vitro replication?
c. Describe the editing (proofreading) function of DNA polymerases.
d. How is the Klenow fragment prepared from DNA pol I, and what might be an advantage of this product in the molecular lab?
e. What is the technique “nick translation” commonly used for in the molecular lab?
f. Describe the activity that DNA pol III enzyme has that allows it to perform a nick translation operation on DNA.
g. What is the enzyme “terminal transferase” commonly used for in the molecular lab?
7. Compare what we know about DNA replication in prokaryotes with what we know about this process in eukaryotes.
8. How are thymine dimers (pyrimidine dimers) formed in cells, and what are the consequences of such a mutation?
9. What is the difference between the activity of endonucleases, compared to exonucleases?
a. Restriction enzymes are site-specific endonucleases. Describe the differences between the DNA recognition sites of Type I,
Type II, and Type III restriction enzymes.
b. Type II restriction enzymes are used most frequently in the molecular lab. Describe the 3 ways that different Type II enzymes
cut the DNA strands.
c. What are some drawbacks and some advantages of using blunt ends for in vitro recombination reactions?
d. How can blunt ends be converted to sticky ends?
e. How can sticky ends be converted to blunt ends?
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10. Make a table to compare the activities of the following nucleases: Exonuclease I, Exonuclease III, Exonuclease VII, Nuclease
Bal31, mung bean nuclease, S1 nuclease, recBC nuclease, micrococcal nuclease, deoxyribonuclease I (DNAse I), DNA pol I
(exonuclease II). In one column, list the substrate requirements for each enzyme, and in other list the nature of the products of the
reaction and the types of applications each enzyme has in the molecular lab.
11. What are the two types of helicases, and how do their activities differ?
12. How does the role of methylation differ in prokaryotes compared to eukaryotes?
13. Describe the events by which genetic recombination naturally occurs during sexual reproduction.
a. How does recombination occur in asexually reproducing organisms and what are the conditions necessary for this to occur?
b. Why are transformations with closed circular plasmid DNA more efficient than linear DNA?
14. What effect does supercoiling have on the shape of a plasmid?
a. How can supercoiling be easily relaxed in vitro?
b. What effect does supercoiling have on the transformation efficiency of plasmid DNA?
c. How do large plasmids differ from small plasmids?
KEYWORDS
nucleotides
nucleosides
nitrogen base
adenine
cytosine
guanine
thymine
deoxyribose
phosphister bond
nuclein
pyimidines
purines
hydrogen bonds
complementary
mismatches
modified bases
nucleic acid
polarity
5’3’
supar-phosphate backbone
antiparallel
template strand
semiconservative replication
replication fork
Okazaki fragments
leading/lagging strand
DNA polymerase I (pol I)
DNA polymerase II (pol II)
DNA polymerase III (pol III)
replisome
helicase
primase
ligase
Rnase H
pyrophosphatase
Klenow fragment
exonuclease/endonucleases
mutations
proof reading/editing
nick/single-stranded break
nick translation
teminal transferase
thymine dimers (pyrimidine dimers)
repair enzymes
restriction enzymes (Type I-III)
palindromic/bilateral symmetry
stagger cut
overhangs/sticky ends
blunt end cut
Exonuclease I
Exonuclease III
Exonuclease VII
Nuclease Bal31
mung bean nuclease
S1 nuclease
recBC nuclease
micrococcal nuclease
deoxyribonuclease I (DNAse I)
DNA pol I (exonuclease II)
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topoisomerases
gyrases (type II topoisomerases)
methyltransferases
hemimethylated
5-methyl cytosine
recombination
recombinant DNA technology
meiosis
crossing over
conjugation
transduction
transformation
F+ /Fbacterial mating
fertility factor/F factor
Hfr bacteria
chromosomal integration
bacteriophages
telomeres
plasmids
episomal
resistance/ R factors
bacteriocins
large/small plasmids