Questions on Genetics: Answers and Explanations

Running head: QUESTIONS ON GENETICS 1
Questions on Genetic
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QUESTIONS ON GENETIC 2
Questions on Genetics
a) Why is it easier to ligate sticky ends than blunt ends?
The sticky ends consist of regions of unpaired nucleotides called overhang which readily pairs
with another sticky end with a complementary overhang whereas the blunt ends have their
nucleotides already paired between the two strands of the DNA hence cannot be easily ligated
with other nucleotides (Pedersen et al., 2014).
b) How many bases in a restriction site sequence need to be methylated to protect that site from
restriction enzymes?
Only two, that is, Cytosine and Adenine of the four base pairs of the DNA can be methylated
therefore enough to repress restriction. The methylation of cytosine is common in both
eukaryotes and prokaryotes and differs among species while adenine methylation is common in
bacteria, plants, and mammals (Wu et al., 2016).
c) What does a DNA substrate require [or have to have] to be ligated?
A DNA substrate requires four deoxyribonucleotide triphosphates (dNTPs) which are; dATP,
dGTP, dTTP as well as dCTP which are cleaved forming deoxynucleotide monophosphates that
get inserted into a new DNA strand. Also, the substrate requires ribonucleoside triphosphates
(NTPs) which initiate and sustain the synthesis of DNA by synthesizing RNA primers and ATP.
The ATP provides energy for the activation of the enzymes at the replication fork.
d) What is the value of using phosphatase when preparing substrates for a ligation?

QUESTIONS ON GENETIC 3
The process of sealing the nicks between adjacent DNA residues of a single-stranded break on
the double strand substrate is done by the formation of phosphodiester bonds between 3’
hydroxyl and 5’ phosphate residues. Therefore, phosphates are important in the formation of the
insert and plasmid to avoid blunt end ligating into the vectors.
e) We do not use DNA ligase without ensuring we have first removed phosphatase from the
reaction. Why??
It’s essential to remove phosphates to effectively inactivate the 5’ phosphate group to prevent
self-ligation of the DNA using alkaline phosphatase enzyme for easy manipulation into the
desired DNA prior to re-ligation.
f) The specific example of TOPO cloning given in class involved an additional technique
called TA cloning where the T and the A refer to specific bases on the insert and/or vector. What
is TA cloning and how is it used here?
TA (thymine and adenine) cloning is a simple convenient procedure for subcloning the PCR
products through the amplification of Taq polymerase. In this type of cloning, both thymine and
adenine have the ability to hybridize on separate DNA fragments in the presence of ligase
enzymes. Therefore, they become ligated without the use of restriction enzymes.
g) Could TOPO cloning in theory or practice be done without using TA cloning?
TOPO cloning is feasible both in theory and practice without using the TA cloning method
which does not use restriction enzymes. However, some restriction enzymes such as Phusion can
be used to form blunt end TOPO vectors (Fontes, 2013).

QUESTIONS ON GENETIC 4
h) What is one advantage (OTHER THAN EFFICIENCY) of cloning PCR fragments via the
combined TA- and TOPO-cloning over the use of inserts isolated using restriction fragments?
In cases for the absence of viable restriction sites cloning is feasible through TA and TOPO
cloning which produces linearized vectors making the procedure simple and faster.
i) Imagine you were given a mixture of a single plasmid but some of that plasmid had been cut
in one spot by a restriction enzyme, some had been nicked in one strand, and some had not been
touched at all and so was supercoiled just like when it came from the bacterium. Would these
run differently on an agarose gel, or would some or all run at the same position? Explain your
answer.
The mixture can be run in a single gel electrophoresis at the same position. The DNA molecules
are separated by the pores on the gel electrophoresis in respect to their size and shape whereby
the smaller molecules navigate faster in the pores than the large ones. So, for the case of the
mixture plasmid the DNA molecules will separate with the following decreasing rates; the
supercoiled DNA, the linear cut DNA and finally the nicked circles.
j) Name two ways that a recombinant clone can be put into a bacterial cell from a ligation
reaction tube?
The recombinant clone can be put into a bacterial cell through the process of transformation and
electroporation.
k) What is meant by the term “vector” as it relates to this course?