Continuous Assessment

NAME- Debapriya Hazra; Roll No.-218

1.Name a useful and quick method for increasing the ligation efficiency of blunt-ended
DNA molecules.
 Blunt-ended DNA molecule's ligation can be up to less efficient than their sticky-end
counterparts because of the absence of the hydrogen bonding from sticky ends. This
absence means that the interactions between the vector and insert ends are fleeting.
So, to do a successful ligation via ligase relies on a transient association between 5’
phosphate and 3’hydroxyl groups. Here we come across several ways by which the
ligation efficiency of blunt-ended DNA molecules can increase.

o Concentration’s Increment in Case of Insert and Ligase - Increasing the

concentrations of the insert and ligase effectively increases the likelihood of collisions
between the vector and insert ends.
o Use Longer Incubation Times- Allowing the ligation reaction to occur over
a longer interval up to 24 hours increases the probability of two blunt ends bumping
into each other and joined by the ligase.
o Dephosphorylate the Vector- If both ends of the fragment to be ligated into a vector
are blunt-ended, then the vector needs to be dephosphorylated to minimize self-
ligation. Several enzymes are available for this step, including shrimp alkaline
phosphatase (SAP), calf intestinal phosphatase (CIP), or bovine alkaline phosphatase
(BAP).
o Use of a Peg-Rich Ligation Mixture- Polyethylene glycol (PEG) increases the
concentration in the sample by occupying space in the solution, ensuring that the
reagents have less space to move about, increasing the likelihood that blunt ends will
meet.
2. Yeast artificial chromosomes must have which three features of normal chromosomes
to be maintained in cells?
 Artificial chromosomes are DNA molecules assembled in vitro from defined
constituents, that can function like natural chromosomes. YACs (Yeast artificial
chromosomes) has some interesting features very similar to normal chromosomes,
that allow them to sustain in host cells. Here they are –
o The basic structure of a YAC resembles a telocentric chromosome. It contains a
functional telomere, a centromere, an origin of replication. Hence, YACs deal with all
the cis-acting elements required for chromosome replication and proper segregation
inside the yeast host.
o It has two arms just like normal chromosomes- One short arm and one long arm.
o It also contains some markers like any other chromosomes. YACs accommodate one
selectable marker trp1 (tryptophan biosynthesis) on the short arm and the other arm
contains a second selectable marker ura3 (biosynthesis of pyrimidine nucleotides)
o Yeast Artificial Chromosome has sequences to exist inside E. coli as a circular
plasmid and contains sequences, to maintain as linear nuclear chromosomes in yeast.

3. How do the primers determine the specificity of a PCR?

 Continuous Assessment
NAME- Debapriya Hazra; Roll No.-218

 Primers are short nucleotide sequences (approximately 15–30 bases) that base pair to
a specific portion of the DNA i.e. replicated. PCR primers are the main determinants
of PCR specificity.
 To amplify a specific DNA sequence, the sequence of the primer annealing
sites on the target DNA must be known.
 For hybridization to occur, the primer nucleotides must have a sequence that
is complementary to the 3′ end of each strand of the DNA target sequence,
and the 3′ ends of the hybridized primers should point toward one another.
 The sequences of the primers are very important for the polymerase chain
reaction because the reaction cycle has the specific temperatures used in the
heating and cooling stages.
 Besides, a great excess of the primers in the PCR reaction mixture causes
them more likely to encounter a partially complementary primer than a
perfectly complementary DNA template. So, primer complementarity has to
be avoided.

In the PCR method, a pair of primers is used to hybridize with the sample DNA
and define the region of the DNA that will be amplified and it is also determined
in-silico by various tools like PCR-blast.

4. What is the function of the 3` to 5` exonuclease activity of a DNA polymerase?

 Exonuclease activity is the ability of an enzyme to remove one nucleotide at a time

from the end of a polynucleotide chain. Exonuclease activity of DNA polymerase is
of two types based on the direction of DNA synthesis:3’ → 5’ exonuclease activity &
5’ → 3’ exonuclease activity.
 But here we discuss the 3’ → 5’ exonuclease activity of DNA polymerase.
This is a proofreading function of DNA polymerase. It proceeds in the reverse
direction of DNA synthesis. In the DNA elongation, if a mistake has been
made, the DNA polymerase enzyme moves backward, removes the wrong
nucleotide, and adds the correct nucleotide in the 3’ to 5’ direction.
5. This cloning vector possesses a bacterial origin of replication, a selectable marker
(antibiotic resistance gene), and cos sites from a bacteriophage. What type of cloning
vector is this, and what size insert molecule can it carry?

 It is an example of cosmid vector.

 It can carry up to ~40 kb base pairs as an insert.

6. What type of bond is synthesized by DNA ligase? (a) The hydrogen bonds between
bases (b) The phosphodiester bonds between nucleotides (c)The bonds between the
bases and deoxyribose sugars (d) The peptide bonds between amino acids

 (b) The phosphodiester bonds between nucleotides

7. What is the type of reaction that is shown in this figure? Label the steps in the process
and give the temperatures for each step.

 The below reaction showed here is Polymerase Chain Reaction.

 Continuous Assessment
NAME- Debapriya Hazra; Roll No.-218

 labelling steps and inputting temperature required for those are-

Steps Temperature

Initialization 94–96 °C

Denaturation 94–98 °C for 20-30 sec.

Annealing 50–65 °C for 20-40 sec.

Extension/elongation 72°C

8. A. Which of the following types of vector would be most suitable for introducing DNA
into a human cell? (a) Plasmid. (b) Bacteriophage (c) Cosmid. (d) Adenovirus.

 (d) Adenovirus.

8.B. PCR is advantageous to gene cloning for all of the following reasons except: (a)
PCR does not require that the sequence of the gene be known (b) PCR is a very rapid
technique for the isolation of a gene (c) PCR requires very small amounts of starting
DNA compared to gene cloning (d) PCR is very useful for mapping DNA markers

 (a)PCR does not require that the sequence of the gene be known.

9.A. Which of the following is NOT a reason why biochemical phenotypes were
commonly used to create human genetic maps? (a) Humans have no visual
characteristics that are useful for genetic mapping. (b) There are biochemical
phenotypes that are easily screened by blood typing. (c) Some easily characterized
biochemical phenotypes are specified by genes with very large numbers of alleles. (d)It
is unethical to perform controlled breeding experiments with humans.

 (a)Humans have no visual characteristics that are useful for genetic mapping.

9.B. Which of the following genetic markers are present in the highest numbers within
the human genome? (a) RFLP (b) Minisatellites (c) Microsatellites (d) Single nucleotide
polymorphisms.

 (d) Single nucleotide polymorphisms.

10. “Interphase chromosomes are useful for fine-scale mapping by fluorescent in situ
hybridization” – Justify with reasons. (Least condensed type of chromosome)
 Continuous Assessment
NAME- Debapriya Hazra; Roll No.-218

 The principle of the FISH methodology is based on the ability of single-stranded

DNA to anneal to complementary DNA. Interphase fluorescence in situ hybridization
(I-FISH) is a useful technique for detecting chromosomal numerical abnormalities in
tumors and is gaining acceptance as a tool in cytogenetics and clinical diagnoses.
There are certain reasons behind choosing the interphase chromosomes to be the ideal
for FISH mapping. This is because of some interesting features of this phase itself
like -

 The chromatin in interphase is less condensed than are metaphase chromosomes,

making the resolving power of fluorescence in situ hybridization (FISH) more
magnitude is two times higher in interphase nuclei than on metaphase chromosomes
for studies. Interphase chromosomes are most unpacked. Resolution down to 25 kb is
possible.
 In this phase the chromosomes are all separated from each other, making them
more easily visible and easy to study in comparison to other stages.
 In mammalian species, it has been demonstrated that within a certain range
the interphase distance between two FISH sites can be used to estimate the linear
DNA distance between the two probes.

11. How has PCR made the analysis of RFLPs much faster and easier? What was
required to map RFLPs prior to the utilization of PCR?

 RFLPs were the predominant form of DNA variation used for linkage analysis until
the advent of PCR. Even now, in the PCR age, RFLPs provide a convenient means for
turning an uncharacterized DNA clone into a reagent for the detection of a genetic
marker. The main advantage of RFLP analysis over PCR-based protocols is that no
prior sequence information, nor oligonucleotide synthesis, is required. Furthermore, in
some cases, it may not be feasible to develop a PCR protocol to detect a particular
form of allelic variation.
Although PCR detection of RFLPs is an improvement over Southern blot detection,
the real advantage of PCR lies within its nearly-universal ability to discriminate
alleles differing by single base changes even when they do not create or destroy any
known restriction site. Most random base pair changes will be of the non-RFLP type,
and before the advent of PCR, there was no efficient means by which these alleles
could be easily followed in large numbers of samples.

 A genome map provides a guide for the sequencing experiments by showing the
positions of genes and other distinctive features. Before the invention of PCR,
RFLPs were typed by Southern hybridization, which is time-consuming.
In this method, the DNA is digested with the appropriate restriction
enzyme and separated in an agarose gel and a smear of restriction fragments
is transferred to a nylon membrane and probed with a piece of DNA that spans the
polymorphic restriction site. With Southern blot analysis, the sensitivity at which
target sequences can be detected within a defined sample is directly proportional to
the length of the probe.
12. Genetic mapping techniques require at least two alleles for a given marker, while
physical mapping techniques do not rely on the presence of alleles to map genomes.
 Continuous Assessment
NAME- Debapriya Hazra; Roll No.-218

Discuss how the technique of fluorescent in situ hybridization can be used to map
genome locations even if there is no genetic variation present at a given position.

 Fluorescence in situ hybridization provides us a way to visualize and map the

genetic material in an individual's cells, including specific genes or portions of
genes. It can be used to map genome locations even if there is no genetic variation
present at a given position. In FISH, the first step is to prepare short sequences
of single-stranded DNA (Probe) that match a portion of the gene that we are
looking for. The next step is to label these probes by attaching fluorescent dye.
Since the probes are single-stranded, they can bind to the complementary strand of
DNA, wherever it may reside on chromosomes and when a probe binds to
chromosomes, its fluorescent tag provides a way for us to see its location. So,
even without no genetic variation present at a given position, we can map the
certain genome location we are looking for.
In this way, FISH uses a fluorescently labelled DNA fragment as a probe to bind to an intact
chromosome. The binding position can be determined and this information used to create a
physical map of the chromosome.

13. Why do plasmids contain genes for antibiotic resistance?

Presence of an antibiotic resistance gene give the bacteria following facilities-

 Antibiotic resistance gene is usually present in plasmid DNA. These genes code
for a protein that inactivates the effect of antibiotics by reacting with it. Thus, the
bacteria can grow even in presence of that particular antibiotic.
o It also plays a very important role as a marker gene in recombinant DNA technology.
When a foreign DNA is successfully inserted in a marker gene, that gene is no longer
expressed. This is used to identify organisms that have recombinant DNA or not.

14. How might you determine the positions of the restriction sites in a DNA molecule,
other than by working out the sequence of the molecule?

 It is possible to find out the positions of restriction sites without sequencing the
DNA molecule. Performing a restriction mapping may give us an idea of
restriction sites in a DNA molecule.
A restriction map is a map of all known restriction sites within a sequence of DNA
and it requires the use of several restriction enzymes. To find the relative positions of
restriction sites on a plasmid, a technique involving single and double restriction
digests is used, and based on the sizes of the resultant DNA fragments the positions of
the sites can be inferred. After the digestion, the data we get from several fragment
sizes of DNA molecules is very helpful to determine the restriction sites of the DNA
molecule. Restriction enzyme mapping is a powerful tool for the analysis of DNA.
15. E. coli cells take up plasmid DNA in laboratory experiments by which of the
following methods? a. Conjugation. b. Electrophoresis. c. Transduction. d.
Transformation.
 Continuous Assessment
NAME- Debapriya Hazra; Roll No.-218

 (d) Transformation.

16. How does a scientist prepare a clone library of DNA from just a single chromosome?

Cloning libraries are collections of DNA fragments that have been cloned into vectors so that
researchers can identify and isolate the DNA fragments that interest them for further study. A
scientist can break the genome or isolated chromosomes into fragments and clone each one in
a high-capacity vector that can handle large fragments of DNA and by this process, a clone
library is created.
A clone library can be prepared from a specific chromosome if the starting DNA comes from
just one type of chromosome. For that, we need to do –
Separation of individual chromosomes can be by flow cytometry.

 To carry out this technique, dividing cells (ones with condensed chromosomes) are
carefully broken open so that a mixture of intact chromosomes is obtained.
 The chromosomes are then stained with a fluorescent dye as their requirements.
 The chromosome preparation is diluted and passed through a fine aperture, producing
a stream of droplets, each one containing a single specific chromosome.
 The droplets pass through a detector that measures the amount of fluorescence and
hence identifies which droplets contain the particular chromosome.
 An electric charge is applied to these drops, and no others, enabling the droplets
containing the desired chromosome to be deflected and separated from the rest.

Digest the DNA with a restriction enzyme.

This creates fragments that are similar in size, each containing one or more genes.
Several mixtures of restriction enzymes can be used.

Insert the fragments of DNA into vectors.

For this, the same restriction enzyme is used which was used for cut. This will
ensure that every specific gene gets its vectors.

Ligation
Using the enzyme DNA ligase to seal the DNA fragments into the vector. This will
create a large pool of recombinant molecules.
Transformation
These recombinant molecules will be taken up by a host bacterium by transformation,
creating a DNA library.
17. What are the ideal features of a DNA marker that will be used to construct a genetic
map? To what extent can RFLPs, SSLPs, or SNPs be considered “ideal” DNA markers?
 Continuous Assessment
NAME- Debapriya Hazra; Roll No.-218

The Ideal Features of a DNA marker are presented below –

Polymorphism

Markers should exhibit high level of polymorphism. There should be variability in the
markers. It should demonstrate measurable differences in expression between trait types
and/or gene of interest.
Multi-Allelic

The marker should be multi-allelic. There must be at least 2 useful alleles.

Co-Dominant

Marker should be co-dominant. There should be absence of intra-locus interaction. It helps in

identification of heterozygotes from homozygotes.
Neutral

The marker should be neutral. The substitution of alleles at the marker locus should not alter
the phenotype of an individual. This property is found in almost all the DNA markers.
No Epistasis

There should be absence of epistasis. It makes Identification of all phenotypes easy.

No Effect of Environment

Markers should be insensitive to environment. This property is also found in almost all the
DNA markers.

 RFLPs, SSLPs and SNPs as DNA markers –

Restriction fragment length polymorphism (RFLPs) is a type of polymorphism that results

from variation in the DNA sequence recognized by restriction enzymes. They are used as
markers on genetic maps abundantly and gel electrophoresis is used to visualize RFLPs.
Simple Sequence Length Polymorphisms (SSLPs) are also used as genetic markers with
polymerase chain reaction (PCR). An SSLP is a type of polymorphism: a difference in DNA
sequence amongst individuals and these SSLPs are repeated sequences over varying base
lengths in intergenic regions of DNA. Unlike RFLPs, SSLPs can be multiallelic as each SSLP
can have several different length variants.
Single nucleotide polymorphisms (SNPs), which belong to the last-generation molecular
markers, occur at high frequencies in both animal and plant genomes. The development of
SNP markers allows to automatize and enhance the effectiveness of genotype analysis. These
markers are most abundant and most of them are biallelic.
 Continuous Assessment
NAME- Debapriya Hazra; Roll No.-218

From the above info's we can say that SSLPs should be the “ideal” markers, but in
reality, SNPs are more popular.