07. Molecular Biology & Recombinant DNA Technology

Question 1

What are the steps of prokaryotic packaging?

Answer 1

The DNA molecule is initially coiled into loops

These loops then independently supercoil into domains identifiable in electron micrograph.

The supercoiled DNA can be relaxed by introducing
single strand nicks.

Question 2

What do heterochromatin contribute to?

Answer 2

Gene regulation,
epigenetic inheritance and protection from chromosomal integrity.

Question 3

What are the four levels of packaging of eukaryotic chromosomes?

Answer 3

nucleosomes
looped domains
mitotic chromosome
metaphase chromosome

Question 3

What are the four levels of packaging of eukaryotic chromosomes?

Answer 3

nucleosomes
looped domains
mitotic chromosome
metaphase chromosome

Question 4

Importance of DNA replication?

Answer 4

 The essential information for life is stored in DNA. Therefore, the new cells produced
must receive the DNA from their parent cells as, each cell in the body of a diploid organism
should contain the same genetic information as was in the zygote. A multicellular organism
grows by addition of new cells.
 Damaged or dead cells are also replaced by new cells.
 In asexual reproduction, the off spring is identical to the parent cell. This is possible only
because DNA has the ability to replicate and provide an identical set of genetic information
stored in DNA to each daughter cell by mitosis.
 In sexually reproducing organisms, meiosis occurs at some point of life cycle in order to
maintain a constant number of chromosomes. DNA replication occurs prior to meiosis.
 DNA replication is a very accurate process, so that it makes identical copies. However, rare
errors occur in DNA replication, introducing mutations which results in variation. Variation
leads to evolution of organisms.
 Therefore, DNA replication is important in maintenance of the life of an individual organism
and continuation of a species.

Question 5

Overall process of DNA replication?

Answer 5

Relaxation of tightly wound DNA
Unwinding/ unzipping of the double helix
Stabilization of single stranded DNA
Priming of DNA synthesis by a RNA primer
Extension of new DNA strand - Leading strand: Continuous
- Lagging strand – Discontinuous
Removal of RNA primer and replacing RNA (ribonucleotides) with DNA
(deoxyribonucleotides)
Sealing gap between the adjacent nucleotides

Question 6

Types of mutations are based on?

Answer 6

scale of alterations in the genetic material;
small-scale changes occurring in the sequence of nucleotides in a gene or large-scale changes in
the number or the structure of chromosomes.

Question 7

How does the gene technology begins?

Answer 7

Gene technology begins with isolation of the target DNA sequence from the total genome of the
donor cells

Question 8

Why is purified DNA required?

Answer 8

studying DNA structure and
chemistry,

examining DNA-protein interactions,

carrying out DNA hybridizations,

sequencing,
PCR, performing various genetic studies or gene cloning.

Question 9

Why can’t the entire length of the DNA molecule be isolated?

Answer 9

Since DNA molecules are very long, the entire length of a DNA molecule cannot be isolated, except for shorter DNA molecules such as plasmid DNA and viral DNA.

However, breaking of DNA or shearing has to be minimized during the extraction process.

Question 10

What are the basic principles of DNA isolation?

Answer 10

Homogenization or disruption of cells:
Inhibition of DNAse
Dissociation of nucleoprotein complexes:’
Removal of contaminating materials
Precipitation of DNA

Question 11

What involves in the homogenization or disruption of cells?

Answer 11

DNA is located in the nucleus of the eukaryotic
cells and it is concentrated in nucleoid in prokaryotic cells. The fi rst step in DNA isolation
is releasing the DNA from the cells by breaking cells or lysis of the cells.

Question 12

How can the cells be lyzed?

Answer 12

Cells can be
lysed by mechanical methods such as grinding and homogenization or enzymatically by
lysozyme to break bacterial cell wall.

Question 13

What happens during inhibition of DNase?

Answer 13

When the cells are broken, the DNA may get in touch with DNA
degrading enzymes such as Deoxyribonuclease (DNAse).

Therefore, DNA must be protected from such enzymes causing shearing. Chelating agents are added to remove metal ions required for nuclease activity.

Question 14

What happens during dissociation of nucleoprotein complex?

Answer 14

DNA needs to be freed from the proteins that
bind to DNA.

DNA-protein interactions are disrupted with SDS, phenol, or proteolytic
enzymes.

Question 15

What happens during Removal of contaminating materials?

Answer 15

All other molecules in a cell are contaminants of
DNA.

Removal of this contaminants is required for some applications.

Question 16

What happens during precipitation of DNA?

Answer 16

*Here the DNA dissolved in aqueous phase is precipitated with
cold (0 oC) ethanol.

*The precipitate is usually re-dissolved in a buffer.

• RNA is removed by limited treatment with DNAse free RNAase (ribonuclease).

Question 17

Why are enzymes of DNA required?

Answer 17

Enzymes are required in cutting, joining and copying of DNA in vitro

Question 18

Explain the function of restriction endonuclease enzyme?

Answer 18

There are several different types of nucleases in cells having
different functions. In gene technology, cutting DNA at precise locations is important.
The enzymes that recognize specific sequences of DNA and cuts at or near these sites are
called restriction endonucleases. The position where the DNA sequence is cut is called as restriction site or cleavage site (Figure 7.28).
eg. EcoRI- Source: E. coli

Question 18

Explain the function of restriction endonuclease enzyme?

Answer 18

There are several different types of nucleases in cells having
different functions. In gene technology, cutting DNA at precise locations is important.
The enzymes that recognize specific sequences of DNA and cuts at or near these sites are
called restriction endonucleases. The position where the DNA sequence is cut is called as restriction site or cleavage site (Figure 7.28).
eg. EcoRI- Source: E. coli

Question 19

Explain about DNA ligase?

Answer 19

Cut DNA fragments from different sources are joined together by forming a
phosphodiester bond by DNA ligase to obtain recombinant DNA molecules

T4 DNA ligase is the most commonly used DNA joining enzyme in gene technology.

The source of this enzyme is T4 bacteriophage.

Question 20

Explain about DNA polymerase?

Answer 20

These are enzymes that add complementary deoxyribonucleotides to
the template strand in a growing DNA strand, so that they copy DNA.

These are therefore
very important in gene technology especially in PCR and DNA sequencing.

The most
widely used DNA polymerase is Taq DNA polymerase. This is a heat stable enzyme isolated
originally from the thermophilic bacterium Thermus aquaticus.

Question 21

What is agarose gel electrophoresis?

Answer 21

Electrophoresis is a technique that separates large charged
molecules, (such as DNA, RNA and proteins) according to their mobility in an electric fi eld.

Question 22

What does the speed of a molecule in agarose gel electrophoresis depend on?

Answer 22

The speed of a molecule moving in an electric fi eld depends on its net charge and the size.

Question 23

In gel electrophoresis, what happens?

Answer 23

the molecules are moving through small pores of a matrix of gel. This restricts the movement of the molecules and helps in separation according to the size; larger molecules
move slowly compared to smaller molecules. In the case of nucleic acids, the net charge depends
on the length of the molecule, and therefore the separation is based on the size.

Question 24

why is gel electrophoresis used?

Answer 24

For the separation
of DNA, Agarose Gel Electrophoresis is the most frequently used technique.

Question 25

what is agarose?

Answer 25

Agarose is purified
agar, obtained from a type of seaweed.

This forms a polysaccharide matrix.

Question 26

explain the agarose gel electrophoresis process?

Answer 26

In the Agarose gel electrophoresis equipment, the gel is placed in buff er and a cathode and an anode are placed
on either end of the gel

Question 27

What happens when a current is supplied?

Answer 27

When a current is applied using a power supply, the
negatively charged DNA molecules migrate towards the anode through the gel

Question 28

When preparing the gel, what are formed?

Answer 28

When preparing
the gel, wells are formed, and the DNA is loaded into these wells.

Question 29

What is seperated DNA stained by?

Answer 29

stained by ethidium bromide and be visualized by exposing to UV light

Question 30

What does stains like ethidium bromide show?

Answer 30

Although stains like ethidium bromide show the presence of double stranded DNA band on an
agarose gel, those stains cannot diff erentiate a band with a specifi c nucleotide sequence from the
others

Question 31

To identify such bands among many bands, what are used?

Answer 31

DNA probes

Question 32

What is a DNA probe?

Answer 32

A DNA probe is a fragment of single stranded labeled DNA used to detect the presence of complementary nucleic acid sequences by hybridization.

Question 33

What is DNA labelling of a probe?

Answer 33

Labeling is a modification of the DNA strand in such a way that it gives a signal which enables the detection of that DNA strand.

Question 34

How can labelling be done?

Answer 34

incorporation of a radioactive
isotope, or addition of a fluorescent molecule to the structure of the probe.

Question 35

What can the single stranded DNA stretch do?

Answer 35

This single stranded DNA stretch can hybridize to complementary single strand of DNA or RNA.

Therefore before hybridizing with the probe, the double stranded DNA needs to be denatured to make room for the probe

Question 36

What should we do to the denatured bands on the gel?

Answer 36

The denatured bands on the gel need to be transferred to a nitrocellulose or nylon filter membrane by a process called Southern Blotting. The bands are then fixed to the membrane.

Question 37

What happens to the labelled probe added to the membrane?

Answer 37

Then the labeled probe is added to the membrane and allowed to renature. The probes will bind strongly only to the complementary sequences, which are fixed to the membrane

Question 38

What will the probes bind to?

Answer 38

only to the complementary sequences, which are fixed to the membrane.

Question 39

When the probe is washed, what is removed?

Answer 39

The probe is removed except the probes bound to the bands with target nucleotide sequence.

Question 40

If the probe is radioactively labelled, how can the band with the target sequence can be identified?

Answer 40

If the probe is radioactively labelled, the band with the target sequence can be identifi ed
by autoradiography of the membrane.

Question 41

If the probe is labelled with a florescent dye, how can the band be identified?

Answer 41

If the probe is labeled with a fl uorescent dye, the band can
be identifi d using UV light.

Question 42

What is the basis of recombinant DNA technology?

Answer 42

All organisms on Earth have evolved from a common
ancestor and their genetic information is stored in DNA,
except in some viruses.

At the chemical level, DNA is the same in all organisms. Further, all organisms share the same genetic code, so the same peptide is encoded by a gene whether it is expressed in a bacterium, a plant or an animal.

This forms the basis of recombinant DNA technology, where
DNA from two or more diff erent species are joined together
and inserted into a host to obtain a new genetic combination
which is of value to science, medicine, agriculture, industry
and environmental applications.

Question 43

What are the techniques used to make a rDNA molecule?

Answer 43

 Isolation of DNA from different sources.
 Restriction digestion of isolated DNA with restriction enzymes.
 Separation of DNA fragments by gel electrophoresis.
 Identification of the correct fragments with desired nucleotide sequences using probes.
 joining the DNA fragments from multiple sources using DNA ligase.

Question 44

Why was DNA cloning introduced?

Answer 44

Inserting DNA molecules to host cells is a difficult step. Cells show resistance to DNA uptake.

This is important for the survival of organisms because invading DNA can cause genetic alterations which are usually harmful.

Therefore, a large number of copies of the recombinant DNA molecules are necessary to make sure at least a few host cells will receive a copy.

If the desired DNA fragment is a shortone, in vitro
multiplication can be done by a technique called DNA cloning.

Question 45

What is DNA cloning?

Answer 45

In DNA cloning, the DNA replication machinery of a host cell is used to copy the desired DNA.

Question 46

What is the requirement for host cell to be copied in DNA cloning?

Answer 46

However, inserting a fragment of DNA into a host cell will not be copied, unless an origin of
replication (Ori) is present in that.

Question 47

To replicate recombinant DNA molecule, what should be there?

Answer 47

Therefore, in order to replicate the recombinant DNA molecule
or DNA of interest, it has to be combined with DNA with Ori and that can be replicated independent
of the chromosomal DNA (chromosomal DNA replicates only once in a cell division)

Question 48

Describe the nature of a bacterial host

Answer 48

In a bacterial host, there are several copies of plasmids and also when infected with a bacteriophage, a large number of copies of viral DNA per bacterial cell are present.

As such the DNA molecule of
interest can be integrated into these self-replication units, which are called vectors.

Question 49

Briefly describe about vectors

Answer 49

The vectors are vehicles to carry the DNA of interest into a host for multiplication or cloning.T

he vectors used in cloning of DNA are called cloning vectors.When the vector is carrying the foreign DNA, it is called a recombinant vector.

Question 50

How is a recombinant vector made?

Answer 50

Making a recombinant vector also follows the same procedure for making the recombinant DNA
molecule. Here the gene of interest has to be cut with a restriction enzyme and the vector (plasmid
or the viral DNA) should also be cut with the same enzyme. These two should be mixed and
allowed to integrate and ligated using DNA ligase (Figure 7.30). The site in the vector, where the
DNA to be cloned is inserted is a called cloning site. In order to be able to use several restriction
enzymes to cut DNA (both the vector and the DNA to be cloned), the cloning site contains a
sequence which carry sites for many restriction enzymes. Therefore, this is called multiple cloning
site. A host cell, usually a bacterial host which can copy the vector, is then transformed by the
recombinant vector. The host then will copy the plasmid carrying the DNA of interest. A number
of recombinant plasmid is present in each descendent cell of the colony of bacterial host.

Question 51

What is the function of restriction maps?

Answer 51

Restriction maps are very important in construction of cloning vectors: Cloning vectors are cut by
restriction enzymes at the cloning site to insert the fragments of DNA from other sources to the
cloning site.

Question 52

Advantages of STR markers in DNA fingerprinting?

Answer 52

• they occur frequently in genome
• easily amplifi ed by PCR
• highly variable polymorphisms and
• a large number of characterized STRs are available.

Question 53

How does DNA technology start?

Answer 53

DNA isolation, through identification of desired DNA sequences to gene technology or recombinant DNA technology.