BCH 226: BASIC MOLECULAR BIOLOGY (JIBRIL

LIMAN)

PROKARYOTIC AND EUKARYOTIC CHROMOSOMES
The genome of an organism encompasses all of the genes of that organism. Gene is a
distinct sequence of nucleotides forming part of a chromosome, the order of which
determines the order of monomers in a polypeptide or nucleic acid molecule. Thus a
protein-coding gene is defined as a region of DNA that encodes a single polypeptide or a
set of closely related polypeptides. Genes are contained in chromosomes. Chromosomes
are thus structures within cells that contain hundreds to thousands of genes.

PROKARYOTIC CHROMOSOMES

The DNA of a bacterial cell, such as Escherichia coli, is a circular double-stranded

molecule often referred to as the bacterial chromosome. The circular DNA is packaged
into a region of the cell called the nucleoid where it is organized into 50 or so loops or
domains that are bound to a central protein scaffold, attached to the cell membrane. The
DNA is negatively supercoiled, that is, it is twisted upon itself. It is complexed with
several DNA-binding proteins, the most common of which are proteins HU, HLP-1 and
H-NS. These are histone-like proteins.

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EUKARYOTIC CHROMOSOMES

The large amount of genomic DNA in a eukaryotic cell is tightly packaged in

chromosomes contained within a specialized organelle, the nucleus. With the exception
of the sex chromosomes, diploid eukaryotic organisms such as humans have two copies
of each chromosome, one inherited from the father and one from the mother.
Chromosomes contain both DNA and protein. Most of the protein on a weight basis is
histones, but there are also many thousands of other proteins found in far less abundance
and these are collectively called non- histone proteins (NHP). This nuclear DNA–
protein complex is called chromatin. In the nucleus, each chromosome contains a single
linear double-stranded DNA molecule. The length of the packaged DNA molecule varies.
In humans, the shortest DNA molecule in a chromosome is about 1.6 cm and the longest
is about 8.4 cm. The extensive packaging of DNA in chromosomes results from three
levels of folding involving nucleosomes, 30 nm filaments and radial loops.

NUCLEOSOMES

The first level of packaging involves the binding of the chromosomal DNA to histones.
Overall, in chromosomes, the ratio of DNA to histones on a weight basis is
approximately 1:1. There are five main types of histones called H1, H2A, H2B, H3 and
H4. Histones are very basic proteins; about 25% of their amino acids are lysine or
arginine so histones have a large number of positively charged amino acid side-chains.
These positively charged groups therefore bind to the negatively charged phosphate
groups of DNA

30NM FIBER

If nuclei are lysed very gently, the chromatin is seen to exist as a 30 nm diameter fiber.
The fiber is formed by a histone H1 molecule binding to the linker DNA of each
nucleosome at the point where it enters and leaves the nucleosome. The histone H1
molecules interact with each other, pulling the nucleosomes together.

RADIAL LOOPS

When chromosomes are depleted of histones, they are seen to have a central fibrous
‘protein scaffold’ (or nuclear matrix) to which the DNA is attached in loops. Therefore,
in vivo it seems likely that the next order of packaging involves the attachment of the 30
nm fiber to multiple locations on this central protein scaffold in a series of radial loops.
The mitochondria and chloroplasts of eukaryotic cells also contain DNA but, unlike the
nuclear DNA, this consists of double-stranded circular molecules resembling bacterial
chromosomes.

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 MORDERN MOLECULAR BIOLOGY STUDY AND
TECHNIQUES
Modern molecular biology is a term used for the study and applications of molecular
tools and techniques of the 21st century. Even some of the molecular studies of the late
20th century are till date still considered as modern because of their relevance to our
modern day science. Molecular biology can be traced as far back as the discovery of
Frederick Griffith in the early 1900s to a latter finding as that of Sanger in DNA
sequencing and a more recent one as next generation sequencing.
Some of the few applications of molecular biology can be seen as stated in the context
below
1. Gel electrophoresis
2. SDS Polyacrylamide gel electrophoresis (SDS-PAGE)
3. Western blotting
4. Southern blotting
5. Northern blotting
6. DNA cloning
7. Polymerase chain reaction (PCR)
8. Enzyme-linked immunosorbent assay (ELISA)
9. Sanger sequencing
10. Next generation sequencing (NGS)

GEL ELECTROPHORESIS
Gel electrophoresis is a technique used to separate DNA fragments (or other
macromolecules, such as RNA and proteins) based on their size and charge.
Electrophoresis involves running a current through a gel containing the molecules of
interest. Based on their size and charge, the molecules will travel through the gel in
different directions or at different speeds, allowing them to be separated from one
another.
All DNA molecules have the same amount of charge per mass. Because of this, gel
electrophoresis of DNA fragments separates them based on size only. Using
electrophoresis, we can see how many different DNA fragments are present in a sample
and how large they are relative to one another. We can also determine the absolute size of
a piece of DNA by examining it next to a standard "yardstick" made up of DNA
fragments of known sizes.

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POLYMERASE CHAIN REACTION (PCR)
PCR is a revolutionary method developed by Kary Mullis in the 1980s. PCR is based on
using the ability of DNA polymerase to synthesize new strand of DNA complementary to
the offered template strand. Because DNA polymerase can add a nucleotide only onto a
preexisting 3'-OH group, it needs a primer to which it can add the first nucleotide. This
requirement makes it possible to delineate a specific region of template sequence that the
researcher wants to amplify. At the end of the PCR reaction, the specific sequence will be
accumulated in billions of copies (amplicons).
Components of PCR
DNA template is the sample DNA that contains the target sequence. At the beginning of
the reaction, high temperature is applied to the original double-stranded DNA molecule
to separate the strands from each other.
DNA polymerase is a type of enzyme that synthesizes new strands of DNA
complementary to the target sequence. The first and most commonly used of these
enzymes is TaqDNA polymerase (from Thermis aquaticus), whereas PfuDNA
polymerase (from Pyrococcus furiosus) is used widely because of its higher fidelity when
copying DNA. Although these enzymes are subtly different, they both have two
capabilities that make them suitable for PCR: 1) they can generate new strands of DNA
using a DNA template and primers, and 2) they are heat resistant.
Primers are short pieces of single-stranded DNA that are complementary to the target
sequence. The polymerase begins synthesizing new DNA from the end of the primer.
Nucleotides (dNTPs or deoxynucleotide triphosphates) are single units of the bases A,
T, G, and C, which are essentially "building blocks" for new DNA strands.
RT-PCR (Reverse Transcription PCR) is PCR preceded with conversion of sample RNA
into cDNA with enzyme reverse transcriptase

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Limitations of PCR and RT-PCR
The PCR reaction starts to generate copies of the target sequence exponentially. Only
during the exponential phase of the PCR reaction is it possible to extrapolate back to
determine the starting quantity of the target sequence contained in the sample. Because of
inhibitors of the polymerase reaction found in the sample, reagent limitation,
accumulation of pyrophosphate molecules, and self-annealing of the accumulating
product, the PCR reaction eventually ceases to amplify target sequence at an exponential
rate and a "plateau effect" occurs, making the end point quantification of PCR products
unreliable. This is the attribute of PCR that makes Real-Time Quantitative RT-PCR so
necessary.
WESTERN BLOTTING
The western blot (sometimes called the protein immunoblot), or western blotting, is a
widely used analytical technique in molecular biology and immunogenetics to detect
specific proteins in a sample of tissue homogenate or extract.
Western blot technique uses three elements to achieve its task of separating a specific
protein from a complex: separation by size, transfer of protein to a solid support, and
marking target protein using a primary and secondary antibody to
visualize. A synthetic or animal-derived antibody (known as the primary antibody) is
created that recognizes and binds to a specific target protein. The electrophoresis
membrane is washed in a solution containing the primary antibody, before excess
antibody is washed off. A secondary antibody is added which recognizes and binds to the
primary antibody. The secondary antibody is visualized through various methods such
as staining, immunofluorescence, and radioactivity, allowing indirect detection of the
specific target protein.
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)
ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed
for detecting and quantifying soluble substances such as peptides, proteins, antibodies,
and hormones. Other names, such as enzyme immunoassay (EIA), are also used to
describe the same technology. In an ELISA, the antigen (target macromolecule) is
immobilized on a solid surface (microplate) and then complexed with an antibody that is
linked to a reporter enzyme. Detection is accomplished by measuring the activity of the
reporter enzyme via incubation with the appropriate substrate to produce a measurable
product. The most crucial element of an ELISA is a highly specific antibody-antigen
interaction.

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Direst ELISA: an antibody that has been directly conjugated to an enzyme detects an
antigen coated to a multi-well plate. This detection method is a good option if there are
no commercially available ELISA kits for your target protein.
Indirect ELISA: The antigen coated to a multi-well plate is detected in two stages or
layers. First an unlabeled primary antibody, which is specific for the antigen, is applied.
Next, an enzyme-labeled secondary antibody is bound to the first antibody.
Sandwich ELISA: These typically require the use of matched antibody pairs, where each
antibody is specific to a different, non-overlapping part (epitope) of the antigen molecule.
A first antibody (known as capture antibody) is coated to the wells.
Competitive ELISA: Competitive ELISA also known as inhibition ELISA is a
surface/plate based assay, where the plate is coated with capture antibodies reactive to the
molecule of interest.
DNA CLONING
DNA cloning is the process of making multiple, identical copies of a particular piece of
DNA. In a typical DNA cloning procedure, the gene or other DNA fragment of interest
(perhaps a gene for a medically important human protein) is first inserted into a circular
piece of DNA called a plasmid. The insertion is done using enzymes that “cut and paste”
called “restriction enzymes and DNA ligase respectively” DNA, and it produces a
molecule of recombinant DNA, or DNA assembled out of fragments from multiple
sources.
Next, the recombinant plasmid is introduced into bacteria. Bacteria carrying the plasmid
are selected and grown up. As they reproduce, they replicate the plasmid and pass it on to
their offspring, making copies of the DNA it contains.
What is the point of making many copies of a DNA sequence in a plasmid? In some
cases, we need lots of DNA copies to conduct experiments or build new plasmids. In
other cases, the piece of DNA encodes a useful protein, and the bacteria are used as
“factories” to make the protein. For instance, the human insulin gene is expressed in E.
coli bacteria to make insulin used by diabetics.

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Steps in DNA cloning
1. Cut open the plasmid and "paste" in the gene. This process relies on restriction
enzymes (which cut DNA) and DNA ligase (which joins DNA).
2. Insert the plasmid into bacteria. Use antibiotic selection to identify the bacteria
that took up the plasmid.
3. Grow up lots of plasmid-carrying bacteria and use them as "factories" to make the
protein. Harvest the protein from the bacteria and purify it.

OMICS
The word omics refers to a field of study in biological sciences that ends with -omics,
such as genomics, transcriptomics, proteomics, or metabolomics. The ending -ome is
used to address the objects of study of such fields, such as the genome, proteome,
transcriptome, or metabolome, respectively. More specifically genomics is the science
that studies the structure, function, evolution, and mapping of genomes and aims at
characterization and quantification of genes, which direct the production of proteins with
the assistance of enzymes and messenger molecules. Transcriptome is the set of all
messenger RNA molecules in one cell, tissue, or organism. It includes the amount or
concentration of each RNA molecule in addition to the molecular identities. The term
proteome refers to the sum of all the proteins in a cell, tissue, or organism. Proteomics is
the science that studies those proteins as related to their biochemical properties and
functional roles, and how their quantities, modifications, and structures change during
growth and in response to internal and external stimuli. The metabolome represents the
collection of all metabolites in a biological cell, tissue, organ, or organism, which are the
end products of cellular processes. Metabolomics is the science that studies all chemical
processes involving metabolites. More specifically, metabolomics is the study of
chemical fingerprints that specific cellular processes establish during their activity; it is
the study of all small-molecule metabolite profiles. Overall, the objective of omics
sciences is to identify, characterize, and quantify all biological molecules that are
involved in the structure, function, and dynamics of a cell, tissue, or organism.
Genomics is the new science that deals with the discovery and noting of all the sequences
in the entire genome of a particular organism. The genome can be defined as the
complete set of genes inside a cell. Genomics, is, therefore, the study of the genetic
make-up of organisms.
Determining the genomic sequence, however, is only the beginning of genomics. Once
this is done, the genomic sequence is used to study the function of the numerous genes
(functional genomics), to compare the genes in one organism with those of another
(comparative genomics), or to generate the 3-D structure of one or more proteins from
each protein family, thus offering clues to their function (structural genomics).

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GENOMICS
Genomics is an entry point for looking at the other ‘omics’ sciences. The information in
the genes of an organism, its genotype, is largely responsible for the final physical
makeup of the organism, referred to as the “phenotype”. However, the environment also
has some influence on the phenotype.
DNA in the genome is only one aspect of the complex mechanism that keeps an organism
running – so decoding the DNA is one step towards understanding the process. However,
by itself, it does not specify everything that happens within the organism.
The basic flow of genetic information in a cell is as follows. The DNA is transcribed or
copied into a form known as “RNA”. The complete set of RNA (also known as its
transcriptome) is subject to some editing (cutting and pasting) to become messenger-
RNA, which carries information to the ribosome, the protein factory of the cell, which
then translates the message into protein.
PROTEOMICS
Proteins are responsible for an endless number of tasks within the cell. The complete set
of proteins in a cell can be referred to as its proteome and the study of protein structure
and function and what every protein in the cell is doing is known as proteomics. The
proteome is highly dynamic and it changes from time to time in response to different
environmental stimuli. The goal of proteomics is to understand how the structure and
function of proteins allow them to do what they do, what they interact with, and how they
contribute to life processes.
An application of proteomics is known as protein “expression profiling” where proteins
are identified at a certain time in an organism as a result of the expression to a stimulus.
Proteomics can also be used to develop a protein-network map where interaction among
proteins can be determined for a particular living system.
Proteomics can also be applied to map protein modification to determine the difference
between a wild type and a genetically modified organism. It is also used to study protein-
protein interactions involved in plant defense reactions.
METABOLOMICS
Metabolomics is one of the newest ‘omics’ sciences. The metabolome refers to the
complete set of low molecular weight compounds in a sample. These compounds are the
substrates and by-products of enzymatic reactions and have a direct effect on the
phenotype of the cell. Thus, metabolomics aims at determining a sample’s profile of
these compounds at a specified time under specific environmental conditions.
Genomics and proteomics have provided extensive information regarding the genotype
but convey limited information about phenotype. Low molecular weight compounds are
the closest link to phenotype.
Metabolomics can be used to determine differences between the levels of thousands of
molecules between a healthy and diseased plant. The technology can also be used to

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determine the nutritional difference between traditional and genetically modified crops,
and in identifying plant defense metabolites.

Example of a metabolic network model for E. Coli

Genomics provides an overview of the complete set of genetic instructions provided by
the DNA, while transcriptomics looks into gene expression patterns. Proteomics studies
dynamic protein products and their interactions, while metabolomics is also an
intermediate step in understanding organism’s entire metabolism.
TRANSCRIPTOMICS
Meet another member of the 'omics family: transcriptomics – the study of all the RNA
molecules within a cell, otherwise known as the transcriptome. Many studies of the
transcriptome focus on messenger (m)RNA molecules only, which reflect the genes that
are being actively expressed (as protein products) in a cell or tissue at a given time or in a
given situation. However, over 95%of the RNAs in a cell are not translated into a protein,
so transcriptomics also includes the study of these non-coding RNAs, which have a
dizzying variety of forms and functions.
Understanding and categorising the whole transcriptome, including discovering the role
of many of the RNA molecules in the cell, is a daunting scientific and analytical
challenge.
Analysis of RNAs relies on expertise in next generation sequencing technologies, all of
which currently require RNA to be reverse transcribed (copied) into complementary
DNA (cDNA) for sequencing. Until recently, microarrays were the most common
technique in use in research laboratories. Microscopic spots containing DNA sequences
of interest are attached to a solid surface like a microscope slide. cDNA for analysis,
labeled with fluorescent markers, is washed over the slide; cDNAs attach to any
complementary strands on the slide. Higher levels of binding give a higher fluorescent
signal and indicate a higher level of gene activity. This technique is commonly used, for
example in panel assays that test for the presence of a known subset of gene transcripts.