Department of Animal and Environmental Biology, University of Cross River State

Semester: 2nd Semester 2021/2022

Course code: BIO 4204

Course Title: Molecular Biology & Genetic Engineering

Biological macromolecules: protein, carbohydrates. The genetic code and protein synthesis;
Gene function in prokaryotes and eukaryotes; Recombinant DNA: restriction enzymes,
vectors, analysis of cloned gene sequence; Genetic engineering in animals: transgenic
animals. The human genome project. DNA probes and genetic disorders: examples.
Synthesis of human insulin. Prerequisite: AEB 2102.
Course Objectives: To expose students to the theories of Molecular Biology and Genetic
Engineering.
Policies for attendance, withdrawal, late assignments: Refer to Students’ Handbook
Reading Materials: Library Materials on subject area and Internet websites.

S/N TOPIC CRHR LECTURER

1 Introduction; Biological Macromolecules: 3 hr [Link]. Hannah Etta
Protein, Carbohydrate.
2 The Genetic code and Protein synthesis: 2hrs Jude Ogbeche

3 Gene function in prokaryotes and 3hrs Jude Ogbeche

eukaryotes
4 Recombinant DNA: restriction enzymes, 2hrs Dr. E. Ayim
vectors, analysis of cloned gene sequence

5 PRACTICAL 2hrs ALL LECTURERS

5 Genetic engineering in animals: transgenic 3hrs Dr. E. Ayim
animals.

6 The human genome project. 3hrs Dr. E. Ayim

7 DNA probes and genetic disorders 2hrs Dr. Mrs. Hannah Etta
Course Coordinator: Etta, Hannah Edim PhD

TOPIC 1: RECOMBINANT DNA

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Recombinant DNA, or rDNA, is the term used to describe the combination of two DNA
strands that are constructed artificially by the combination of two or more gene sequences.
Genetic scientists can do this to create unique DNA strand for different purposes, using
several types of techniques. In most cases, rDNA is created in a laboratory setting using a
process of molecular cloning. This method allows in vivo DNA replication, in the living cells
of the subject.

A cloning vector is a DNA molecule that replicates inside a living cell and is used to form
rDNA. The cloning vector is usually a small part of a DNA strand that holds the genetic
information that is needed for the replication of cells. Polymerase chain reaction (PCR) is
another method that can be used to replicate a specific DNA sequence and create rDNA,
which is used to replicate DNA in a laboratory test tube.

The standard method of making recombinant DNA involves:

 Choosing the appropriate host organism and cloning vector.

 Preparation of vector DNA and DNA to be cloned.
 Creation of recombinant DNA.
 Introduction of rDNA to host organism.
 Screening for rDNA with specific properties sought from host organisms.

 Restriction enzymes
A restriction enzyme is a protein produced by bacteria that cleaves DNA at specific sites
along the molecule. In the bacterial cell, restriction enzymes cleave foreign DNA, thus
eliminating infecting organisms. Restriction enzymes are one class of the broader
endonuclease group of enzymes. The use of restriction enzymes is critical to certain
laboratory methods, including recombinant DNA technology and genetic engineering. To be
able to sequence DNA, it is necessary to cut it into smaller fragments. Restriction enzymes
can be isolated from bacterial cells and used in the laboratory to manipulate fragments of
DNA, such as those that contain genes; for this reason they are indispensable tools
of recombinant DNA technology (genetic engineering).
 Vectors
In molecular cloning, a vector is any particle (e.g., plasmids, cosmids, Lambda phages) used
as a vehicle to artificially carry a foreign nucleic sequence – usually DNA – into another cell,
where it can be replicated and/or expressed. A vector containing foreign DNA is
termed recombinant DNA. The four major types of vectors are plasmids, viral
vectors, cosmids, and artificial chromosomes. Of these, the most commonly used vectors are
plasmids. Common to all engineered vectors are an origin of replication, a multicloning site,
and a selectable marker.
The vector itself generally carries a DNA sequence that consists of an insert (in this case
the transgene) and a larger sequence that serves as the "backbone" of the vector. The purpose

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of a vector which transfers genetic information to another cell is typically to isolate, multiply,
or express the insert in the target cell. All vectors may be used for cloning and are
therefore cloning vectors, but there are also vectors designed specially for cloning, while
others may be designed specifically for other purposes, such as transcription and protein
expression. Vectors designed specifically for the expression of the transgene in the target cell
are called expression vectors, and generally have a promoter sequence that drives expression
of the transgene. Simpler vectors called transcription vectors are only capable of being
transcribed but not translated: they can be replicated in a target cell but not expressed, unlike
expression vectors. Transcription vectors are used to amplify their insert.
The manipulation of DNA is normally conducted on E. coli vectors, which contain elements
necessary for their maintenance in E. coli. However, vectors may also have elements that
allow them to be maintained in another organism such as yeast, plant or mammalian cells,
and these vectors are called shuttle vectors. Such vectors have bacterial or viral elements
which may be transferred to the non-bacterial host organism, however other vectors termed
intragenic vectors have also been developed to avoid the transfer of any genetic material from
an alien species.
(a) Plasmid vector
Plasmids are double-stranded extra chromosomal and generally circular DNA sequences that
are capable of replication using the host cell's replication machinery. Plasmid vectors
minimalistically consist of an origin of replication that allows for semi-independent
replication of the plasmid in the host. Plasmids are found widely in many bacteria, for
example in Escherichia coli, but may also be found in a few eukaryotes, for example in yeast
such as Saccharomyces cerevisiae. Bacterial plasmids may be conjugative/transmissible and
non-conjugative.

(b) Viral vector

Viral vectors are genetically engineered viruses carrying modified viral DNA or RNA that
has been rendered noninfectious, but still contain viral promoters and also the transgene, thus
allowing for translation of the transgene through a viral promoter. However, because viral
vectors frequently are lacking infectious sequences, they require helper viruses or packaging
lines for large-scale transfection. Viral vectors are often designed for permanent
incorporation of the insert into the host genome, and thus leave distinct genetic markers in the
host genome after incorporating the transgene. For example, retroviruses leave a
characteristic retroviral integration pattern after insertion that is detectable and indicates that
the viral vector has incorporated into the host genome.
(c) Artificial chromosomes
Artificial chromosomes are manufactured chromosomes in the context of yeast artificial
chromosomes (YACs), bacterial artificial chromosomes (BACs), or human artificial
chromosomes (HACs). An artificial chromosome can carry a much larger DNA fragment
than other vectors. YACs and BACs can carry a DNA fragment up to 300,000 nucleotides
long. Three structural necessities of an artificial chromosome include an origin of replication,
a centromere, and telomeric end sequences.
Analysis of cloned gene sequence
Gene cloning is a common practice in molecular biology labs that is used by researchers to create
copies of a particular gene for downstream applications, such as sequencing, mutagenesis,
genotyping or heterologous expression of a protein. The traditional technique for gene cloning
involves the transfer of a DNA fragment of interest from one organism to a self-replicating genetic

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 element, such as a bacterial plasmid. This technique is commonly used today for isolating long or
unstudied genes and protein expression. A more recent technique is the use of polymerase chain
reaction (PCR) for amplifying a gene of interest. The advantage of using PCR over traditional gene
cloning, as described above, is the decreased time needed for generating a pure sample of the gene
of interest. However, gene isolation by PCR can only amplify genes with predetermined sequences.
For this reason, many unstudied genes require initial gene cloning and sequencing before PCR can
be performed for further analysis.

DNA Sequencing
DNA sequencing is typically the first step in understanding the genetic makeup of an organism,
which helps to:

 Locate regulatory and gene sequences

 Compare homologous genes across species
 Identify mutations

Sequencing uses biochemical methods to determine the order of nucleotide bases (adenine,
guanine, cytosine, and thymine) in a DNA oligonucleotide. Knowing the sequence of a particular
gene will assist in further analysis to understand the function of the gene. PCR is used to amplify
the gene of interest before sequencing can be performed. Many biotechnology companies offer
sequencing instruments, however, these instruments can be expensive. As a result, many
researchers usually perform PCR in-house and then send out their samples to sequencing labs.

Site-Directed Mutagenesis
Site-directed mutagenesis is a widely used procedure for the study of the structure and function of
proteins by modifying the encoding DNA. By using this method, mutations can be created at any
specific site in a gene whose wild-type sequence is already known. Many techniques are available
for performing site-directed mutagenesis. A classic method for introducing mutations, either single
base pairs or larger insertions, deletions, or substitutions into a DNA sequence, is the Kunkel
method.

Tutorial questions
1. What is recombinant DNA and state the standard methods of making
recombinant DNA???
2. Explain any two of the following; (i) restriction enzymes (ii) vectors
(iii) Analysis of cloned gene sequence, in recombinant DNA technology and
genetic engineering.
TOPIC 2: GENETIC ENGINEERING IN ANIMALS: TRANSGENIC ANIMALS.
A transgenic animal is one whose genome has been altered by the transfer of a gene or genes
from another species or breed.
Many initial problems, such as low efficiencies, random transgene insertions and unexpected
and undesirable behaviour of transgenic animals, have been overcome or are at least
understood in more detail. Furthermore, an ever-increasing knowledge of genes, gene
function and regulation of gene expression facilitates the planning and creation of transgenic
animals with desired traits.
The main interest of modern agricultural research with regard to transgenic animals can be
divided into two broad categories: (1) production of animals with improved intrinsic traits,
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such as higher growth rates, improved milk production, disease resistance etc., transgene has
been applied in cattle and pigs etc., to obtain improved milk products. It has also been applied
in rats for improved meat production.
(2) The production of animals that produce novel products, such as pharmaceuticals, proteins
of medical relevance, vaccines etc
 Experiments in cattle are focusing on the myostatin gene, a negative regulator of
muscle mass, resulting in a high increase in muscle mass in animals with a myostatin
mutation or deletion. Transgenesis is also employed for fish; injection of embryos
with constructs containing either the bovine or Chinook salmon growth hormone has
been reported, with the aim of improving fish growth in general and especially under
adverse conditions, e.g. low water temperatures. This has resulted in an up to 5-11
fold increase in weight after one year of growth for transgenic salmon and 30-40
percent increased growth of transgenic catfish .
All these studies demonstrate the fundamental feasibility of applying transgenesis to
agricultural animals for improved food production, but so far no transgenic food producing
animal has been released for commercial use.
 Transgenic animals for production of human therapeutics which is one major
application of animal transgenesis nowadays, also known as animal pharming. The
costs for producing transgenic animals are high, but since the pharmaceutical industry
is a billion-dollar market the input is likely to be a feasible and economically
worthwhile investment.
Pharmaceutical proteins or other compounds can be produced in a variety of body fluids,
including milk, urine, blood, saliva, chicken egg white and seminal fluid, depending on the
use of tissue-specific promoters diseases, compared with human-derived material.

Importance of transgenic animals

(1) Transgenic animals are routinely used in the laboratory as models in biomedical
research. Over 95 per cent of those used are genetically modified rodents,
predominantly mice. They are important tools for researching human disease, being
used to understand gene function in the context of disease susceptibility, progression
and to determine responses to a therapeutic intervention.
(2) Mice have also been genetically modified to naturally produce human antibodies for
use as therapeutics. Seven out of the eleven monoclonal antibody drugs approved by
the FDA between 2006 and 2011 were derived from transgenic mice.
(3) Transgenic farm animals are also being explored as a means to produce large
quantities of complex human proteins for the treatment of human disease.

Tutorial questions

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 1. How can the agricultural demands of an increasing world population be met
through genetic engineering?
2. Define transgenic animals.
3. State three importance of transgenic animals to the society.
TOPIC 3: THE HUMAN GENOME PROJECT
The Human Genome Project (HGP) was an international scientific research project with the
goal of determining the base pairs that make up human DNA, and of
identifying, mapping and sequencing all of the genes of the human genome from both a
physical and a functional standpoint. It started in 1990 and was completed in 2003. It remains
the world's largest collaborative biological project. Planning for the project started after it
was adopted in 1984 by the US government, and it officially launched in 1990. It was
declared complete on April 14, 2003, and included about 92% of the genome. The final
gapless assembly was finished in January 2022.
Funding came from the United States government through the National Institutes of
Health (NIH) as well as numerous other groups from around the world. A parallel project was
conducted outside the government by the Celera Corporation, or Celera Genomics, which
was formally launched in 1998. Most of the government-sponsored sequencing was
performed in twenty universities and research centres in the United States, the United
Kingdom, Japan, France, Germany, and China, working in the International Human Genome
Sequencing Consortium (IHGSC).
The Human Genome Project originally aimed to map the complete set
of nucleotides contained in a human haploid reference genome, of which there are more than
three billion. The "genome" of any given individual is unique; mapping the "human genome"
involved sequencing samples collected from a small number of individuals and then
assembling the sequenced fragments to get a complete sequence for each of 24 human
chromosomes (22 autosomes and 2 sex chromosomes). Therefore, the finished human
genome is a mosaic, not representing any one individual. Much of the project's utility comes
from the fact that the vast majority of the human genome is the same in all humans.

State of completion
Notably, the project was not able to sequence all of the DNA found in human cells; rather,
the aim was to sequence only euchromatic regions of the nuclear genome, which make up
92.1% of the human genome. The remaining 7.9% exists in
scattered heterochromatic regions such as those found in centromeres and telomeres. These
regions by their nature are generally more difficult to sequence and so were not included as
part of the project's original plans.
The Human Genome Project (HGP) was declared complete in April 2003. An initial rough
draft of the human genome was available in June 2000 and by February 2001 a working draft
had been completed and published followed by the final sequencing mapping of the human
genome on April 14, 2003. Although this was reported to cover 99% of the euchromatic
human genome with 99.99% accuracy, a major quality assessment of the human genome
sequence was published on May 27, 2004, indicating over 92% of sampling exceeded 99.99%
accuracy which was within the intended goal.

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In March 2009, the Genome Reference Consortium (GRC) released a more accurate version
of the human genome, but that still left more than 300 gaps, while 160 such gaps remained in
2015.
Though in May 2020, the GRC reported 79 "unresolved" gaps, accounting for as much as 5%
of the human genome, months later, the application of new long-range sequencing
techniques and a hydatidiform mole-derived cell line in which both copies of each
chromosome are identical led to the first telomere-to-telomere, truly complete sequence of a
human chromosome, the X chromosome. Similarly, an end-to-end complete sequence of
human autosomal chromosome 8 followed several months later.
In 2021, it was reported that the Telomere-to-Telomere (T2T) consortium had filled in all of
the gaps except five in repetitive regions of ribosomal DNA. Months later, those gaps had
also been closed. The full sequence did not contain the Y chromosome, which causes the
embryo to become male, being absent in the cell line that served as the source for the DNA
analyzed. About 0.3% of the full sequence proved difficult to check for quality, and thus
might have contained errors, which were being targeted for confirmation. In April 2022, the
complete non-Y chromosome sequence was formally published, providing a view of much of
the 8% of the genome left out by the HGP. In December, 2022, a preprint article claimed that
the sequencing of the remaining missing regions of Y chromosome had been performed, thus
completing the sequencing of all 24 human chromosomes.

Application of proposed benefits

The sequencing of the human genome holds benefits for many fields, from molecular
medicine to human evolution. The Human Genome Project, through its sequencing of the
DNA, can help researchers understand diseases including: genotyping of specific viruses to
direct appropriate treatment; identification of mutations linked to different forms of cancer;
the design of medication and more accurate prediction of their effects; advancement
in forensic applied sciences; biofuels and other energy applications; agriculture, animal
husbandry, bioprocessing; risk assessment; bioarcheology, anthropology and evolution.
Another proposed benefit is the commercial development of genomics research related to
DNA-based products, a multibillion-dollar industry.
Findings
Key findings of the draft (2001) and complete (2004) genome sequences include:

1. There are approximately 22,300 protein-coding genes in human beings, the same
range as in other mammals.
2. The human genome has significantly more segmental duplications (nearly identical,
repeated sections of DNA) than had been previously suspected.
3. At the time when the draft sequence was published, fewer than 7% of protein
families appeared to be vertebrate specific.
Tutorial questions

1. Discuss the human genome project.