General Biology

BIOL201
Biotechnology
Dr Diana Hatoum

PhD in Cancer Research, UTS, Sydney

University Lecturer, College of Public Health and College of Arts and Sciences, PU
Principal Scientist in COVID-19 diagnosis, Department of Laboratory Medicine, RHUH
 Outline
• DNA Cloning
• DNA Sequencing
• Genomics
• Genetic Engineering
• Gene Therapy
 DNA Cloning
• Making identical copies of a piece of DNA
• A restriction enzyme cuts DNA wherever a specific
nucleotide sequence occurs
– For example, the enzyme EcoRI cuts DNA at the
nucleotide sequence G-A-A-T-T-C
• Other restriction enzymes cut at different sequences
• Making recombinant DNA – 1st step
• DNA cloning - a set of laboratory methods that uses living
cells to mass-produce specific DNA fragments
 Restriction
Enzymes and
Recombinant
DNA
Recombinant DNA - A
DNA molecule that
contains genetic
material from more
than one organism.
 Why Clone?
• An mRNA cannot be cut with restriction enzymes (RE) or pasted
with DNA ligase
– RE work only on double-stranded DNA - cloning mRNA requires
reverse transcriptase (RT)
• RT is made by some viruses
– RT uses mRNA as a template to make a strand of cDNA
– A cDNA contains all of the protein-building information encoded
in a eukaryotic mRNA but in a DNA form
– Vector - A DNA molecule that can accept foreign DNA and be
replicated inside a host cell
 Why Clone?
• cDNA is inserted into an appropriate vector and host → synthesis of an mRNA
→ synthesis of a protein product
• Advantages:
1. It allows researchers to manipulate eukaryotic genes
– for example by changing them, knocking them out, or inserting them into
other species
2. It allows genes from complex eukaryotes to be expressed in simpler organisms
such as bacteria and yeast
– This bypasses normal gene expression controls that would otherwise limit
the protein’s production in its eukaryotic cell of origin
– Gene expression is also much more easily adjusted in these single-celled
organisms than it is in complex eukaryotes - Particularly in bacteria
 Cloning
Making cDNA for cloning
 The Genome
• The entire set of genetic material—the genome—of
most organisms consists of thousands of genes
• Genomic and cDNA libraries are DNA libraries - sets of
cells that host various cloned DNA fragments
Polymerase Chain Reaction
(PCR)
Exponential Amplification by PCR
DNA Sequencing
The Human Genome
 Genomics
• The study of whole-genome structure and function
• Insights into evolution
– Studying the products of the human genome is even more
difficult than interpreting the genome itself
• Genome is constant but gene expression is not
• Proteomics - the study of proteins on a large scale → proteins
produced by an organism or a cellular system in it
• Bioinformatics - computational tools for analyzing and
interpreting biological data
 Gene Therapy
• Gene therapy is the transfer of DNA into an individual’s body cells - correct a genetic
disorder or treat a disease
– 15,000 serious genetic disorders
– Drugs and other treatments can minimize the symptoms of some genetic
disorders but gene therapy is the only cure
– Treatment for AIDS, muscular dystrophy, heart attack, sickle-cell anemia, cystic
fibrosis, hemophilia A, Parkinson’s disease, Alzheimer’s disease, inherited
diseases of the eye, the ear, and the immune system, and several types of cancer
 “Bubble Boy”
• SCID-X1, a severe X-linked genetic disorder - mutated
allele of the IL2RG gene
• ‘Bubble boy’ - Can survive only in germ-free isolation
tents
• Genetically engineered virus inserted unmutated
copies of IL2RG into cells taken from the bone marrow
of 20 boys with SCID-X1 then modified cells were
infused back into each boy’s bone marrow
• 18 of the boys left their isolation tents
• 5 boys later developed leukemia and died
• Gene was inserted into chromosomes at a site near a
proto-oncogene → Leukemia
 Genetic Testing Application
• Genetic testing revealed that
celebrity Angelina Jolie carries
a BRCA1 mutation associated
with an 87% lifetime risk of
developing breast cancer
• Even though she did not yet
have cancer, Jolie underwent
a double mastectomy, thereby
reducing her risk of breast
cancer to 5%