Genetic engineeringggggggggggggggggggg.pdf

Biochemistry of genetic engineering

1. GENETIC
ENGINEERING
By
TaiwoT. Adeniyi
Medical Biochemistry Dept.
OAU, Ile-Ife

2. •Two types;
•DNA
•RNA
•The building
blocks of
nucleic
acids are
called
NUCLEOTIDES
Nucleic acids

3. DNA is used to store genetic information
◦It is replicated before cell division
DNA is very important so it is stored in the
nucleus.
It never leaves the nucleus
DNA stores the code for proteins, which exhibit
the “traits”
The DNA gets converted to RNA in order to
move out into the cytoplasm where it can
synthesize proteins
DNA Stores genetic information.
Maintains growth and repair.
Controls all cellular activities.
Contains protein codes.
Ensures each daughter cell & gamete receives
exact genetic information.

4. Chromosome
Packaged and organized chromatin, a complex of
macromolecules found in cells, consisting of DNA, protein
and RNA
DNA
A molecule that encodes the genetic instructions used in
the development and functioning of all known living
organisms and many viruses.
Gene
A segment of chromosome that contain information for
one heritable character in the organism. It is the unit of
heredity
Heredity
The study of heredity in biology is called genetics, which
includes the field of epigenetics

5. A set of techniques capable to allow the
identification, manipulation and multiplication of
genes of living organisms.
Changing of genes by using in-vitro processes
Other terms:
-gene manipulation, gene cloning, genetic
modification, Recombinant DNA technology
Objectives:
Basic research on gene structure and function
Production of useful products and services
Generation of transgenic plants and animals
Investigation of human genome for gene therapy

6. This is the technology of combining DNAs in vitro by cutting
up DNA molecules from more than one organisms and
splicing the fragments together to make a new DNA
molecule(rDNA)
This means: Modifying the genetic make up of an organism
by
a)adding new genes
b)changing the existing genes
Objectives:
-artificially synthesize new genes
-altering the genome of an organism
-create new gene combinations not found in nature
-understanding hereditary diseases and their cure
-Improving human genome

7. Genetic inheritance was first discovered by GregorMendel
in 1865 following experiments crossing peas. Although
largely ignored for 34 years he provided the first evidence
of hereditary, segregation and independent assortment
In 1889 Hugo de Vriescame up with the name "(pan)gene"
for after postulating that particles are responsible for
inheritance of characteristics
In 1928 Frederick Griffith proved the existence of a
"transforming principle" involved in inheritance, which
Avery, MacLeod and McCarty later (1944) identified as
DNA.
Edward LawrieTatum and George Wells Beadle developed
the central dogma that genescode for proteins in 1941.

8. The double helix structure of DNA was identified by James
Watson andFrancis Crick in 1953.
In 1970 Hamilton Smiths lab discovered restriction
enzymes that allowed DNA to be cut at specific places and
separated out on an electrophoresis gel. This enabled
scientists to isolate genes from an organism's genome
DNA ligases, that join broken DNA together, had been
discovered earlier in 1967 and by combining the two
enzymes it was possible to "cut and paste" DNA sequences
to create recombinant DNA.
Plasmids, discovered in 1972, becameimportant tools for
transferring information between cells and replicating DNA
sequences

9. Frederick Sanger developed a method for sequencing DNA
in 1977, greatly increasing the genetic information
available to researchers
Polymerase chain reaction (PCR), developed by KaryMullis
in 1983, allowed small sections of DNA to be amplified
and aided identification and isolation of genetic material.
In 1972 Paul Berg utilisedrestriction enzymes and DNA
ligasesto create the first recombinant DNA molecules
Herbert Boyer and Stanley N. Cohen took Bergs work a
step further and introduced recombinant DNA into an
bacterial cell

10. Foreign DNA/Synthetic DNA
Enzymes
Vehicle/Vector DNA
Host cells
Culture media, buffers, reagents

11. DNA: The Raw Material
Isolated from specific sources
Heat to denature DNA
DNA strands separate if heated to just below
boiling
Exposes nucleotides
When slowly cooled, strands will renature(anneal)

12. DNA or RNA polymerase: Replicating or annealing a
DNA chain
Reverse Transcriptase: Synthesize cDNAfrom RNA
template
DNA ligase: Joining DNA strands together
Nucleases: Breaks phosphodiesterbonds within free
ends (exonucleases) or in an interior
position(endonucleases)
Restriction Endonucleases: Recognisea specific base
sequence where they cut the DNA

13. oA special class of sequence-specific enzymes
oFound in bacteria, used for protecting its genetic
material from the invasive attacks of viruses
oSite specific: Cleave DNA molecules at specific
nucleotide sequences
oRecognize DNA base sequences(4-10 bp) that are
palindromes. (A palindrome is a word, phrase,
number or other sequence units that can read the
same way in either direction)
oMake two stranded cuts, one in each strand. May cut
to create
o -cohesive or sticky ends(overhanging ssends or
o -blunt ends(dsnon-overhanging ends)
oThey cut DNA to fragments of different length called
Restriction fragments

14. Exameples of RE
1. EcoRI: Escherichia coli strain R,1st enzyme
2. BamHI:Bacillus amyloliquefaciensstrain H,1st
enzyme
3. DpnI: Diplococcuspneumoniae, 1st enzyme
4. HindIII:Haemophilusinfluenzae, strain D, 3rd
enzyme
5. BglII:Bacillus globigii, 2nd enzyme
6. PstI :Providencia stuartii 164, 1st enzyme
7. Sau3AI :Staphylococcus aureusstrain 3A, 1st
enzyme
7. KpnIKlebsiellapneumoniae, 1st enzyme
Over 3000 restriction enzymes have been
studied in detail, and more than 600 of
these are available commercially

15. 'CTGATCTGACTGATGCTp -5
'GACTAGACTGACTACGA -3
Applications of R.Endonucleases
1-preparing a restriction map of DNA (useful in
RFLP analysis and DNA Finger printing)
2-fragmenting genomic DNA prior to southern
blotting
3-generating DNA fragments for cloning in vectors
4-generating DNA fragments for labelledprobes

17. The DNA that acts as a carrier is a vehicle or vector DNA e.g.
-Plasmid DNA, -BacteriophageDNA, -Cosmid
-Bacterial DNA(BAC), -Yeast DNA(YAC)
-Human DNA(HAC) -Expression vectors
Characteristics of Cloning Vectors
• Capable of carrying a significant piece of the
donor DNA
• Readily accepted by the cloning host
• Must have a promoter in front of the cloned gene
• Vectors (such as plasmids and bacteriophages) should have three
important attributes:
–An origin of replication somewhere on the vector
–Must accept DNA of the desired size, –Contain a gene that
confers drug resistance to their cloning host.

18. Plasmid
present in bacteria
A small, circular, dsDNA
Confer antibiotics resistance against the
bacteria
many copies of plasmid in a bacterium
replicate independent of the bacterial DNA
Bacteriophage
is a virus that can infect bacteria
e.g. λand M13 phages
Cosmid
plasmid + phage
Has Cos site from λ-phage
can carry larger DNA fragments
for binding to bacteriophages

19. Plasmid vector
PractcalFeatures of Cloning Vectors
-Origin of replication
-Multiple cloning sites
-Selectable marker genes
-RNA Polymerase Promoter sequences
-DNA sequencing primers

20. Prokaryotic hosts: Bacteria(e.gE.coli, Bacillus
sp., Pseudomonas sp., Streptomycessp.)
Eukaryotic hosts: Yeast(e.g. Saccharomyces)
Algae(e.g. Chlamydomonas) Fungi(e.g.
Aspergillus, Neurospora)
Note: 2 types of host-vectors
a) cloning vector-for propagation of DNA
inserts
b) Expression vector-for production of
proteins

21. Isolate desired DNA
Cut with a suitable R.E
Ligateinto suitable cloning vector (chimericDNA)
Transform rDNAinto a suitable host cell
Screening/selection: to select the cells that were
successfully transfectedwith the new DNA
Grow the successfully transfectedcells from which to
harvest the desired products
Examples: fine chemicals, hormones, enzymes,
vaccines, antibodies, antibiotics, blood factors etc.
Molecular cloning/DNA cloning refers to the process
of making multiple identical DNA molecules

22. Basics of rDNAtechnology

23. Gene Cloning

24. Transformationis the genetic alteration of a
cell(bacteria) resulting from the introduction,
uptake and expression of foreign DNA. This can be
by
Transfection: chemical or physical tricks to make a
cell take up DNA from the culture medium
Transduction: Virus mediated transfer
Direct Transfer: physically inserting the DNA e.g.
microinjection
Natural gene transfer: a receptor-mediated lateral
binding. Fusogenicproteins are used.

25. Calcium phosphate-co precipitate method-is a
transfectionmethod described by Graham and Van
derEb(1973)
Procedure:
-treat bacteria cells with ice-cold CaCl2.
-add plasmid DNA to cells chilled on ice which
forms Ca-PO4-DNA precipitate.
–Heat the mixture to 42oC.
–membrane becomes fluid and plasmid DNA enters
bacterial cells.

26. Selection is a process designed to facilitate the
identification of recombinant bacteria while
preventing the growth of non-transformed
bacteria
The bacterial are challenged with antibiotic (such
as ampicillin)
If the E.colihave taken up and expressed an
ampicillinresistant gene on a plasmid, they will
live,otherwisethey will die.
Those that survive ampicillincan be subcultured
on tetracyclinmedium to detect the plasmids
that take up the DNA(The DNA is inserted within
this gene)

27. DNA hybridization assay: The target DNA is denatured at
80oC and bound to a nitrocellulose filter discs. Such filters
are hybridized with radioactive DNA probes. The result are
monitored by autoradiography
Colony immunoassay(ELISA): The transformed colonies are
transferred to a nitrocellulose filter. The colonies are lysed
and the released proteins are attached to the matrix. The
matrix is treated with a primary antibody which specifically
binds to the proteins encoded by the target gene. The matrix
is then washed to remove any unbound antibody. The matrix
is treated with a second antibody attached to an enzyme(e.g.
alkaline phosphatase). A colorless substrate is then added
which is hydrolysedby the enzyme to give a colored complex
Other method is
-screening by protein activity

28. Cloning Libraries
R.E Mapping
Polymerase Chain Reaction(PCR)
Nucleic Acid Hybridization(Southern Blotting)
DNA Probes
Gel Electrophoresis
DNA sequencing

29. a)Genomic or Gene Libraries: Collection of cloned
DNA fragments from the genome or a
chromosome of a particular organism
contained within bacteria or viruses as the host.
Screening, identification and characterisationof
cloned fragments are possible with suitable
probes.
b) cDNALibraries: Contain only complementary
DNA molecules synthesized from mRNA in a cell.
mRNA from tissue of interest is isolated and
converted to ds-DNA using Reverse transcriptase
Use: i)for protein expression. ii)as probe for
analytical techniques

30. Synthesizing
of cDNA

31. Frequently it is important to have a restriction
enzyme site map of a cloned gene for further
manipulations of the gene.
Restriction Map: diagram showing a map of known
restriction sites within a sequence of DNA
Restriction mapping: a physical mapping technique
which is used to determine the relative location of
restriction sites on a DNA fragment to give a
restriction map.
A particular R.E generates a unique set of DNA
fragments, and another R.E will generate a different
set of DNA fragments from the same original DNA.
Steps: a) Preparation of DNA for restriction analysis.
b) Restriction digestion of DNA. c) separation of
restricted DNA on AGE. b) Collection of data. c)
Construction of restriction map

34. Procedure:
-Extraction of Human chromosome and PCR
-Digestion with one or more Res
-Electrophoresis of fragments
-Southern blot
-Visualization
-Application : molecular analysis of genes
involved in disease, DNA fingerprinting.

35. Developed in 1983 by KaryMullis. It’s a technique for
making copies or amplifying a specific sequence of DNA in
a short period of time
It is automated using a thermocycler. Repeatitiveprocess
consist of 3 steps(Denaturation, Annealing of Primrers,
Extension). Amount of DNA is doubled at the end of a
cycle.
Denaturation
Heat to 94°C to separate into two strands. Cool to
between 50°C and 65°C
Priming
Primers added in a concentration that favors binding to
the complementary strand of test DNA. Prepares the two
strands (amplicons) for synthesis
Extension
72°C. TaqDNA polymerase(mg2+) and nucleotides(4
dNTp) are added. Polymerases extend the molecule. The
amplified DNA can then be analyzed
Number of cycles: 20-45.

37. when insufficient DNA molecules arepresent in test
samples for DNA analytical techniques.
Diagnostic uses used to quickly detect microbial
infections, when the number of microbes is less in
the sample.
Prenatal diagnosis of genetic disorders Sections of
genes, having particular mutations known to cause a
disease are Amplified, Analysed, Diagnosis
Forensic Uses:
Samples used : Blood, saliva, semen, hair
Obtained from : a victim or suspect
Volume of the sample : is insufficient
Sample PCR Amplification of DNA
Amplified DNA is analysedby techniques. i.e., DNA
fingerprinting

38. A Southern blot allows the detection of a gene of interest
by probing DNA fragments that have been separated by
electrophoresis with a “labeled” probe.
Northern Blot (probe RNA on a gel with a DNA probe)
Western Blot (probe proteins on a gel with an antibody)
Southern Blot Technique Process : 6 steps
1) Extraction of DNA from the test sample/cells
2) Digestion by a suitable RE to DNA fragments
3) Electrophoresis of the digest toseparation fragments
4) Denaturationof DNA and blotting onto a membrane
(nitrocellulose membrane)
5) Adding a radiolabeledDNA probe
6) Autoradiography : Visualization on X-ray film . DNA
fragments hybridized with the DNA probes radiolabeled.
The pattern observed on Southern blot analysis depends
on : the specific RE used, location of the restriction site in
the DNA sample and the probe used.

39. Blood
stain
DNA
extracte
d
RE treated
DNA fragments
separated
by gel electrophoresis
DNA
denaturation
with alkali
Nitrocellulose
membrane
Transfer to
membrane
DNA Probes
(radiolabeled) to
the
membrane
Exposure to
X-ray film
DNA pattern

40. -Single stranded, fragments / pieces of DNA
-Contain nucleotide sequence
complimentary to the target sequence
-Radiolabeledwith radioisotopes (usually
32P) to visualize on an X-ray film
-Use : for detecting a target sequence e.g.in
Southern and Northern blot techniques
Examples for probes
-RNA
-Synthetic oligonucleotides
-Antibodies (protein) -as a probe for
protein molecule -in Western blot
technique.

42. Gel electrophoresis –DNA fragments of
different sizes can be separated by an
electrical field applied to a “gel”. The
negatively charged DNA migrates away from
the negative electrode and to the positive
electrode. The smaller the fragment the
faster it migrates. E.gagarosegel
electrophoresis

43. Technique used in determining nucleotides sequence of DNA
Methods of sequencing are developed by Maxamand Gilbert, and
Fredrick Sangersin 1970s. Sangersmethod is more commonly used
DNA chains are synthesized on a template strand(one to be
sequenced), on which chain growth stop when one of 4 possible
ddNTPsis incorporated
Population of truncated DNA molecules are produced that represent
each of the sites of that particular nucleotide in the template DNA
The synthesized fragments are separated by gel electrophoresis and
the sequence is read from bottom to top of the gel
This process has been fully computerised.
HUMAN GENOME PROJECT
Started in 1990 and completed in april2003
i. Sequencing of human genomes(about
20,000 to 25,000 protein-coding
genes)
ii. Mapping of human inherited diseases
iii. Development of new DNA
technologies
iv. Development of bio-informatics
v. ComparitiveGenomics
vi. Functional Genomics

44. 1. Basic research for understanding structure and functions
of DNA and proteins. E.g
i)Complete sequencing of the human genome (Human
Genome Project), Gene localizing and defining the map of the
human genome.
ii) Isolation and detailed molecular analysis of genes
involved in diseases (using RFLP analysis).
2. Diagnosis of diseases -genetic and microbial. E.g.
-Prenatal diagnosis of disease(sickle cell)
-Techniques used : PCR, Southern blot & RFLP
-Test sample : Amniotic fluid, chorionic villi
3. Forensic Uses:
-For identifying dead bodies, Settling parental disputes,
Identifying criminals.
-Samples used : Blood, saliva, semen, hair
-Obtained from : a victim or suspect. Volume of the sample
is usually insufficient
-P C R Amplification of DNA is carried out and used for
analytical techniques ( i.eDNA fingerprinting)

45. 4. Production of Proteins Using Recombinant DNA Technique
-Proteins, especially human proteins produced in large amounts
and are not antigenic when administered to humans
-Proteins produced are used for:
a. Replacement therapy and other treatments (e.g. insulin,
growth hormone, interleukins, antihemophilicfactors etc.)
b. Disease prevention(e.g. vaccines, such as hepatitis B antigen)
c. Diagnostic tests (e.g. monoclonal antibodies).
5. Treatment of genetic diseases :
Example : Gene therapy which Involves
-Introduction of normal foreign gene into somatic cells of the
patient having the genetic disease to compensate for the
defective protein which is the product of the mutant gene.
Genetic disorders treated by Gene therapy (attempt) :
-Severe Combined ImmunoDeficiency (SCID)Adenosine
deaminase(ADA) Deficiency
-Cystic Fibrosis Chloride channel
-Familial Hypercholesterolemia Receptor for LDL
-HemophiliaClotting factor (factor VIII or IX)

46. Agriculture:
Production of transgenic organisms: recombinant plants
and animals altered by addition of genes from other
organisms e.g.
Herbicide tolerance
(glyphosateresistance by a salmonella gene)
Pest Resistance
(gene for Bt toxin harmful only to insects)
Nutritional value improvement
(increased shelve life for tomatoes by suppressing
gene that breaks pectin, BGH gene enables meat
production in chicken for example, gene for β-carotene
inserted in rice to produce yellow vitamin A rice)
Ecological Application:
Recombinant Bacteria-bacteria which can be
engineered to “eat” oil spills.

48. Genomic
DNA
mRNA
OR
CLONING FROM