4. OUTLINE
rDNA
⢠rDNA technology
â˘Basic techniques
â˘Restriction
enzymes
â˘Vectors
â˘applications
Site directed
mutagenesis
⢠About random
mutagenesis
⢠Description of site
directed
mutagenesis
DNA sequencing
⢠Definition
⢠Sanger method
⢠Applications
⢠references
5. Genetic engineering & Biotechnology
â˘The modification of DNA of
an organism to produce new
genes with new characters
Genetic
Engineering
â˘Use of living organisms to
perform practical tasksBiotechnology
9. Carry genetic
information.
Components of DNA
are universal
(Nucleotides), only
the difference exists
in arrangement of
bases
rDNA is artificial
new DNA that can
be synthesized in
vivo or vitro through
biological
techniques e.g.
molecular cloning
DNA
rDNA
10. History of rDNA
Idea: Peter Lobban, Student of Biochemistry Department
at Stanford University Medical School in 1972 -1973
Insulin (First drug for human), developed by Eli Lilly and
Company 1980
Stanley N. Cohen and Herbert W. Boyer
11. Stanley N. Cohen (1935â) (top)
and Herbert Boyer (1936â)
(bottom), who constructed the
first recombinant DNA using
bacterial DNA and plasmids.
Stanley N. Cohen , who
received the Nobel Prize in
Medicine in 1986 for his
work on discoveries of
growth factors.
12. Creating recombinant DNA
Molecular cloning
⢠Molecular cloning is the laboratory process used to create
recombinant DNA molecular cloning involves replication of the
DNA within a living cell.
Polymerase chain reaction(PCR)
⢠PCR is the laboratory process used to create recombinant DNA
that replicates DNA in the test tube, free of living cells.
13. Recombinant DNA Technology
DEFINITION
Intentional modification of organismsâ
genomes for practical purposes
Three goals
Eliminate undesirable phenotypic traits
Combine beneficial traits of two or more organisms
Create organisms that synthesize products humans need
14. How is Recombinant DNA made?
There are three different methods by
which Recombinant DNA is made.
Transformation
Phage
Introduction
Non-Bacterial
Transformation
15.
16. Transformation(Steps)
*select a piece of DNA to be inserted into a
vector.
*cut that piece of DNA with a restriction enzyme
*ligate the DNA insert into the vector with DNA
Ligase
*The vector is inserted into a host cell
One example of a possible host cell is E. Coli.
17.
18. Phage Introduction
Phage introduction is the process equivalent
to transformation, except a phage is used
instead of bacteria.
E.g. lambda or MI3 phages to produce phage
plaques which contain recombinants.
19.
20. Non-Bacterial Transformation
very similar to Transformation
But does not use bacteria such as E. Coli for the host.
In microinjection, the DNA is injected directly into the nucleus of the
cell being transformed.
or the host cells are bombarded with high velocity micro projectiles,
such as particles of gold or tungsten that have been coated with DNA.
21.
22. Applications of rDNA technology
Better Crops (drought & heat resistance)
Recombinant Vaccines (Hepatitis B)
Prevention and cure of sickle cell anemia
Prevention and cure of cystic fibrosis
Production of clotting factors
Production of insulin
Production of recombinant pharmaceuticals
Plants that produce their own insecticides
Germ line and somatic gene therapy
23. Insect-resistant tomato plants
The plant on the left contains a gene that encodes a
bacterial protein that is toxic to certain insects that
feed on tomato plants. The plant on the right is a
wild-type plant. Only the plant on the left is able to
grow when exposed to the insects.
26. A transgenic
mouse
Mouse on right is
normal; mouse on
left is transgenic
animal expressing
rat growth hormone
27. Cloned gene
Retrovirus
capsid
Bone
marrow
cell from
patient
Inject engineered
cells into patient.
Insert RNA version of normal allele
into retrovirus.
Viral RNA
Let retrovirus infect bone marrow cells
that have been removed from the
patient and cultured.
Viral DNA carrying the normal
allele inserts into chromosome.
Bone
marrow
Somatic cells
Only!
Not for
reproductive
cells !!
30. Tools of Recombinant DNA Technology
â˘Bacterial enzymes that cut DNA
molecules only at restriction
sites
RESTRICTION
ENZYMES
â˘Two groups based on type of cut
â˘Cuts with sticky ends
â˘Cuts with blunt ends
31.
32. The Tools of Recombinant DNA Technology
⢠Nucleic acid molecules that deliver a gene into
a cell
⢠Include viral genomes, transposons, and
plasmids
Vectors
⢠Small enough to manipulate in a lab
⢠Survive inside cells
⢠Contain recognizable genetic marker
⢠Ensure genetic expression of geneare
4 different type of vectors:
Plasmid vectors
Lamda (Îť) phage vectors
Cosmids
Expression vectors
33.
34.
35. The Tools of Recombinant DNA Technology
⢠A collection of bacterial or phage
clones
⢠Each clone in library often contains
one gene of an organismâs genome
Gene
Libraries
⢠Library may contain all genes of a
single chromosome
⢠Library may contain set of cDNA
complementary to mRNA
Cont âŚ.
36.
37. Multiplying DNA in vitro:
⢠Large number of identical
molecules of DNA produced in vitro
⢠Critical to amplify DNA in variety
of situations in vivo
The Polymerase
Chain Reaction
(PCR)
⢠Epidemiologists use to amplify
genome of unknown pathogen
Cont âŚ.
38. Multiplying DNA in vitro:
⢠Separates molecules based on electrical
charge, size, and shape
⢠Allows scientists to isolate DNA of interest
Gel
Electrophoresis
and the Southern
Blot
⢠Negatively charged DNA drawn toward
positive electrode
⢠Smaller fragments migrate faster than
larger ones
Cont âŚ.
39.
40.
41. What Is a Mutation?
Genetic information is encoded by the
sequence of the nucleotide bases in
DNA of the gene. The four nucleotides
are: adenine (A), thymine (T), guanine
(G), and cytosine (C), a mutation is a
change in the order of these
nucleotides.
A change in the order can cause the
gene to encode for wrong proteins and
inhibit the function of the gene or cause
the gene to be virtually inactive.
42. Random mutagenesis
⢠based on process of natural evolution
⢠NO structural information required
⢠NO understanding of the mechanism
required
Random
Mutagenesis
â˘Generation of genetic diversity
⢠Screening and natural
selection
Cont âŚ..
43. Site-directed Mutagenesis
Or
Site Directed Mutagenesis
is a powerful technique
where site specific
changes in DNA sequence
are produced in vitro-for
instance to change an
amino acid residue into
another by changing the
codon sequence within
the gene sequence
Site Directed Mutagenesis is
a molecular biology
technique in which a
mutation is created at a
defined site in a DNA
molecule known as a
plasmid. Wild-type gene
sequence must be known.
44. CAG
GTC
CAG
+ primer
CAG
primer GCC
+ polymerase
CAG
GCC
replication
CAG
GTC
Wild type
translation
Val
Mutant
Wild type protein
CGG
GCC
translation
Thr
Mutant protein
Only one amino acid changed
Val â Thr
(1)
(2)
(3)
(4)
(5)
(6)
Smith (1993)
45. Site directed Vs Random mutagenesis
-> site-directed mutagenesis
-> point mutations in particular known area
-> random mutagenesis
-> point mutations in all areas within DNA of interest
46. INVENTION
Site Directed Mutagenesis using oligonucleotide
was first described in 1978 by Michael Smith &
shared Nobel Prize in chemistry in October 1993
with Kary B. Mullis who developed the PCR
technique.
ď Site âdirected mutagenesis
Requirements:
-> Knowledge of sequence and preferable Structure
(active site,âŚ.)
-> Understanding of mechanism
(knowledge about structure â function relationship)
47. Site Directed Mutagenesis
Cassette mutagenesis
Oligonucleotide directed mutagenesis
Using M13 DNA
Using Plasmid DNA
3. PCR amplified Oligonucleotide directed mutagenesis
4. Random mutagenesis
With Degenerate Oligonucleotide primers or
Using Nucleotide Analogues
48. CASSETTE MUTAGENESIS
ďśCleavage by a Restriction
Enzyme (RE) at a particular site
in the plasmid.
ďśLigation of an Oligonucleotide
containing Mutation in the gene
of interest to the plasmid.
ďRE that cuts at the plasmid
and Oligonucleotide is same,
permitting sticky ends of the
plasmid & inserts to ligate to
one another.
49. Site-directed mutagenesis â
Oligonucleotide - directed method
ďśSynthetic single-stranded fragments of DNA used for the
mutated clones.
ďśIn order to work, the primers must meet the following
criteria:
ďś-must contain desired mutation.
ďś-mutation should be in the middle of the primer.
ďś-the GC content should be at a minimum of 40% and should
terminate in one or more of C or G bases.
Oligonucleotides areâŚ
53. 1. Synthesize an Oligonucleotide
containing the changed sequence.
Ex.---ATT---Wild type sequence
(Codon for ILe)
---CTT---Desired Change (Leu)
---GAA---Mutagenic
Oligonucleotide (MO)
2.Hybridize MO ss form of
gene cloned into M13.
3.Synthesize second strand of
DNA with KLENOW fragment
&dNTPs.
4.Seal nick in new strand
with T4 DNA ligase.
5.Introduce into E. Coli.
6.ss + phage isolated from
plaques & screened by
hybridization.
Oligonucleotide directed
mutagenesis with M13 DNA:
55. Uses of Site Directed Mutagenesis
1.Site Directed Mutagenesis is also used to âengineerâ
commercially important Proteins for many different
purposes, for example
â˘Improve stability
â˘Change specificity
â˘Reduce toxicity
56. Site Directed Mutagenesis enabled new
approaches to drug designing â
particularly in order to improve
FUNCTION.
Random Mutagenesis is used to construct
large & diverse clone libraries of mutated
DNA fragments.
57. DNA sequencing
DNA sequencing is the process of determining the precise
order of nucleotides within a DNA molecule.
It includes any method or technology that is used to
determine the order of the four basesâadenine,
guanine, cytosine, and thymineâin a strand of DNA.
58. Applications of DNA sequencing
Knowledge of DNA
sequences has
became valuable
for basic biological
research
virology
medical diagnosis
biological
systematics
forensic biology
59. DNA sequencing helps to
Molecular biology:
to study genomes and the proteins they encode.
allows researchers to identify changes in genes
identify potential drug targets.
Evolutionary biology:
to study how different organisms are related and how they evolved.
60. DNA sequencing helps to
Metagenomics: The field of metagenomics
identification of organisms present in a body
of water, sewage, dirt, debris filtered from
the air, or swab samples from organisms.
Sequencing enables researchers to
determine which types of microbes may be
present in a microbiome.
61. Sanger Sequencing/ chain termination
method
The most popular method for doing this is
called the dideoxy method or Sanger method
(named after its inventor, Frederick Sanger,
who was awarded Nobel prize in chemistry in
1980[his second] for this achievment).
62.
63. The Procedure
The DNA to be sequenced is prepared as a single strand.
This template DNA is supplied with
a mixture of all four normal (deoxy) nucleotides in ample quantities
dATP ,dGTP ,dCTP ,dTTP.
a mixture of all four dideoxynucleotides, each present in
limiting quantities and each labeled with a "tag" that
fluoresces a different color:
ddATP ,ddGTP ,ddCTP ,ddTTP
64. DNA polymerase I
Because all four normal nucleotides are present, chain elongation proceeds
normally until, by chance, DNA polymerase inserts a dideoxy nucleotide
instead of the normal deoxynucleotide.
At the end of the incubation period, the fragments are separated by length
from longest to shortest. The resolution is so good that a difference of one
nucleotide is enough to separate that strand from the next shorter and next
longer strand. Each of the four dideoxynucleotides fluoresces a different color
when illuminated by a laser beam and an automatic scanner provides a
printout of the sequence.
65.
66. Other methods
⢠Maxam-Gilbert sequencing
⢠Advanced methods and de novo sequencing
⢠Shotgun sequencing
⢠Bridge PCR