Basic Principles of Recombinant DNA Technology (173865)
Basic Principles of Recombinant DNA Technology (173865)
Basic Principles of Recombinant DNA Technology (173865)
PRINCIPLES
OF
RECOMBINAN
T DNA
TECHNOLOGY
PRINCIPLES & PROCEDURES OF GENETIC
ENGINEERING (RECOMBINANT DNA
TECHNOLOGY)
Genetic engineering procedures involve type of DNA
called recombinant DNA
RECOMBINANT DNA (rDNA)
is a form of artificial DNA that is created by combining two
or more sequences that would not normally occur together
through the process of gene splicing.
is DNA that contains genetic material from 2 different
organisms
is made by inserting a gene obtained from DNA of one
organism into the DNA from another kind of organism.
Encompasses a number of experimental protocols
leading to the transfer of genetic information(DNA)
from one organism to another. Involves the
manipulation of genetic material(DNA) to achieve the
desired goal in a pre-determined way.
PRINCIPLES & PROCEDURES OF GENETIC
ENGINEERING (RECOMBINANT DNA
TECHNOLOGY)
What is Recombinant DNA Technology?
Recombinant DNA technology is a technology
which allows DNA to be produced via artificial
means. The procedure has been used to change
DNA in living organisms and may have even more
practical uses in the future.
It is an area of medical science that is just
beginning to be researched in a concerted effort.
Recombinant DNA technology is one of the recent
advances in biotechnology, which was developed
by two scientists named Boyer and Cohen in
1973.
PRINCIPLES & PROCEDURES OF GENETIC
ENGINEERING (RECOMBINANT DNA
TECHNOLOGY)
PRINCIPLES & PROCEDURES OF GENETIC
ENGINEERING (RECOMBINANT DNA
TECHNOLOGY)
Recombinant DNA technology works by taking
DNA from two different sources and combining
that DNA into a single molecule. That alone,
however, will not do much.
Recombinant DNA technology only becomes
useful when that artificially-created DNA is
reproduced. This is known as DNA cloning.
PRINCIPLES & PROCEDURES OF GENETIC
ENGINEERING (RECOMBINANT DNA
TECHNOLOGY)
Basic principles of rDNA technology:
Generation of DNA fragments & selection
of the desired piece of DNA.
Insertion of the selected DNA into a
cloning vector to create a rDNA or
chimeric DNA.
Introduction of the recombinant vectors
into host cells.
Multiplication & selection of clones
containing the recombinant molecules.
Expression of the gene to produce the
desired product.
PRINCIPLES & PROCEDURES OF GENETIC
ENGINEERING (RECOMBINANT DNA
TECHNOLOGY)
PRINCIPLES & PROCEDURES OF GENETIC
ENGINEERING (RECOMBINANT DNA
TECHNOLOGY)
PRINCIPLES & PROCEDURES OF GENETIC
ENGINEERING (RECOMBINANT DNA
TECHNOLOGY)
PRINCIPLES & PROCEDURES OF GENETIC
ENGINEERING (RECOMBINANT DNA
TECHNOLOGY)
The bacterium E. coli w/c is found in the
intestinal tract of humans an animals, is
the organism most often used in
recombinant DNA experiments.
Steps in obtaining E. coli cells that
contain recombinant DNA
step1 : Cell Membrane Dissolution
step2: Isolation of Plasmid fraction
step3: Cleavage of Plasmid DNA
step4: Gene removal from another organism
step5: Gene-plasmid splicing
THE POLYMERASE CHAIN REACTION
A technique called the Polymerase chain reaction (PCR) has revolutionized recombinant
DNA technology. It can amplify DNA from as little material as a single cell and from very
old tissue such as that isolated from Egyptian mummies, a frozen mammoth, and insects
trapped in ancient amber.
Is a method for rapidly producing multiple copies of a DNA nucleotide
sequence.
method is used to amplify DNA sequences
The polymerase chain reaction (PCR) can quickly clone a small sample of DNA in a test
tube
Billions of copies of a specific DNA sequence (gene) can be produced in a few hours via
this reaction.
Is easy to carry out, requiring only a few chemicals, a container, and a source of heat.
The PCR process, devised in 1983, has become a valuable tool for diagnosing diseases
and detecting pathogens in the body.
It is now used in the prenatal diagnosis of a number of genetic disorders, including
muscular dystrophy and cystic fibrosis, and in the identification of bacterial pathogens
It is also the definitive way to detect the AIDS virus.
The PCR process has also proved useful in certain types of forensic investigations.
A DNA sample may be obtained from a single drop of blood or semen or a single strand
of hair at the crime scene and amplified by the PCR process.
Work with DNA in the forensic area is often referred to as DNA fingerprinting.
THE POLYMERASE CHAIN REACTION
TERMS & DEFINITIONS
Applications in medicine
Human Gene Therapy
Diagnosis of genetic disorders
Forensic Evidence
Industrial Application
Enzymes
Protein Products
ANALYSIS OF GENE STRUCTURE
AND EXPRESSION
Usingspecialized recombinant DNA techniques,
researchers have determined vast amounts of DNA
sequence including the entire genomic sequence of
humans and many key experimental organisms.
This enormous volume of data, which is growing at
a rapid pace, has been stored and organized in two
primary data banks:
the GenBank at the National Institutes of Health,
Bethesda, Maryland,
and the EMBL Sequence Data Base at the European
Molecular Biology Laboratory in Heidelberg, Germany.
PHARMACEUTICAL
PRODUCTS
Some pharmaceutical applications of DNA technology:
Large-scale production of human hormones and other proteins
with therapeutic uses
Production of safer vaccines
A number of therapeutic gene products insulin, the
interleukins, interferons, growth hormones, erythropoietin,
and coagulation factor VIIIare now produced
commercially from cloned genes
Pharmaceutical
companies already are
producing molecules
made by recombinant
DNA to treat human
diseases.
Recombinant bacteria
are used in the
production of human
growth hormone and
human insulin
Use recombinant cells to mass produce proteins
Bacteria
Yeast
Mammalian
Insulin
Hormone required to properly Human Growth hormone deficiency
process sugars and fats Faulty pituitary and
Treat diabetes regulation
Now easily produced by Had to rely on cadaver source
bacteria Now easily produced by
bacteria
SUBUNIT HERPES VACCINE
NOT ALWAYS USED FOR GOOD...
Mouse on right is
normal; mouse on
left is transgenic
animal expressing
rat growth hormone
FARM ANIMALS AND PHARM
ANIMALS
Trangenicplants and animals
have genes from other
organisms.
overshadow
humanitarian efforts
APPLICATION IN MEDICINE
Human Gene Therapy
Diagnosis of genetic disorders
Forensic Evidence
HUMAN GENE THERAPY
Human gene therapy seeks to repair the
damage caused by a genetic deficiency
through introduction of a functional version
of the defective gene. To achieve this end, a
cloned variant of the gene must be
incorporated into the organism in such a
manner that it is expressed only at the proper
time and only in appropriate cell types. At this
time, these conditions impose serious
technical and clinical difficulties.
HUMAN GENE THERAPY
Viral RNA
Somatic cells
Only!
Viral DNA carrying the normal
allele inserts into chromosome.
Not for
reproductive
Bone
marrow cells !!
cell from
patient
Bone
Inject engineered marrow
cells into patient.
DIAGNOSIS OF GENETIC
DISORDERS
2 Step Process:
1. Pentose sugar and N-containing base reacts to form
a 2 sub unit entity called a NUCLEOSIDE
2. The nucleoside reacts with a phosphate group to
form the three sub-unit entity called a
NUCLEOTIDE
(A,C,G,T & U)
MP for monophosphate
Deoxyadenosine monophosphate,
also known asdeoxyadenylate,
ordAMP, is a derivative of the
commonnucleic acidAMP, or
adenosine monophosphate, in which
the -OH (hydroxyl) group on the 2'
carbon on the
nucleotide'spentosehas been reduced
to just a hydrogen atom (hence the
"deoxy-" part of the name).It is
amonomer used inDNA.
Molecular Formula C10H14N5O6P
DNA NUCLEOTIDE
DGMP ( DEOXYGUANOSINE 5-MONOPHOSPHATE)
Deoxyguanosine monophosphate,
also known asdeoxyguanylate,
ordGMP, is a derivative of the
commonnucleic acid Guanosine
triphosphate(GTP), in which the
OH (hydroxyl) group on the 2' carbon
on the nucleotide'spentosehas been
reduced to just a hydrogen atom
(hence the "deoxy-" part of the name).
It is used as amonomerinDNA.
Molecular Formula C10H14N5O7P
DNA NUCLEOTIDE
DCMP ( DEOXYCYTIDINE 5-MONOPHOSPHATE)
Deoxycytidine monophosphate,
also known asdeoxycytidylate,
ordCMP, is adeoxynucleotide, and
one of the fourmonomers that make
upDNA. In a DNA double helix, it
will base pair withdeoxyguanosine
monophosphate.
Molecular Formula C9H14N3O7P
DNA NUCLEOTIDE
DTMP ( DEOXYTHYMIDINE 5-MONOPHOSPHATE)
Deoxythymidine 5-
monophosphate also known as5-
thymidylate,thymidylate,TMP,
ordTMP, is anucleotidethat is used
as a monomerinDNA. It is
anesterofphosphoric acidwith
thenucleosidethymidine. dTMP
consists of aphosphategroup,
thepentose sugardeoxyribose, and
thenucleobasethymine. As
asubstituent, it takes the form of the
prefixthymidylyl-.
Molecular Formula C10H14N2O8P1
RNA NUCLEOTIDE
CMP ( CYTIDINE 5-MONOPHOSPHATE)
Cytidine monophosphate, also
known as5'-cytidylic acidor
simplycytidylate, and
abbreviatedCMP, is
anucleotidethat is used as
amonomerinRNA.It is
anesterofphosphoric acidwith
thenucleosidecytidine. CMP consists
of thephosphategroup, the
pentosesugarribose, and
thenucleobasecytosine; hence,
aribonucleoside monophosphate. As
asubstituentit takes the form of the
prefixcytidylyl-.
Molecular formula C9H14N3O8P
RNA NUCLEOTIDE
AMP ( ADENOSINE 5-MONOPHOSPHATE)
Adenosine
monophosphate(AMP), also known
as5'-adenylic acid, is
anucleotidethat is used as
amonomerinRNA. It is an
esterofphosphoric acidand the
nucleosideadenosine. AMP consists
of aphosphategroup, the
sugarribose, and the nucleobase
adenine. As asubstituentit takes the
form of the prefixadenylyl-
Molecular formula C10H14N5O7P
RNA NUCLEOTIDE
GMP ( GUANOSINE 5-MONOPHOSPHATE)
Guanosine monophosphate, also known as5'-
guanidylic acidorguanylic acidand
abbreviatedGMP, is anucleotidethat is used as
amonomerinRNA. It is anesterofphosphoric
acidwith thenucleosideguanosine. Guanosine
monophosphate is commercially produced by microbial
fermentation.
Guanosine monophosphate in the form of itssalts,
such asdisodium guanylate(E627),dipotassium
guanylate(E628) andcalcium guanylate(E629),
arefood additivesused asflavor enhancersto provide
theumamitaste. It is often used in synergy
withdisodium inosinate; the combination is known
asdisodium 5'-ribonucleotides. Disodium guanylate is
often found in instant noodles, potato chips and
snacks, savoury rice, tinned vegetables, cured meats,
and packet soup.
As it is a fairly expensive additive, it is usually not
used independently ofglutamic acidormonosodium
glutamate(MSG), which also contribute umami. If
inosinate and guanylate salts are present in a list of
ingredients but MSG does not appear to be, the
glutamic acid is likely provided as part of another
ingredient, such as a processed soy protein complex
(hydrolyzed soy protein),autolyzed yeastorsoy sauce.
As anacylsubstituent, it takes the form of the
prefixguanylyl-.
Molecular Formula C10H14N5O8P
RNA NUCLEOTIDE
UMP ( URIDINE 5-MONOPHOSPHATE)
Uridine monophosphate, also known as5'-uridylic
acidand abbreviatedUMP, is anucleotidethat is
used as amonomerinRNA. It is
anesterofphosphoric acidwith
thenucleosideuridine. Another common shorthand for
the molecule isuridylate- the deprotonated form of
the molecule, which is predominant in aqueous
solution. As asubstituentit takes the form of the
prefix uridylyl-. The deoxy form is abbreviateddUMP.
Effects on animal intelligence
In a study,gerbilsfed a combination of uridine
monophosphate,choline, anddocosahexaenoic
acid(DHA) were found to have significantly improved
performance in running mazes over those not fed the
supplements, implying an increase in cognitive
function.
In brain research studies, uridine monophosphate is
used as a convenient delivery compound foruridine.
Uridineis the active component of this compound.
Uridine is present in many foods, mainly in the form of
RNA. However, uridine in RNA is not bioavailable,
since it is almost entirely destroyed in the liver and
gastrointestinal tract. Thus no food, when consumed,
has ever reliably been shown to raise blood uridine
levels except mothers' milk or infant formulas which
contain uridine in the form of uridine monophosphate
instead of as RNA.
Molecular formula C9H13N2O9P