1.
GENETIC ENGINEERING

2.
The modification of the genetic information of living organism by direct manipulation
of their DNA.
GENETIC ENGINEERING
 A gene of know function can be transferred from its normal location into
cell via a suitable mobile genetic element called vector.(plasmid.virusers
etc)
 Gene-cloning methods are central to genetic engineering, the direct
manipulation of genes
for practical purposes.
 Genetic engineering has
launched a revolution in
biotechnology, the use of
organisms or their
components to make useful
products.

3.
TOOLS OF GENETIC ENGINEERING
 ENZYME:exonuclease restrcition enzyme SI ENZYME (to change
cohesive end of single stranded DNAfragement into blunt ) DNA
Ligsaes ,alkaline phosphatase,reverse transcriptase,DNA
Polymerases.
 Foreign DNA :it is a fragment of DNA molecule which is enzymatically
isolated and cloned.the gene is identified on genome and pulled out
from it either before or after cloning.the cloned foreign DNA fragment
expresses normally as In parents cell.
 Cloning vectors:vector or cycle DNA are those DNA that can be a
foregin DNA fragment when inserted into it .based on natural resource
,the vector are grouped bacterial plasmids,bacteriophage and cosmids.

4.
PLASMID HAVE VARIOUS CURIOUS PROPERTIES
1.THE GENE THEY CARRY ARE NOT BE ABSOLUTELY ESSENTIAL FOR AND SO
PLASMID CAN SOMETIMES HAVE ONE BACTERIAL CELL AND ENTER THERE
BY TRANSFERRING GENETIC BETWEEN CELL.
2.THE PLAMID REPRODUCE ITSELF INSIDE THE BACTERIUM INDEPENDENTLY
OF THE MAIN BACTERIAL DNA.
3.A PLASMID CAN SOMETIMES FUSE WITH THE MAIN DNA AND CAN BE
DEPART FROM THE MAIN GENOME IN SUCH A MANNER AS TO DRAG A PIECE
OF THE MAIN DNA WITH IT.
TOOLS OF GENETIC ENGINEERING
 DNA library:The first step in gene cloning experiment is to
obtain DNA carrying the specific gene that we have
intersting in cloning.There are two obtained for experiment
designed to identify an unknown gene-genomic DNA and
cDNA.These DNA sources are used in construction of two
kind of DNA library and cDNA library.

5.
TOOLS OF GENETIC ENGINEERING
 Genomic library:A genomic library is a collection of clones representing the complete
genome of an organism.this mean that the entire genome of an organism is represnted as
a set of DNA fragments insersted or cloned into vector molecule that can be propagated in
suitable host.
 TO PREPARED A GENOMIC LIBRARY THE TOTAL GENOMIC DNA ISOLATED FROM
TISSUE.
(A).AND IS FRAGEMENTED INTO RANDOM PIECES OF SUITABLE SIZE FOR
INSERTION IN AN APPROPIATE VECTOR
B.THE GENOMIC DNA FRAGMENT ARE INSERETED INTO SUITABLE VECTOR CUT
WITH RESTRCITION ENZYME
C.EACH VECTOR MOLECULE WILL CONTAIN DIFFERENT FRAGEMENT DNA
AND RECOMBINANT DNA MOLECULES
THUS GENERATED ARE INTRODUCED INTO AN APPORIATED HOST SUCH
AS BACTERIAL CELL OR PHAGE PARTICLES.

6.
Entire genome from donor cells
Random DNA FRAGMENTS VECTOR DNA
Fragmentation by restrction enzyme
Ligation by Cohesive ends Blunt ends
Using linker Homopolymer tailing
CIRCULAR RECOMBINANT DNA
Intro into host cell by Transformation,
Transfection, in vitro packaging with phage.
HOST CELL CONTANING RECOMBINANT DNA
CLONES OF GENE LIBRARY
Selection of cell by Direct phenotypic
selection Nucleic acid hybridization
Immunlogical test Hybrid arrest translation
METHOD OF CONSTRUCTION OF GENE LIBRARY

7.
CDNA LIBRARY:
ALTHOUGH A GENOMIC LIBRARY REPRESENTS THE ENTIRE GENOME OF
AN ORGANISM IT MAY NOT USEFUL IN CASE OF EUKARYOTIC ORGANISM.
GENOMIC DNA OF EUKARYOTES CONTAIN NON CODING DNA SUCH AS
INTRON CONTROL REGION AND REPETITIVE SEQUENCES. THEREFORE
ANOTHER LIBRARY CALLED DNA LIBRARY CAN BE MADE USING
MESSENGER RNA AS THE STARTING MATERIAL.
THIS LIBRARY HAS TWO MAJOR ADVANTAGE OVER A GENOMIC LIBRARY

8.
FIRSTLY; IT REPRESENTS ONLY THOSE GENES THAT ARE BEING
EXPRESSED BY PARTICULAR CELL OR UNDER A SPECIFIC
CONDITION .
SECONDLY: SINCE MRNA LACKS INTRON THIS LIBRARY WOULD
REPRESENT ONLY THE CODING SEQUENCE OF THE GENE.
HOWEVER IS NOT POSSIBLE TO DIRECTLY CLONE THE MRNA
SINCE THE RNA IS UNSTABLE.
HENCE IT IS CONVERTED INTO DNA COPY BEFORE CLONING INTO A
SUITABLE VECTOR.
SUCH A LIBRARY MADE FROM CDNA OR COMPLEMENTARY DNA IS
CALLED A CDNA LIBRARY.
MOLECULARPROBE.A PROBE IS EITHER A RADIOACTIVE LABELLED
P32 OR NON RADIOACTIVE LABELLED (BIOTIN OR DIOXIGENIN)

9.
IDENTIFICATION OF FOOD
CONTAMINANTS AND IN FORENSIC TEST.
ANTIBODIES ARE ALSO OCCASIONALLY
USED AS PROBES TO RECONGNIZE
SPECFIC PROTEIN SEQUENCES.

10.
CERTAIN GENERAL TECHIQUES OF GENETIC
ENGINEERING
 ISOLATION AND USE OF RESTRICTION
ENZYME: Recombinant DNA technology make
a frequenty use of restrction endonuclease
which cut the DNA double helix in very percise
way.they have the capacity to recongnize
specific base sequenes on DNA and then cut
each strand at given place.these enzyme are
called restrcition enzyme because they restrict
Infection of bacteria by certain viruses by
degrading the viral DNA without affecting the
bacterial DNA.

11.
Each bacterium has its own unique restrcition
enzyme and each enzyme recognize only one type
of sequence.
The DNA sequence recongnized by restricition
enzyme are called palindromes.
Palindromes are the base sequences that read the
same on the two strand but n opposite directions.
Example if the sequenceon one strand is GAATTC
read in 5”to3” direction the sequence on opposite
strand is CTTAAG read in the 3”to 5” direction but
when both strands are read in the 5” to 3”
direction the sequence is the same.
5” GAATTC 3”
3” CTTAAG 5”

12.
IN ADDITION THERE IS A POINT OF SYMMETRY WITHIN
THE PALINDROME.IN OUR E.G THIS POINT IS THE
CENTRE BETWEEN THE AT/AT.THE VALUE OF
RESTRUCITION ENZYME IS THAT THEY MAKE CUT IN
THE DNA MOLECULE AROUD THIS POINT OF
SYMMETRY.
IN THIS E.G WE HAVE USED THE PALINDROME
SEQUENCE RECOGNIZED BY ONE OF THE MOST
POPULAR RESTRCTION ENZYME CALLED ECO RI FROM
E.COIL.

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• Southern blotting involves the transfer of DNA
from a gel to a membrane, followed by detection
of specific sequences by hybridization with a
labeled probe.
• Northern blotting, RNA is run on a gel.
• Western blotting entails separation of proteins on
an SDS gel, transfer to a nitrocellulose membrane,
and detection proteins of interest using antibodies.

14.
Southern blotting

15.
NORTHERN BLOTTING
NORTHERN HYBRIDIZATION
Northern blotting is similar to Southern blotting,
but involves the transfer of RNA from a gel to a
membrane
RNA

16.
BASIS STEPS OF NB
1. Isolation of intact mRNA
2. Separation of RNA according to size (through a
denaturing agarose gel e.g. with Glyoxal/formamide)
Transfer of the RNA to a solid support
Fixation of the RNA to the solid matrix
Hybridization of the immobilized RNA to probes
complementary to the sequences of interest
Removal of probe molecules that are nonspecifically
bound to the solid matrix
Detection, capture, & analysis of an image of the
specifically bound probe molecules.

17.
WESTERN BLOTTING
 Western blotting entails separation of proteins on an SDS
gel, transfer to a nitrocellulose membrane, and detection
proteins of interest using antibodies.
 Blotting is the transfer of separated proteins from the
gel matrix into a membrane, e.g., nitrocellulose
membrane, using electro- or vacuum-based transfer
techniques.
wikipedia
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WESTERN BLOTTING

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 Gel electrophoresis: produces a
readable pattern of DNA fragments

20.
GEL ELCTROPHORESIS
 APPLICATIONS:
 Estimation of the size of DNA molecules
following restriction enzyme digestion, e.g.
in restriction mapping of cloned DNA.
 Analysis of PCR products, e.g. in
molecular genetic diagnosis or genetic
fingerprinting.
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• Some techniques to analyze DNA and RNA
are limited by the small amounts of test
nucleic acid available
• Polymerase chain reaction (PCR) rapidly
increases the amount of DNA in a sample
• So sensitive- could detect cancer from a
single cell
• Can replicate a target DNA from a few copies
to billions in a few hours

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THREE BASIC STEPS THAT CYCLE
 Denaturation
 Heat to 94°C to separate in to 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
 DNA polymerase and nucleotides are added
 Polymerases extend the molecule
 The amplified DNA can then be analyzed

24.
VECTOR TRANSFORMATION AND MOLECULAR
CLONING:
 Plasmid: are extra-chromosomal DNA element found
mostly in bacteria.these plasmid contain DNA sequence
coding for drug resistance sex factor etc.when the
bacterium multiplies the plasmid multiply along with
chromosomal It is possible to isolated these plasmid in
large quantity.
 The foreign DNA fragement can be made to recombine
DNA with the plasmid DNA and product is referred to as
recombinant DNA.the plasmid DNA carrying the foreign
DNA fragement can be put back into a suitable reciepient
bacterium.this bacterium grow in large qunatities and
recombinant plasmid are isolated from such bacteria this
is called molecular cloning.

25.
VECTOR OR VEHICLE:
PLASMIDS AND VIRUSES WHICH ARE USED AS CARRIES OF FOREIGN ARE
REFERRED TO AS VECTOR OR VEHICLE DNAS.BY INTERCHANGING
PLASMID DNA AND VIRAL DNA FRAGEMENTS SEVERAL NEW VECTORS
HAVE BEEN SYNTHESIZED WHICH CARRY NEW GENE INTO BACTERIA
YEAST INSECT PLANTS AND ANIMALS CELL.
WHEN SUCH NEW DNA FRAGEMENT ARE INTRODUCED INTO RELEVANT
HOST CELL.SUCH AS CELL ARE SAID TO BE TRANSFORMED (IN CASE
BACTERIA)OR TRANGENIC (IN CASE OF PLANT AND ANIMALS) AND THE
PROCESS IS CALLED TRANSFORMATION.IN ANIMALS THE TERM
TRANSFECTION IS USED IN PLACE OF TRANSFORMATION.

26.
 The gene can be synthesized by following two methods
 Organochemical synthesis of polynucleotides (Chemical synthesis
of tRNA genes)
 Synthesis of gene from mRNA(enzymatic synthesis of gene)
ORGANOCHEMICAL SYNTHESIS OF POLYNUCLEOTIDES
 When the structure of gene become procurable then such a gene
can be synthesized by purely chemical method.
 The structure of gene could be inferred from its product.
 Synthesis of genes initially considered to be long to be
synthesized because an average gene contain 1500 base pairs.
 The tRNA molecules are fairly small in size up to 80 nucleotides,
so it could be easily synthesized.
SYNTHESIS OF GENE

27.
SYNTHESIS OF GENE FOR YEAST ALNYL TRNA
 The process of synthesis of gene for yeast tRNA
involves the following steps:
 Synthesis of oligonucleotides
 Synthesis of three duplex fragments
 Synthesis of tRNA gene from three duplex fragments

28.
SYNTHESIS OF OLIGONUCLEOTIDES
 15 oligonucleotides ranging from pentanucleotide (5 bases)
to an iscosannucleotide (20 bases) were synthesized.
 Synthesis can be conducted through condensation between
hydroxyl group at the 3’ position of one nucleotide and
phosphate group at 5’ position of second nucleotide.
 In order to bring about condensation, all other functional
groups, not taking part in condensation ,were protected
using specific protective groups.
 Subsequent condensation was done between groups of two,
three or four nucleotide

29.
Mode of condensation b/w 2 nucleotides with protected 5’OH and 3’OH groups
in the
sugars and protected amino groups in the nitrogen bases.
Mesitylene
sulphonyl
chloride
Anisoyl
group

30.
SYNTHESIS OF THREE DUPLEX FRAGMENTS
 Fifteen single stranded oligonucleotides were used to
prepare three duplex fragments, each containing a single
stranded ends. each fragments are characterized as follows:
 Fragment A consisted of first 20 nucleotides in which 17-20
single stranded. Fragment B contained nucleotide residues
from 17-50,in which single stranded region being 17-20 and
46-50.Fragment C included nucleotide residues 46-77 with
single stranded region 46-50.

31.
SYNTHESIS OF TRNA GENE FROM THREE
DUPLEX FRAGMENTS
 The concluding steps involved the joining (ligation by
enzyme) of 3 duplex fragments A,B and C to give a
complete gene in following two ways
1. In one scheme, fragment A was joined to B taking
advantage of overlap in residues 17-20, fragment C was
then added, with the overlap in the region of 46-50
residues.
2. In the alternative scheme, B fragment was first added to C
and to this A fragment was added in the end to get the
complete gene.

32.
SYNTHESIS OF COMPLETE GENE
 In 1976, Khorana and co-workers started the synthesis of a
gene for E. coli tyrosine tRNA precursor. In 1979,he
completely synthesized a biologically functional tyrosine tRNA
suppressor gene of E. coli which was 207 base pairs long and
contained
 (i) a 51 base pairs long DNA corresponding to promoter
region,
 (ii) a 126 base pair long DNA corresponding to precursor
region of tRNA,
 (iii) a 25 base pair long DNA including 16 base pairs
contained restriction site for EcoRI.
• This complete synthetic gene was joined in phage lambda
vector which in turn was allowed to transfect E. coli cells.
• After transfection phage containing synthetic gene was
successfully multiplied in E. coli.

33.
SYNTHESIS OF GENE FROM MRNA(ENZYMATIC
SYNTHESIS OF GENE)
 RNA directed DNA polymerase enzyme, which was
discovered by Temin and Baltimore(1970),can synthesize
DNA from RNA template.
 Most important genes are the genes for sea urchin histone
proteins,ovalbumin gene in chicken and globin genes in
mammals.
 One of the leasing discoveries, in which the technique of
enzymatic synthesis of gene has been utilized, is the
synthesis of rat insulin cDNA isolated from pancreatic islets
of Langerhans(Ullrich et al,1997)

34.
RESTRICTION MAPS AND RFLPS
 Restriction mapping is a method used to
map an unknown segment of DNA by
breaking it into pieces and then identifying
the locations of the breakpoints. This
method relies upon the use of proteins
called restriction enzymes, which can cut,
or digest, DNA molecules at short,
specific sequences called restriction sites.
After a DNA segment has been digested
using a restriction enzyme, the resulting
fragments can be examined using a
laboratory method called gel
electrophoresis, which is used to separate
pieces of DNA according to their size.
 For example, when the radioactive
nucleotide tritiated was added for a short
period at the beginning of DNA replication
in SV40 viruses, the radioactivity always
reappeared in only one restriction
fragment.

35.
RESTRICTION MAPS AND RFLPS
 Restriction map often allows researchers to correlate the
genetic map and physical map of chromosome.
 The difference in fragment sizes are called restriction
fragment length polymorphisms(RFLPs) and have proved
valuable in pinpointing the exact location of genes and
determining the identity or relatedness of individuals.
 Once a disease gene was localized, RFLP analysis of other
families could reveal who was at risk for the disease, or who
was likely to be a carrier of the mutant genes.
 RFLP analysis was also the basis for early methods of genetic
fingerprinting, useful in the identification of samples retrieved
from crime scenes, in the determination of paternity, and in
the characterization of genetic diversity or breeding patterns
in animal populations.