Recombinant DNA technology involves isolating DNA from different species, cutting it with restriction enzymes, and splicing the pieces together to form new recombinant molecules. These molecules are then inserted into host cells like bacteria or yeast where they can be replicated in large quantities. Key aspects of the process include using restriction enzymes to cut DNA at specific recognition sequences, producing DNA fragments with cohesive or blunt ends, and inserting the fragments into plasmids - small extrachromosomal DNA molecules found in bacteria. Plasmids are often used as vectors to carry foreign DNA, and they allow selection of cells containing the recombinant DNA through the use of antibiotic resistance genes on the plasmid.

1. Recombinant DNA
Technology
Represented By: Faisal-Ur-Rehman
Roll Number: 16103
BS Bioinformatics 5th (evening)

2. Recombinant DNA Technology
 Recombinant DNA technology procedures by which DNA
from different species can be isolated, cut and spliced
together -- new "recombinant " molecules are then
multiplied in quantity in populations of rapidly dividing
cells (e.g. bacteria, yeast).
Other Definition
 Recombinant DNA technology, joining together of DNA
molecules from two different species that are inserted into
a host organism to produce new genetic combinations that
are of value to science, medicine, agriculture, and
industry.

3. Recombinant DNA Technology
 The term gene cloning, recombinant DNA technology and
genetic engineering may seems similar, however they are
different techniques in Biotechnology and they are
interrelated.
 Human gene therapy, genetically-engineered crop plants
and transgenic mice have become possible because of the
powerful techniques developed to manipulate nucleic
acids and proteins.
Gene Therapy: the introduction of normal genes into cells
in place of missing or defective ones in order to correct
genetic disorders.

4. Recombinant DNA Technology
History
 In the early 1970s it became possible to isolate a specific
piece of DNA out of the millions of base pairs in a typical
genome.
 Currently it is relatively easy to cut out a specific piece of
DNA, produce a large number of copies , determine its
nucleotide sequence, slightly alter it and then as a final
step transfer it back into cell in.

5. Recombinant DNA technology is based on
a number of important things:
 Bacteria contain extra chromosomal molecules of DNA
called plasmids which are circular.
 Bacteria also produce enzymes called restriction
endonucleases that cut DNA molecules at specific places
into many smaller fragments called restriction fragments.

6. Restriction Enzymes and plasmid
 A restriction enzyme is a protein that recognizes a
specific, short nucleotide sequence and cuts the DNA only
at that specific site, which is known as restriction site or
target sequence. More than 400 restriction enzymes have
been isolated from the bacteria that manufacture them.

7. Restriction Enzymes and plasmid
 There are many different kinds of Restriction Enzymes but
here five restriction enzymes are given:
Enzyme Source
Recognition
Sequence
Cut
EcoRI Escherichia coli
5'GAATTC
3'CTTAAG
5'---G AATTC---3'
3'---CTTAA G---5'
EcoRII Escherichia coli
5'CCWGG
3'GGWCC
5'--- CCWGG---3'
3'---GGWCC ---5'
BamHI
Bacillus
amyloliquefaciens
5'GGATCC
3'CCTAGG
5'---G GATCC---3'
3'---CCTAG G---5'
HindIII
Haemophilus
influenzae
5'AAGCTT
3'TTCGAA
5'---A AGCTT---3'
3'---TTCGA A---5'
TaqI Thermus aquaticus
5'TCGA
3'AGCT
5'---T CGA---3'
3'---AGC T---5'

8. Restriction Enzymes and plasmid
 A restriction enzyme cuts only double helical segments
that contain a particular sequence, and it makes its
incisions only within that sequence--known as a
"recognition sequence".
 The recognition sequence, sometimes also referred to
as recognition site, of any DNA-binding protein motif that
exhibits binding specificity, refers to the DNA sequence to
which the domain is specific. Recognition sequences are
palindromes .
 In Biochemistry. a region of DNA in which the sequence of
nucleotides is identical with an inverted sequence in the
complementary strand: GAATTC is a palindrome of
CTTAAG.

9. Restriction Enzymes and plasmid
 Sticky end and blunt end are the two possible configurations
resulting from the breaking of double-stranded DNA .
 If two complementary strands of DNA are of equal length, then they
will terminate in a blunt end, As the following Example:
5‘-CpTpGpApTpCpTpGpApCpTpGpApTpGpCpGpTpApTpGpCpTpApGpT-3'
3'-GpApCpTpApGpApCpTpGpApCpTpApCpGpCpApTpApCpGpApTpCpA -5'

10. Restriction Enzymes and plasmid
 However, if one strand extends beyond the complementary
region, then the DNA is said to possess an ‘overhang’.
5'-ApTpCpTpGpApCpT-3'
3'-TpApGpApCpTpGpApCpTpApCpG-5'

11. Restriction Enzymes and plasmid
 If another DNA fragment exists with a complementary
overhang, then these two overhang, will tend to associate
with each other and each strand is said to possess a
‘sticky end’
5'-ApTpCpTpGpApCpT pGpApTpGpCpGpTpApTpGpCpT-3'
3'-TpApGpApCpTpGpApCpTpApCpGp CpApTpApCpGpA-5'
Becomes
5'-ApTpCpTpGpApCpT pGpApTpGpCpGpTpApTpGpCpT-3'
3'-TpApGpApCpTpGpApCpTpApCpGp CpApTpApCpGpA-5'

12. Restriction Enzymes and plasmid
 Restriction Enzymes are primarily found in bacteria and are
given abbreviations based on genus and species of the
bacteria.
 One of the first restriction enzymes to be isolated was
from EcoRI
 EcoRI is so named because it was isolated from Escherichia
coli strain called RY13.

13. Digestion of DNA by EcoRI to produce
cohesive ends (Figure)

14. Creating Recombinant DNA
 The first Recombinant DNA molecules
were made by Paul Berg at Stanford
University in 1972
 In 1973 Herbert Boyer and Stanley
Cohen created the first recombinant
DNA organisms.

15. Summary of Recombinant DNA
technology process:
 Recombinant DNA technology requires DNA extraction,
purification, and fragmentation.
 Fragmentation of DNA is done by specific 'restriction'
enzymes and is followed by sorting and isolation of
fragments containing a particular gene.
 This portion of the DNA is then coupled to a carrier
molecule.
 The hybrid DNA is introduced into a chosen cell for
reproduction and synthesis.

16. Plasmids and Antibiotic resistance
 Plasmids were discovered in the late sixties, and it was
quickly realized that they could be used to amplify a gene
of interest.
 A plasmid containing resistance to an antibiotic (usually
ampicillin) or Tetracycline, is used as a vector.
 The gene of interest (resistant to Ampicillin) is inserted
into the vector plasmid and this newly constructed
plasmid is then put into E. coli that is sensitive to
ampicillin.
 The bacteria are then spread over a plate that contains
ampicillin.

17. Plasmids and Antibiotic resistance
 The ampicillin provides a selective pressure because only
bacteria that have acquired the plasmid can grow on the
plate.
 Those bacteria which do not acquire the plasmid with the
inserted gene of interest will die.
 As long as the bacteria grow in ampicillin, it will need the
plasmid to survive and it will continually replicate it,
along with the gene of interest that has been inserted to
the plasmid .

18. Selecting a Gene
in a plasmid and
Antibiotic
selection

19. Thank you for Attention