Recombinant DNA technology uses restriction enzymes to cut DNA at specific sequences. The gene of interest is cut out and placed on a vector for manipulation and cloning. The vector, with the inserted gene, is then inserted into a host cell. The host cell is able to survive selection due to an antibiotic resistance gene on the vector. When the host cell replicates, it produces colonies of recombinant cells containing the inserted gene.

1. Recombinant DNA
Technology

5. Recombinant DNA Technology
Host Organism

14. Restriction enzymes cut DNA at specific sequences.
Molecular cloning involves cutting out the gene of interest from its original location and placing it
on a vector for subsequent manipulation.
Modification enzymes protect DNA from the corresponding restriction enzymes by adding
methyl groups at the recognition site.
Blunt ends Ends of a double-stranded DNA molecule that are fully base paired and have no
unpaired single-stranded overhang
Sticky ends Ends of a double-stranded DNA molecule that have unpaired single-stranded
overhangs, generated by a staggered cut
DNA ligase Enzyme that joins DNA fragments covalently, end to end.
Molecular cloning

15. After cloning, the chimeric DNA is normally inserted into an appropriate
host cell.
First DNA from some particular source is cut to liberate a gene or other fragment
of interest.
This fragment is then “cloned” by inserting it into another DNA
macromolecule, known as a vector.

16. A chimera is any hybrid molecule of DNA, such as a vector plus a cloned
gene, which has been engineered from two different sources of DNA.
First we will consider the enzymes used to cut and join fragments of DNA. Then
vectors are used in cloning and how DNA fragments are inserted into them.
Cloned genes may be used in the manufacture of high levels of recombinant
protein or may be applied in gene therapy to cure inherited defects.
Application

17. Nucleases are enzymes that degrade nucleic acids. Ribonucleases (RNases)
attack RNA and deoxyribonucleases (DNases) attack DNA.
Some nucleases will only attack single stranded nucleic acids, others will only
attack double-stranded nucleic acids and a few will attack either kind.
Exonucleases attack at the end of nucleic
acid molecules and usually remove just a
single nucleotide, or sometimes a short
oligonucleotide. Any particular exonuclease
attacks either the 3’-end or the 5’-end but
not both.
Endonucleases cleave the
nucleic acid chain in the
middle.

18. How our cell distinguish cell’s own DNA
from DNA of foreign origin
Methylation of DNA by bacteria is used to distinguish the cell’s own DNA from
DNA of foreign origin.
Bacteria make restriction and modification enzymes that respectively cut and methylate DNA, ensuring
than the foreign DNA is recognized and destroyed. Whenever a bacterial cell makes a restriction
enzyme, it also makes the corresponding modification enzyme that modifies and protects its own DNA.

19. Restriction enzymes are
endonucleases that cut double-
stranded DNA. They bind to DNA at a
specific sequence of bases, called
the recognition site and then proceed
to cut the DNA. Modification enzymes bind to the DNA
at the same recognition site as the
corresponding restriction enzymes and
methylate the DNA.
Modification enzymes usually add the
methyl group to adenine or cytosine
within the recognition site.

20. Restriction and Modification Systems

21. How EcoR1 produce Zigzag cut?
EcoRI recognizes the sequence, 5’-GAATTC-3’, and cuts after the G.
Since this sequence is an inverted repeat, the enzyme also cuts the other
strand after the corresponding G, giving a zig-zag cut.
Modification enzymes are paired with restriction enzymes and recognize the same sequence. Modification
enzymes methylate the recognition sequence, which prevents the restriction enzyme from cutting it.

22. If two restriction enzymes from different species share the same
recognition sequence they are known as isoschizomers.
Note that isoschizomers may not always cut in the same place even though
they bind the same base sequence. For example, the sequence GGCGCC is
recognized by four enzymes, each of which cuts in different places: NarI
(GG/CGCC), BbeI (GGCGC/C), EheI (GGC/GCC), and KasI (G/GCGCC).
Isoschizomers

24. Type I restriction enzyme Type of
restriction enzyme that cuts the DNA a
thousand or more base pairs away from
the recognition site.
Type II restriction enzyme Type of
restriction enzyme that cuts the DNA in
the middle of the recognition site.
Some generate blunt ends, others give
sticky ends.

25. The enzyme DNA ligase is used to join DNA fragments covalently. DNA ligase operates during
DNA replication where it joins up the fragments of the lagging strand.

27. If DNA ligase finds two DNA fragments touching each other end to end, it will
ligate them together.
Ligating blunt ends is very slow and requires a high concentration of DNA ligase,
as well as, a high concentration of DNA.
In fact, bacterial ligase cannot join blunt ends at all. In practice, T4 ligase is
normally used in genetic engineering as it can join blunt ends if need b

28. A diagram that shows the location of restriction enzyme cut sites on a segment
of DNA is known as a restriction map.

29. Basic Structural make-up of cloning Vector

30. Basic Structural make-up of cloning Vector

43. Competent Cell

46. Competent Cell only able to survive
Due to the presence of amp. resistant gene for screening purpose.

49. Formation of colonies of Recombinant Cells

50. Thank you for your Attentions
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