5. Definition of recombinant
DNA
• Production of a unique DNA molecule by
joining together two or more DNA
fragments not normally associated with
each other.
• DNA fragments are usually derived
from different biological sources
6.
7. What is required ?
1. DNA of interest which you want to clone.
2. Vector to carry and multiply the DNA of your interest.
3. Restrictions enzymes to cut the DNA molecules.
4. DNA ligase to seal the DNA’s.
5. AN expression Vector for the production of the protein
of our interest
13. Must have:
1) Ori
2) A dominant selectable
Marker
3) Cleavage sites for cloning
4) (high copy no.)
Figure 8.4 The plasmid cloning
vector pUC19. This plasmid has an
origin of replication (ori), an ampR
selectable marker, and a polylinker
located within part of the -
galactosidase gene lacZ+.
14. Figure 8.5 Insertion of a piece of DNA into the plasmid cloning vector pUC19 to produce a recombinant
DNA molecule. The vector pUC19 contains several unique restriction enzyme sites localized in a
polylinker that are convenient for constructing recombinant DNA molecules. The insertion of a DNA
fragment into the polylinker disrupts part of the -galactosidase (lacZ+) gene, leading to nonfunctional
-galactosidase in E. coli. The blue–white color selection test described in the text can be used to select for
vectors with or without inserts.
16. Origin and function
• Bacterial origin = enzymes that cleave foreign
DNA
• Named after the organism from which they
were derived
– EcoRI from Escherichia coli
– BamHI from Bacillus amyloliquefaciens
• Protect bacteria from bacteriophage
infection
– Restricts viral replication
• Bacterium protects it’s own DNA by
methylating those specific sequence motifs
17. Availability
• Over 200 enzymes identified, many
available commercially from
biotechnology companies
18. Classes
• Type I
– Cuts the DNA on both strands but at a
non-specific location at varying distances
from the particular sequence that is
recognized by the restriction enzyme
– Therefore random/imprecise cuts
– Not very useful for rDNA applications
19. • Type II
– Cuts both strands of DNA within the particular
sequence recognized by the restriction enzyme
– Used widely for molecular biology procedures
– DNA sequence = symmetrical
20. • Reads the same in the 5’ 3’ direction on
both strands = Palindromic Sequence
• Some enzymes generate “blunt ends” (cut
in middle)
• Others generate “sticky ends” (staggered
cuts)
– H-bonding possible with complementary tails
– DNA ligase covalently links the two fragments
together by forming phosphodiester bonds of the
phosphate-sugar backbones
21.
22.
23.
24.
25.
26. Figure 8.1 Restriction
site in DNA, showing
symmetry of the
sequence around the
center point. The
sequence is a
palindrome, reading the
same from left to right
(5’-to-3’) on the top
strand (GAATTC, here)
as it does from right to
left (5’-to-3’) on the
bottom strand. Shown is
the restriction site for
EcoRI.
28. Figure 8.2 Examples of how restriction enzymes cleave DNA. (a) SmaI results in blunt ends.
(b) BamHI results in 5’ overhanging (“sticky”) ends. (c) PstI results in 3’ overhanging
(“sticky”) ends.
29. Figure 8.3 Cleavage of DNA by the restriction enzyme EcoRI. EcoRI makes staggered,
symmetrical cuts in DNA, leaving “sticky” ends. A DNA fragment with a sticky end produced
by EcoRI digestion can bind by complementary base pairing (anneal) to any other DNA
fragment with a sticky end produced by EcoRI cleavage. The gaps can then be sealed by
DNA ligase.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41. Insert that into an expression vector
for the expression of the proteins
42.
43. cDNA Libraries are best for eukaryotes
Figure 8.8 The synthesis of double-stranded complementary DNA (cDNA) from a
polyadenylated mRNA, using reverse transcriptase, RNase H, DNA polymerase I, and DNA
ligase.
