DNA cloning requires five main steps: 1) cutting DNA at specific locations, 2) selecting a replicable DNA fragment, 3) joining DNA fragments, 4) inserting recombinant DNA into host cells, and 5) identifying host cells containing the recombinant DNA. Restriction endonucleases and DNA ligases are used to cut and join DNA fragments for cloning. Plasmids and bacteriophages are common cloning vectors used to replicate and amplify recombinant DNA in host cells such as E. coli.

1. Department of Biochemistry, Nobel College, Nepal
Saturday, June 11,
2016
Rajesh Chaudhary
1

2. DNA cloning requires following five
procedure
1. Cutting DNA at precise locations.
2. Selecting a small molecule of DNA capable of self-
replicating.
3. Joining two DNA fragments covalently.
4. Moving recombinant DNA from a test tube to a host cell.
5. Selecting or identifying host cells that contain
recombinant DNA.
Saturday, June 11,
2016
Rajesh Chaudhary
2

3. “
”
The method used to accomplish these
and related tasks are collectively
referred to as recombinant DNA
technology or more informally, genetic
engineering.
Saturday, June 11,
2016
Rajesh Chaudhary
3
— Lehninger’s Principles of Biochemistry, 6th. Edition

4. Restriction Endonucleases and DNA
ligases yield recombinant DNA
Saturday, June 11,
2016
Rajesh Chaudhary
4

5. • Restriction endonuclease cleaves the DNA at specific
site.
• DNA ligase helps in joining the cut DNA into a vector.
Saturday, June 11,
2016
Rajesh Chaudhary
5

6. Because it is protected by methylation of DNA catalyzed
by DNA methylase.
Saturday, June 11,
2016
Rajesh Chaudhary
6
The restriction endonuclease along with the DNA methylase sometimes
referred to as restriction modification system.

7. Types and function of Endonuclease
Type 1, Type II, and Type III
 Both type I and type III are generally large, multisubunit complexes
containing both endonuclease as well as methylase activity.
 Type I restriction endonuclease cleave DNA at random sites that can
be more than 1000 bp from recognition sequence.
 Type II is simpler, requires no ATP, cleaves phosphodiester bond at
the recognition site.
 Type III cleaves DNA 25 bp from the recognition site.
Saturday, June 11,
2016
Rajesh Chaudhary
7

8. Recognition site
Saturday, June 11,
2016
Rajesh Chaudhary
8
Thousands of restriction endonuclease has been identified so far and one restriction
endonuclease can identify more than 100 of DNA sequence to be cleaved.
Recognition sequence are usually 4 to 6 bp long and are palindromic in nature.

9. List of sequences recognized by
restriction endonuclease type II
Saturday, June 11,
2016
Rajesh Chaudhary
9

10. Saturday, June 11,
2016
Rajesh Chaudhary
10 DNA molecule can be
separated by size using
Gel electrophoresis

11. Sticky ends Vs Blunt ends
Saturday, June 11,
2016
Rajesh Chaudhary
11

12. Polylinkers
Saturday, June 11,
2016
Rajesh Chaudhary
12
• Inserted DNA
fragments with multiple
recognition sequences
for restriction
endonucleases are
called Polylinkers.

13. Cloning vector allows amplification of
inserted DNA segment
Saturday, June 11,
2016
Rajesh Chaudhary
13
• Circular DNA molecule that
replicate separately from a host
cell.
• Sizes: 5000 – 400,000 bp.
• Plasmid incorporate several
specialized sequence that that
enable them to make use of the
cell’s resource for their own
replication & gene expression.
E. coli

14. Plasmids
Circular DNA molecular that replicate separately from the
host cells.
Size: 5,000 – 400,000
Can be introduced into the bacterial cells by the process
called “transformation”.
Saturday, June 11,
2016
Rajesh Chaudhary
14

15. How transformation is done?
The cells (general E.Coli) and plasmid DNA are incubated
together at 0 oC in a calcium chloride solution, then subjected to
shock by rapid shifting the temperature to 37 0C to 43 0C.
Some of the cells uptake the plasmid DNA.
Some species of bacteria, such as “Acinetobacter baylyi”, are
naturally competent for DNA uptake and do not require the
calcium chloride treatment.
Saturday, June 11,
2016
Rajesh Chaudhary
15

16. DNA cloning
Saturday, June 11,
2016
Rajesh Chaudhary
16

17. Use of pBR322 to
clone foreign DNA
into E.Coli
Saturday, June 11,
2016
Rajesh Chaudhary
17

18. Use of pBR322 to clone foreign DNA into
E.Coli
Saturday, June 11,
2016
Rajesh Chaudhary
18

19. pBR322 important features
An origin of replication, ori, a sequence where replication is
initiated by cellular enzymes. This sequence is required to
propogate the plasmid and maintain it at a level of 10-20
copies per cell.
Two genes that confer resistance to different antibiotics (tetR,
ampR), allowing the identification of cells that contain the intact
plasmid or a recombinant version of the plasmid.
Saturday, June 11,
2016
Rajesh Chaudhary
19

20. pBR322 important features
Several unique recognition sequences (PstI, EcorRI, BamHI, SalI,
PvuII) that are targets for different restriction endonucleases,
providing sites where the plasmid can later be cut to insert
foreign DNA.
Small size (4,361 bp), which facilitates entry of the plasmid into
cells and the biochemical manipulation of the DNA.
Saturday, June 11,
2016
Rajesh Chaudhary
20

21. Bacteriophage cloning vector
Saturday, June 11,
2016
Rajesh Chaudhary
21

22. Complementary
DNA (cDNA)
Saturday, June 11,
2016
Rajesh Chaudhary
22

23. Complementary DNA (cDNA)
Saturday, June 11,
2016
Rajesh Chaudhary
23

24. Hybrid selection
Saturday, June 11,
2016
Rajesh Chaudhary
24

25. Hybrid selection
Saturday, June 11,
2016
Rajesh Chaudhary
25

26. Construction of human genomic
library
Saturday, June 11,
2016
26

27. Transgenic animal
Rajesh Chaudhary
27

28. Transgenic animal
Rajesh Chaudhary
28

29. Transgenic animal
Rajesh Chaudhary
29

30. Polymerase Chain Reaction (PCR)
 A technique used in the molecular biology to make number of
copies of DNA using a single DNA.
 Developed in 1983 by Kary Mullis.
Saturday, June 11,
2016
Rajesh Chaudhary
30
Types of PCR
Total types of PCR: 11

31. Types of PCR
 1. Multiplex PCR
 2. Asymmetric PCR
 3. VNTR PCR
 4. Nested PCR
 5. Quantitative PCR
 6. Hot-start PCR
 7. Touchdown PCR
 8. Assembly PCR
 9. RT-PCR
 10. Methylation-specific PCR
Saturday, June 11,
2016
Rajesh Chaudhary
31

32. Components of PCR
 DNA as a template
 Primers
Forward
Reverse
 Taq DNA polymerase
 dNTP (dATP, dGTP, dCTP, dTTP)
 Mg2+ ions
 DMSO (optional)
 H20
Saturday, June 11,
2016
Rajesh Chaudhary
32

33. PCR33

34. PCR in laboratory setup
Saturday, June 11,
2016
Rajesh Chaudhary
34

35. PCR in laboratory setup
Saturday, June 11,
2016
Rajesh Chaudhary
35

36. Rajesh Chaudhary
36