this presentation gives information about cloning technique such as TOPO Cloning, SLIC and, Golden Gate Cloning. این ارایه در مورد تکنیک های کلون کردن می باشد

1. Cloning techniques (Golden Gate, TOPO and SLIC)
By: Shahnam Azizi
Department of Biotechnology, Faculty of
Agriculture, Azarbaijan Shahid madani
University, Tabriz, Iran
Spring 2018

2. Contents
Introduction to DNA cloning
Important characteristic of vectors
Common cloning technique list
Description of three important cloning techniques
I. TOPO Cloning
II. Sequence and Ligation Independent Cloning (SLIC)
III. Golden Gate Cloning
2

3. DNA cloning
DNA cloning is the process of making
multiple, identical copies of a particular
piece of DNA
Different type of vectors , with variety of
features, can be used for this strategy (DNA
cloning)
Selection of cloning technique is commonly
based on speed, cost, availability of starting
materials or just personal preference
3

4. Vectors
Vectors were the first DNA tools used in genetic
engineering, and continue to be cornerstones of
this technology
Common to all engineered vectors are an origin
of replication, a multicloning site, and a
selectable marker
The purpose of a vector which transfers genetic
information to another cell is typically to isolate,
multiply, or express the insert in the target cell
4

5. Common cloning technique
Restriction Cloning
Golden Gate Cloning
TOPO Cloning
Sequence and Ligation Independent Cloning (SLIC)
Gateway Cloning
CcdB-The Toxic Key to Efficient Cloning
Gibson Cloning
5

6. TOPO cloning
6

7. TOPO cloning is the enzyme DNA topoisomerase I, which functions
both as a restriction enzyme and as a ligase.
Its biological role is to cleave and rejoin DNA during replication.
Vaccinia virus topoisomerase I specifically recognizes the pentameric
sequence 5´-(C/T)CCTT-3´ and forms a covalent bond with the
phosphate group attached to the 3´ thymidine. It cleaves one DNA
strand, enabling the DNA to unwind.
The enzyme then religates the ends of the cleaved strand and releases
itself from the DNA.
To harness the religating activity of topoisomerase, TOPO vectors are
provided linearized with topoisomerase I covalently bound to each 3´
phosphate.
This enables the vectors to readily ligate DNA sequences with
compatible ends The ligation is complete in only 5 minutes at room
temperature7

8. TOPO cloning
8

9. Zero Blunt TOPO cloning of blunt-end DNA
9

10. TOPO TA cloning of Taq-amplified DNA
Efficiency rate of 95%10

11. Directional TOPO cloning of blunt-end DNA
Fast and simple directional PCR cloning strategy with >90%
efficiency11

12. 3 Steps in TOPO Cloning
NOTE: Do not add 5´ phosphates to your primers for PCR.12

13. SEQUENCE AND LIGATION-INDEPENDENT CLONING (SLIC)
• Was developed by Li in 2007 and published in
Nature Methods.
13

14. SEQUENCE AND LIGATION-INDEPENDENT CLONING (SLIC)
Simple
Cost-effective
Time-saving
Versatile
Highly efficient and directional cloning can be achieved by direct
bacterial transformation 2.5 min after mixing any linearized vector,
insert(s) prepared by PCR, and T4 DNA polymerase in a tube at room
temperature
One-step sequence- and ligation-independent cloning (SLIC) is a
14

15. SEQUENCE AND LIGATION-INDEPENDENT CLONING (SLIC)
If cloning methods had personalities, SLIC (sequence- and
ligation-independent cloning) would be a true rebel
Unlike other forms of cloning, SLIC does not require
restriction enzymes or a ligase
Ligation-independent cloning (LIC) is based on the 3′-to-5′
exonuclease activity of T4 DNA polymerase (not ligase)
One-step SLIC utilizes only T4 DNA polymerase
15

16. Overview of one-step SLIC. (A) Schematic diagrams of one-step SLIC. (B) Partial sequences
of the vector and insert.
Jae-Yeon Jeong et al. Appl. Environ. Microbiol. 2012;78:5440-5443
16

17. (A) Schematic diagrams of one-step SLIC
17

18. (B) Partial sequences of the vector and insert. The arrows below the
sequences indicate the forward and reverse primers used to amplify
the insert. Homologous regions are in the same color. The BamHI
restriction site is in bold.
18

19. (C) Restriction map of the vector and insert. (D) Analysis of
recombinants. Plasmid DNAs purified from 22 independent colonies
19

20. GOLDEN GATE CLONING (Golden Gate assembly)
20
 First discovered in 1996
Main components of this cloning technique are
• Type IIs restriction enzymes
• T4 DNA ligase
 As digestion and ligation can be done in one 30-minute
reaction.
 It make seamless (scarless) assembly of DNA fragments
 reaction is essentially irreversible

21. GOLDEN GATE CLONING (Golden Gate assembly)
21
 Allows to simultaneously and directionally assemble multiple
DNA fragments
 The accuracy of the assembly is dependent on the length of
the overhang sequences. Therefore, Type IIS REases that
create 4-base overhangs (such as BsaI/BsaI-HF®, BbsI/BbsI-HF,
BsmBI and Esp3I) are preferred

22. Type IIS Restriction Enzymes
Type IIS restriction enzymes recognize asymmetric DNA
sequences and cleave outside of their recognition
sequence. They are useful for many applications, including
Golden Gate Assembly
They can create non-palindromic overhangs
List of some Type IIS Restriction Enzymes found in in below site
https://www.neb.com/tools-and-resources/selection-charts/type-iis-
restriction-enzymes
22

23. Golden gate cloning protocol
23

24. 24

25. GoldenGate.neb.com
25

26. Advantages of Golden Gate Cloning
Scalability (the capacity to be changed in size or
scale)
Unique 4 base overhangs can be used to assemble
multiple fragments - up to 10 fragments are
commonly assembled in a single reaction (scalability)
Less expensive than many commercial cloning
methods
26

27. Advantages of Golden Gate Cloning
Overhangs specify the desired order of fragments
The popular Gateway cloning system produces constructs with an attB
recombination scar encoding eight amino acids, but Golden Gate
assembly can be designed to be scarless
Golden Gate Assembly has been widely used in the construction of
custom-specific TALENs for in vivo gene editing
27

28. Disadvantages of Golden Gate Cloning
• Golden Gate cloning is not 100% sequence-
independent: to avoid undesired digestion, the Type
IIS site used must not be present within the
fragments you seek to assemble
One way to work around this is to “domesticate” your
fragment:
PCR-based amplification can be used to create silent
point mutations at internal recognition sites thus
eliminating these from your gene of interest
 Another important consideration is the design of
flanking overhangs. Although there are theoretically
256 distinct flanking sequences, sequences that
differ by only one base may result in unintended
ligation products.
28

29. References
• Cermak, Tomas, et al. “Efficient design and assembly of custom TALEN and other TAL effector-based
constructs for DNA targeting.” Nucleic acids research (2011): gkr218. PubMed PMID: 21493687.
• Engler, Carola, and Sylvestre Marillonnet. “Golden gate cloning.” DNA Cloning and Assembly Methods
(2014): 119-131. PubMed PMID: 24395361.
• Engler, Carola, Romy Kandzia, and Sylvestre Marillonnet. “A one pot, one step, precision cloning
method with high throughput capability.” PloS one 3.11 (2008): e3647. PubMed PMID: 18985154.
• https://bitesizebio.com
• Lee, Jae H., et al. “Sequential amplification of cloned DNA as tandem multimers using class-IIS
restriction enzymes.” Genetic analysis: biomolecular engineering 13.6 (1996): 139-145.
• Li, Mamie Z., and Stephen J. Elledge. “Harnessing homologous recombination in vitro to generate
recombinant DNA via SLIC.” Nature methods 4.3 (2007): 251-256. PubMed PMID: 17293868.
• Li, Mamie Z., and Stephen J. Elledge. “SLIC: a method for sequence-and ligation-independent
cloning.” Gene Synthesis: Methods and Protocols (2012): 51-59. PubMed PMID: 22328425.
• Novel approach to molecular cloning and polynucleotide synthesis using vaccinia DNA
topoisomerase. Shuman S. J Biol Chem. 1994 Dec 23;269(51):32678-84. PubMed PMID: 7798275.
• Shuman S. “Recombination mediated by vaccinia virus DNA topoisomerase I in Escherichia coli is
sequence specific.” Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10104-8. PubMed PMID: 1658796.
PubMed Central PMCID: PMC52876.
29