different types of cloning,gateway cloning,infusion cloning,golden gate cloning,ta cloning

1. CLONING & TYPES OF CLONING
SWAPNIL MISHRA
M.S.(PHARM) ( 1st Year) PT
NIPER - KOLKATA

2. FLOW OF PRESENTATION
• WHAT IS CLONING ?
• RECOMBINATION CLONING ?
• GATEWAY CLONING ?
• LAMBDA PHAGE RECOMBINATION IN E.COLI
• GOLDEN GATE CLONING
• INFUSION CLONING
• TA CLONING
• GLOSSARY
• REFERENCES

3. WHAT IS CLONING ?
 The process of generating a genetically identical copy of a cell or an organism . It
happens often in nature.
 For example- when a cell replicates itself asexually without any genetic alteration or
recombination.
 Prokaryotic organisms such as bacteria creates genetically identical duplicates of
themselves using binary fission or budding.
 Eukaryotes such a humans, all the cells that undergoes mitosis.
 Such as skin cells & cells lining GI tract are clones , the only exceptions are gametes ,
which undergo meiosis and genetic recombination.

4. RECOMBINATION CLONING ?
It is a universal cloning technique based on site specific recombination that is
independent of the insert DNA sequence of interest , which differentiate this method
from classical restriction enzyme based cloning methods.
ADVANTAGES-
 It enables rapid & efficient parallel transfer of DNA inserts into Multiple expression systems.
 Multiple entry clones of varied sequences can be simultaneously transferred into a single
expression vector to make specific kind.

5. GATEWAY CLONING ?
This system invented & commercialised by
Invitrogen life technologies since the late 1990s.
It is a molecular biology method that enables
researchers to efficiently transfer DNA fragments
between plasmids using an appropriate set of
recombination sequences,the ‘Gateway att’ sites &
two proprietary enzyme mixes called as “LR clonase”
& “BP clonase”.

6. Your Application
Gene1 Gene2 Gene3 Gene4
Your Application
Gene
Protein
Localization
Gene
Gene
Protein
Purification
Gene
RNAi
Gene
Cell-Free
Gene
Protein
interaction
Gene
Gene
Entry Clone
PCR
Gene
synthesis
ORF
collection
Library
Your
Source
GATEWAY CLONING SYSTEM
 Directional cloning
 Maintains Reading frame
 No restriction enzymes
 No ligation
 1hr,room temp reaction with >99%
efficiency
No resequencing
 Compatible with automation
Reversible reactions

7. It enables you to access virtually any expression system in just a
few steps.
It circumvents the roadblocks of traditional restriction enzyme cloning
No need for ligase, sub-cloning steps or the hours spent to screen
countless colonies.

8. GATEWAY SYSTEM relies
on five sets of specific
and non cross reacting
att sequences.
The specificity is given
by the 7 nucleotides of
the core region.

9. BASICS OF GATEWAY CLONING
 BP reaction- to create Invitrogen gateway entry clone.
 LR reaction- to create a gateway expression clone.
 One tube format- to create Gateway expression clone
from a PCR product.
 Gateway vector conversion- converting your favourite
cloning vectors to gateway technology.

10. BP REACTION
• Creating a Gateway entry clone from an attB-flanked PCR product is an easy 1 hour
reaction. See below for an overview of the set-up. For more detailed information, refer
to the manual.
• Add the following components to a 1.5 ml tube at room temperature and mix:
attB-PCR product (=10 ng/µl; final amount ~15–150 ng) 1–7 µl
Donor vector (150 ng/µl) 1 µl
TE buffer, pH 8.0 to 8 µl
• Thaw on ice the Invitrogen BP Clonase II enzyme mix for about 2 minutes. Vortex the BP
Clonase II enzyme mix briefly twice (2 seconds each time).
• To each sample (Step 1, above), add 2 µl of BP Clonase II enzyme mix to the reaction and
mix well by vortexing briefly twice. Microcentrifuge briefly.
• Return BP Clonase II enzyme mix to –20°C or -80°C storage.
• Incubate reactions at 25°C for 1 hour.
• Add 1 µl of the Proteinase K solution to each sample to terminate the reaction. Vortex
briefly. Incubate samples at 37°C for 10 minutes.

11. • Transformation
• Transform 1 µl of each BP reaction into 50 µl of Invitrogen One Shot OmniMAX 2 T1
Phage-Resistant Cells (Catalog no. C8540-03). Incubate on ice for 30 minutes. Heat-
shock cells by incubating at 42°C for 30 seconds. Add 250 µl of S.O.C. Medium and
incubate at 37°C for 1 hour with shaking. Plate 20 µl and 100 µl of each
transformation onto selective plates. Note: Any competent cells with a
transformation efficiency of >1.0 × 10 8 transformants/µg may be used.
• Transform 1 µl of pUC19 DNA (10 ng/ml) into 50 µl of One Shot OmniMAX 2 T1
Phage-Resistant Cells as described above. Plate 20 µl and 100 µl on LB plates
containing 100 µg/ml kanamycin, or the appropriate selection marker for your
donor vector.
Expected results
An efficient BP recombination reaction will produce >1500 colonies if the entire BP
reaction is transformed and plated.

12. BP REACTION

13. LR REACTION
• Transferring your gene from a Gateway entry clone to destination vector is an easy 1 hour reaction. See below for an
overview of the set-up. For more detailed information, refer to the manual.
• Add the following components to a 1.5 ml tube at room temperature and mix:
Entry clone (50-150 ng) 1–7 µl
Destination vector (150 ng/µl) 1 µl
TE buffer, pH 8.0 to 8 µl
• Thaw on ice the Invitrogen LR Clonase II enzyme mix for about 2 minutes. Vortex the LR Clonase II enzyme mix briefly
twice (2 seconds each time).
• To each sample (Step 1, above), add 2 µl of LR Clonase II enzyme mix to the reaction and mix well by vortexing briefly
twice. Micro centrifuge briefly.
• Return LR Clonase II enzyme mix to -20°C or -80°C storage.
• Incubate reactions at 25°C for 1 hour.
• Add 1 µl of the Proteinase K solution to each sample to terminate the reaction. Vortex briefly. Incubate samples at 37°C
for 10 minutes.
• Transformation
Follow the protocol as indicated for the BP reaction, except use the appropriate selection marker for the LB plates suited to your
destination vector (typically 100 µg/ml ampicillin).
Expected results
An efficient LR recombination reaction will produce >5000 colonies if the entire LR reaction is transformed and plated

14. LR REACTION

15. ONE TUBE FORMAT
• If you want to transfer your attB-flanked PCR product directly into an expression clone, you can easily combine the BP and
LR reactions using the following protocol. This will potentially eliminate the transformation and DNA isolation of the
Gateway entry clone.
• In a 1.5 ml micro centrifuge tube, prepare the following 15 µl BP reaction:
attB DNA (50-100 ng) 1.0–5.0 µl
attP DNA (Invitrogen pDONR vector, 150 ng/µl) 1.3 µl
BP Clonase II enzyme mix 3.0 µl
TE Buffer, pH 8.0 add to a final volume of 15 µl
• Mix well by vortexing briefly and incubate at 25°C for 4 hours.
Note: Depending on your needs, the length of the recombination reaction can be extended up to 20 hours. An overnight
incubation typically yields 5 times more colonies than a 1 hour incubation. Longer incubation times are recommended for
large plasmids (=10 kb) and PCR products (=5 kb).
• Remove 5 µl of the reaction to a separate tube and use this aliquot to assess the efficiency of the BP reaction (see below).
• To the remaining 10 µl reaction, add:
Destination vector (150 ng/µl) 2.0 µl
LR Clonase II enzyme mix 3.0 µl
Final volume 15 µl
• Mix well by vortexing briefly and incubate at 25°C for 2 hours.
Note: Depending on your needs, the length of the recombination reaction can be extended up to 18 hours.

16. • Add 2 µl of proteinase K solution. Incubate at 37°C for 10 minutes.
1.Transform 50 µl of the appropriate competent E. coli with 1 µl of the reaction.
2.Plate on LB plates containing the appropriate antibiotic to select for expression clones.
Assessing the efficiency of the BP reaction
1.To the 5µl aliquot obtained from “One-Tube” Protocol, Step 3, above, add 0.5 µl of
proteinase K solution. Incubate at 37°C for 10 minutes.
2.Transform 50 µl of the appropriate competent E. coli with 1 µl of the reaction. Plate
on LB plates containing the appropriate antibiotic to select for entry clones.

17. GATEWAY CONVERSION
• Converting your favorite set of cloning vectors to Gateway Technology is a fairly
straightforward protocol, and will ultimately allow you to streamline your cloning and
expression process.
To convert your cloning vector to a Gateway destination vector, you
will:
• Choose the appropriate reading frame cassette to use depending on your needs.
• Linearize the vector you wish to convert with a restriction enzyme of choice. If you use a
restriction enzyme that generates an overhang, you will need to blunt the ends.
• Remove the 5' phosphates from the vector using calf intestinal alkaline phosphatase.
• Ligate the reading frame cassette into your vector using T4 DNA ligase.
• Transform the ligation reaction into One Shot ccdB Survival Competent E. coli and select for
transformants.
• Analyze transformants.

18. ADVANTAGES OF GATEWAY CLONING
 Fast reactions- 1hour room temperature cloning reactions.
 Accurate result- cloning reactions achieve >95%efficiency to deliver the clone you
need.
 Versatile technology- easily shuttle DNA material / insert from vector to vector.
 Streamlined protocol- no need for resequencing use the same clone from target
identification to validation.

19. GATEWAY RECOMBINATION CLONING VS TRADITIONAL
RESTRICTION ENZYME CLONING
Steps GATEWAY CLONING RESTRICTION ENZYME
CLONING
Existing primers Yes No
Vector ready for cloning Yes No
Ligation reagents included Yes No
Competent cells separately Included Purchase separately: 0 hours
Prepare: upto 6hours
Vector clean up No Yes
PCR FRAGMENT cleanup No Yes
Recombination efficiency up to 95% ~50%

20. GOLDEN GATE CLONING ?
• The efficient and seamless assembly of DNA fragments, commonly referred to as
Golden Gate Assembly has its origins in 1996, when for the first time it was shown
that multiple inserts could be assembled into a vector backbone using only the
sequential or simultaneous activities of a single Type IIS restriction enzyme and T4
DNA ligase. Golden Gate Assembly and its derivative methods exploit the ability of
Type IIS restriction endonucleases (REases) to cleave DNA outside of the recognition
sequence. The inserts and cloning vectors are designed to place the Type IIS
recognition site distal to the cleavage site, such that the Type IIS REase can remove
the recognition sequence from the assembly.

21. • Unlike standard Type II restriction enzymes like EcoRI and BamHI, these enzymes cut
DNA outside of their recognition sites and, therefore, can create non-palindromic
overhangs. Since 256 potential overhang sequences are possible, multiple
fragments of DNA can be assembled by using combinations of overhang sequences.
In practice, this means that Golden Gate cloning is typically scar less. Additionally,
because the final product does not have a Type II restriction enzyme recognition
site, the correctly-ligated product cannot be cut again by the restriction enzyme,
meaning the reaction is essentially irreversible.
• A typical thermal cycler protocol oscillates between 37 °C (optimal for restriction
enzymes) and 16 °C (optimal for ligases) many times . While this technique can be
used for a single insert, researchers have used Golden Gate cloning to assemble
many pieces of DNA simultaneously.

23. Contd…

24. ADVANTAGES OF GOLDEN GATE CLONING
• The overhang sequence created is not dictated by the REase,
and therefore no scar sequence is introduced.
• The fragment-specific sequence of the overhangs allows orderly
assembly of multiple fragments simultaneously.
• The restriction site is eliminated from the ligated product, so
digestion and ligation can be carried out simultaneously.

25. INFUSION CLONING
In-Fusion Cloning is a highly efficient,
ligation-independent cloning method, based on the
annealing of complementary ends of a cloning insert
and linearized cloning vector. This technology ensures
easy, single-step directional cloning of any gene of
interest into any vector at any locus.

26. Contd…

27. BASICS IN INFUSION CLONING
 Think about your final construct-Choose the vector you want to modify and
envision your final, mutated construct (Figure 1; mutation shown in yellow).
 Design your primers-Design inverse primers that overlap each other by 15 bp at
their 5' ends and incorporate your desired deletion, substitution, or addition.
Specific guidelines for mutagenesis primer design are described below.
 Utilize the power of In-Fusion technology-Using an inverse PCR protocol,
amplify the vector with your new primers. Perform the In-Fusion Cloning reaction
using the PCR product. The linear DNA will re-circularize at the site of the 15-bp
overlap and will also contain your mutagenic changes. Use a portion of the In-
Fusion Cloning reaction to transform the Stellar Competent Cells according to the
In-Fusion HD Cloning Plus protocol.
 Obtain your final construct-Recover your mutant from the Stellar cells the
following day.

28. INFUSION CLONING PROTOCOL
Select your vector and identify the mutation site.
Design PCR primers as described above, keeping in mind these general guidelines:
- Primers should be 18–25 bases long. Insertions may require longer primers.
- Primers should be 40–60% GC.
- Primer Tms should be 58–65°C. The difference between forward and reverse primer Tms
should be ≤4°C.

29. Prepare CloneAmp HiFi PCR Master Mix:
CloneAmp HiFi PCR Premix 12.5 μL
Forward primer 200–300 nM
Reverse primer 200–300 nM
Template 0.1–5.0 ng
H2O As needed
Total reaction volume 25 μl

30. Linearize the vector by inverse PCR using a three-step PCR protocol and
CloneAmp HiFi PCR Premix.
Treat the PCR product with Cloning Enhancer to remove the circular double-
stranded template from the reaction. If your PCR product contains multiple
bands, gel-purify instead using the NucleoSpin Gel and PCR Clean-Up kit.

31. Assemble the In-Fusion Cloning reaction
Linear construct containing
your mutation
100 ng
In-Fusion HD Enzyme Premix 2 μl
H2O As needed
Total reaction volume 10 μl

32. Incubate the reaction at 50°C for 15 min.
Transform Stellar Competent Cells with 2.5 μl of the In-Fusion Cloning reaction
The next day, screen for mutants. You have a ≥95% chance of recovering your final
desired construct the very first time.

33. Contd…

34. TA CLONING ?
• TA cloning is one of the simplest and most efficient methods for the cloning of PCR
products. The procedure exploits the terminal transferase activity of certain
thermophilic DNA polymerases, including Thermus aquaticus (Taq) polymerase.
• Taq polymerase has non-template dependent activity which preferentially adds a
single adenosine to the 3'-ends of a double stranded DNA molecule, and thus most
of the molecules PCR amplified by Taq polymerase possess single 3'-A overhangs.
• The use of a linearized "T-vector" which has single 3'-T overhangs on both ends
allows direct, high-efficiency cloning of PCR products, facilitated by
complementarity between the PCR product 3'-A overhangs and vector 3'-T
overhangs.

35. • The TA cloning method can be easily modified so that the same T-vector can be
used to clone any double-stranded DNA fragment, including PCR products amplified
by any DNA polymerase, as well as all blunt- and sticky-ended DNA species.
• This technique is especially useful when compatible restriction sites are not
available for the sub-cloning of DNA fragments from one vector to another.
• Directional cloning is made possible by appropriate hemi-phosphorylation of both
the T-vectors and the inserts. With a single T-vector at hand, any DNA fragment can
be cloned without compromising the cloning efficiency.
• The universal TA cloning method is thus both convenient and labor-saving.

36. TA CLONING FUNCTIONING
 TA cloning is brought about by the terminal transferase activity of certain type of
DNA polymerase such as the Taq polymerase. This enzyme adds a single, 3'-A
overhang to each end of the PCR product. As a result, the PCR product can be
directly cloned into a linearized cloning vector that have single base 3'-T overhangs
on each end. Such vectors are called T- vectors.
 The PCR product with A overhang, is mixed with this vector in high proportion. The
complementary overhangs of a "T" vector and the PCR product hybridize. The result
is a recombinant DNA, the recombination being brought about by DNA ligase.

37. STEPS IN TA CLONING

38. GLOSSARY
att site- A defined length of DNA that constitutes a recombination site.There are 4
classes of att sites called attB,attP,attL,attR.
 ccdB gene- A counterselectable gene that allows for negative selection of
unwanted byproduct plasmid after recombination.
 Entry(pENTR)clone- A vector that contains your gene of interest flanked by attL or
attR sites.
Donor(pDONR)vector- Avector with attP sites flanking a counterselectable gene
that recombines with a gene of interest flanked by attB sites.
Destination(DEST)vector- An application geared vector with attR sites flanking a
counterselectable gene that will recombine with one or more entry clones.
Multisite Gateway Technology- A system that allows simultaneous assembly of
multiple DNA fragments into a single destination vector.

39. REFERENCES
• https://www.embl.de/pepcore/pepcore_services/cloning/cloning_method/recombina
tion/gateway/cloning.html
• https://www.thermofisher.com/in/en/home/life-science/cloning/gateway-
cloning.html
• https://www.takarabio.com/learning-centers/cloning/mutagenesis-with-in-fusion-
cloning

40. THANK YOU