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Gateway Cloning

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Biological and Medical Sciences > Classification by Description > Reagents, Kits and Assays > DNA and RNA > Cloning


Editor:  Christopher Dyer
Branch: Sci-Wiki
Type: General Knowledge
Intention: For Publication
Read/Write Permissions: Open Access
Authors :
17.11.2010 - John Stewart   
27.10.2010 - Christopher Dyer  -  Sci-Mate   
21.09.2010 - Anne Dyer   


Gateway cloning is a very quick and reliable method to clone and sub-clone DNA derived from any source into a wide range of plasmid vectors.

This general knowledge Article and related Protocols (follow links below) are intended as a basic user's guide for getting started with the technique.

Contents

[edit] The Basic Reaction

Gateway is like most directional cloning mechanisms, in which restriction enzymes cut fragments of DNA to leave complementary sticky ends that are then ligated together. The key difference is that digestion and ligation are done in the same tube, saving time, and in combination with some very clever selection strategies, the process works with truly remarkable efficiency.

The technique is based upon the following two reversable (digestion and ligation) reactions:

B1-gene-B2              +   P1-ccdB-P2             <=>       L1-gene-L2       +         R1-ccdB-R2

In which, 'B1', 'B2', 'P1', 'P2', 'L1', 'L2', 'R1' and 'R1' are sequences for Gateway Lambda Phage Restriction Sites, whose complimentary sticky ends ligate fragments in the correct orientation for expression, tagging, sequencing, etc.

B sites recombine with P in the presence of BP clonase to form L and R sites respectively; and in the revese direction, L and R recombine in the presence of LR clonase to form B and P sites.

In terms of inserts and vectors, the above reactions corresponds to the following recombinations:

G-W PCR Product   +   Donor vector            <=>       Entry vector                +         junk fragment

Expression vector   +   Donor vector            <=>       Entry vector                +         Destination vector

Because the reactions are reversable, once a gene or sequence of DNA exists within this system, it can be very easily moved between vectors, see the Basic Gateway Cloning protocol.

The ccdB gene (inhibits growth of DH5-alpha bacteria) and variations in antibiotic resistance genes ensure that only 1 vector product will be viable following transformation (see Designing_a_Gateway_Cloning_Strategy).

[edit] Overview of Gateway Cloning

Essentially, the process is the same as with any cloning experiment:

  1. Design a cloning strategy to put the gene or sequence of interest into the correct context, such as a vector for expression, tagging, sequencing, etc (see Designing a Gateway Cloning Strategy).
  2. Digest and ligate the DNA (see Basic Gateway Cloning).
  3. Transform bacteria and grow overnight on selection plates.
  4. Select a mono-clonal colony from which DNA can be prepared for restriction and/or sequence analysis.

[edit] Key Advantages

Considerable time is saved by not having to digest, separate and purify DNA fragments prior to ligation. This can take anywhere up to 24 hours, depending on how long the digestion is, and how slow the gel needs to run (esp. if using low melting point gels). If the ligation doesn't work, then multiply this time.

More time is saved because the combined reaction takes only 1 hour, without the need for overnight ligation.

The real time saver is, however, that this process works first time virtually everytime, which can save days, weeks and even months of optimising and searching for solutions.

Another important plus is that because it works so well, you can be sure that if it doesn't work, there is something fundamentally wrong. This means it is no longer sensible (in contrast to traditional cloning) to simply repeat the experiment. An impressive number of controls are available to help identify the few variables where a problem could exist (including the user).

BP clonase is expensive, but it works first time almost every time. If you calculated the real costs associated with normal cloning (time required, more reagents, repetition, etc), this would be far far higher than this one reagent.

[edit] Related Articles and Protocols

Basic_Gateway_Cloning

Designing_a_Gateway_Cloning_Strategy

Designing_Gateway_PCR_Primers

Gateway_Lambda_Phage_Restriction_Sites

[edit] Disclaimer:

No money or benefit has accrued to the author, other than those inherent in the technology. I/we believe quite simply in the significant value of this technique to other molecular biologists. Nor does the author take resonsibility for your experimental results or outcomes using the technique.

Discussion, editing and addition to these articles is welcome.





  • This page was last modified 20:11, 17 November 2010.
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