Biomedical Engineering Reference
In-Depth Information
against either the original bait or an irrelevant control. In addition, the isolated prey plasmid
would be sequenced to identify the encoded interaction partner. This process can be signif-
icantly streamlined by incorporating gap repair cloning and YC PCR procedures (Sections
8.2.3 - 8.2.5). The procedure consists of four sequential processes.
•
Construction of bait clones in PJ69-4A (MATa) (as described in Sections 8.2.2 and 8.2.3).
•
Mating of bait clones against a high-complexity prey library. Many good commercial
libraries are now available in Mat
α
yeast strains, such as the Matchmaker libraries
(Clontech). However, if pretransformed libraries are being used, remember the potential
complications of using libraries in the Y187 host strain (Note j, Protocol 8.4).
•
Removal of false positive interactions. This involves testing for the activation of multiple
reporter genes and reconfirming bait specificity in fresh yeast. By repeating the process
in fresh yeast, it is possible to eliminate false positives that arise from spontaneous host
mutations.
•
Identification of true positive prey proteins. This requires the sequencing of the cDNA
insert contained within the prey vector.
As the sequences that flank the inserts in prey libraries are known, it is possible to
design PCR primers that include
30 nucleotides that flank each side of the multiple
cloning site. When these primers are used to perform YC PCR reactions on positive diploid
colonies, the resulting PCR product will include the encoded prey protein flanked by 5
and 3
vector-specific sequences. As such, this PCR product can then be used to perform
a gap repair reaction when co-transfected with an appropriately linearized empty prey
vector. Using this approach, prey vectors can be rapidly regenerated in fresh yeast using
the sequential protocols described in Sections 8.2.1 - 8.2.6. Once prey clones have been
regenerated by gap repair, they can be mated against the original bait clone to reconfirm the
interaction, or against a non-specific bait to establish partner specificity (follow procedures
outlined in Section 8.2.7).
An additional benefit of this approach is that prey-specific PCR products can be directly
sequenced to establish the identity of interacting prey proteins. Using these adaptations, a
single person can perform and process 5 - 10 library screens at a time, which provides a
suitable capacity for most biologically focused research projects.
∼
8.3 Troubleshooting
•
The key to any good Y2H screen is the inclusion of a full spectrum of controls. In
targeted matrix Y2H experiments, the range of different interactions being investigated
often provides good internal controls for both background auto-activation and partner
specificity. However, in library screens this is not possible and it is essential that all
positive interactions must be retested in fresh yeast using the original bait and an irrelevant
partner as control.
•
We have noticed that, in our hands, YC PCR reactions become more problematic as
yeast colonies age. We have also found that when yeast is maintained on plates for
several days, multiple bands may be observed in YC PCR reactions. Therefore, whenever
possible, perform YC PCR reactions on fresh (
∼
2 - 3-day-old) colonies.
•
To reduce the occurrence of spontaneous auto-activating mutations, it is advisable to use
fresh stocks of host yeast when generating bait and prey clones. It is also advisable to
