Deletions can be confirmed in a variety of ways, three of which are shown below. They differ in the number and difficulty of PCR reactions needed, and in the amount of information each gives about the deletion.
The 3-step confirmation amplifies each end of the deletion (custom primer A or B in conjunction with PRY4) to check that the deficiency still has the correct breakpoints as predicted by the progenitor elements. It also involves the amplification of the area of white around the FRT site to determine the correct reconstitution of the white gene.
Three step confirmation.
The PCR's produce small products and the reactions are very reliable and cheap. This is how df's are routinely confirmed at DrosDel, although we are switching over to the 2-step system presently. The main problem with this system is that it assumes the reconstituted white is in the correct place and that a deletion has occurred, which may not always be the case.
The 2-step system confirms the ends of the deletion are okay and also that the white gene has successfully been reconstituted. It also shows that the FRT site you are amplifying is actually connected to the deletion you are looking at and not at some other random point in the genome. Because of the larger sizes of the PCR it's recommended that you use a longPCR with proof-reading DNA polymerase. The PCR reactions are pretty reliable, although we have had some problems with the 'soapier' buffers clogging up the purifiction filters, resulting in incomplete primer removal and poor sequence quality. We are currently investigating different buffers to reduce this problem.
Two step confirmation.
The one step confirmation is the best way of confirming a deletion as it amplifies across the entire region in one go, and the presence of a band suggests a deletion must have taken place. The large size of the PCR however means that longPCR has to be used, which is expensive and does not result in good (if any) amplification in many cases. If you only have one or two confirmations to do however use this method.
One step confirmation.