Learning Module - Experiment 13

Orientation Analysis of topA Subclones by a Complementarity Test

In the last period we propagated recombinant M13mp19topA subclones.

Today, you will test for the orientation of the inserted DNA by a complementarity test (C-test). M13 phage from the supernatant of your cultures will be combined pairwise with "tester" phage of known orientation and heated in the presence of SDS and then cooled. What does the SDS do?

If the two phage have inserts in the same orientation (orientation "A"), no complementary DNA sequences are present on the two phage DNAs and no complementation, or "hybrid" formation occurs. This results in a negative C-test.

Two phages with Orientation A

Likewise, if two orientation "B" phages are combined, a negative C-test results.

Two phages with Orientation B

If, however, the two phage have different inserts, i.e. in the opposite orientation, complementation occurs between the inserted DNAs and a "hybrid" or "figure-8" molecule is formed.

One phage with orientation A and another with orientation B

The "hybrid" DNA is much bulkier than either single-stranded DNA and will migrate slower in agarose gel electrophoresis.

Gel with results of C-test A positive C-test is shown by the band indicated by the star. Sometimes neither tester phage will give a positive C-test (see sample 3).

The results are not always as unambiguous as shown above, and sometimes, neither "tester" phage will complement your unknown.