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.
Likewise, if two orientation "B" phages are combined, a negative
C-test results.
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.
The "hybrid" DNA is much bulkier than either single-stranded DNA
and will migrate slower in agarose gel electrophoresis.
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.
Procede to LON-CAPA to answer the question for this Learning Module.
