genomic library

1. Imagine you have an ordered library of mouse genomic BAC clones (of roughly 200-250 kb each), named BAC-1, BAC-2 etc. ("1", "2" etc. in the table below). You also have a set of STS markers, named a, b, c, d etc. You are trying to order the clones by STS content mapping. The table below presents all of the BAC clones that include STS-a. It is somewhat realistic that a particular sequence might be found in at least 8 clones because the library has to have many copies of any one region to provide good coverage. The presence (+) or absence (-) of an STS in a particular BAC clone (which could be determined experimentally by hybridization or whether you produce a PCR product from the STS primers) is indicated.

a b c d e f g h

1 + + - + + - - -
2 + + - - + - - -
3 + - - + - + - +
4 + - + + - + - +
5 + + - + + - - -
6 + + - - + - + -
7 + - - + + + - -
8 + - - + - + - -

(i) Draw the overlap of the named BAC clones that you can deduce from the above data. Explain your method of getting the results

(ii) The STS markers you use in such a mapping project would essentially be random, unique sequences (you would know nothing about their positions). Does the mapping process (in this example) tell you the exact ORDER of STS markers AND does it tell you their DISTANCE apart?

(iii) I only tabulated the BACs containing STS-a and only showed results for other STS markers where at least one was positive among this group of BACs. In the real mapping experiment there would be thousands of BACs and each would be tested for thousands of STS markers. How would you extend your map to the left or the right using the additional information that would be available? (Do not just say use a computer- you have to tell the computer what to look for first).

(iv) An STS is supposed to be a unique sequence (not a repeated sequence).
(a) Before you use an STS in the mapping experiment described here how could you find out if it is unique?
(b) If you mistakenly used an STS that was not unique in the above experiment it would lead you to group sets of BACs together in an incompatible way. Hence, it would be very useful to eliminate data from such an STS before starting to group BACs. By looking at the data from this type of mapping experiment how might you detect non-unique STSs?

(v) Imagine your BAC library were much smaller and so you only had data for BACs 1, 2 and 3. Would you still be able to determine with near certainty that they overlapped (and crudely how they overlapped)? Think also about how your mapping would go if you only used half as many STS markers. From that, comment on whether this mapping method depends on a critical number of BACs or markers AND what extra information is provided by using more BACs or more markers.