Bob Tacon <bob_tcc at yahoo.com> wrote:
: Is there a systematic approach to restriction mapping? Especially when
: you are working with several enzymes it is getting difficult to find the
: possible combinations and determine the order of the restriction sites.
: All literature I found only uses simple digestions with not more than
: two enzymes.
: Example: A plasmid was cut with four different enzymes. Double and
: triple digestions were also performed. The results are:
: Cut with EcoRI 10
: Cut with HindIII 5 , 3 , 2
: Cut with with HaeIII 6.5 , 3.5
: Cut with BgI II 6 , 4
: Cut with HindIII + BgI II 4 , 3, 2 , 1
: Cut with HindIII + HaeIII 5, 2 , and 2 moles of 1.5
: Cut with HaeIII + BgI II 4 , 3.5 , 2.5
: Cut with HindIII + HaeIII + BgI II 4, 2 , 2 moles of 1.5 , 1
: How would you best approach such a restriction mapping problem?
: Bob
Um, this was actually pretty straightforward:
Hin Hae Hin Bgl
|---2----|---1.5--|---1.5--|--1--|---4---|
(Of course, since this is a plasmid, one would draw a circle, but that's
beyond my capabilities here.)
The way you figure this out: look at HindIII + BglII, for example. You
know that with HindIII alone you get fragments 2 and 3 so most likely
these weren't cleaved again by BglII. The remaining ones, 1 and 4, add to
five, which is the size of the other HindIII fragment. So from this
information you know that the map is:
Hin Hin Bgl
|---3--|--2--|--1--|---4---|
or
Hin Hin Bgl
|--2--|--3--|--1--|---4---|
You use the same logic to figure out the other pairs, then put all your
information together for all three enzymes to get the final map. You can
read more on this in any intro genetics book; mine was _An Introduction to
Genetic Analysis_ by Griffiths et al. These problems are really quite fun
and easy once you get them.
---
Jennifer L. Giel
Graduate Student in Microbiology, University of Wisconsin-Madison
http://www-personal.umich.edu/~jlgiel/
---
