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INTRODUCTION
Restriction endonucleases are powerful tools for the molecular analysis of complex genomes such as those of
mammals. These enzymes can be isolated from a wide variety of micro-organisms and have the property of cutting both
strands of double-stranded DNA only at a specific nucleotide sequence, usually 4-6 base pairs long. A considerable
number of different restriction endonucleases are now available, each one capable of cleaving DNA at a different
specific sequence.
This exercise will show how these enzymes can be used to cleave λ DNA, a linear DNA molecule 48500 bp long, into
various numbers of discrete fragments depending on the enzyme used. By using combinations of different restriction
endonucleases, these fragments can be ordered into a physical map of cutting sites along λ DNA.
BACKGROUND INFORMATION
E E
1 2 3
we then need to be able to resolve the three fragments produced (1, 2, 3) and to measure their sizes. This is most
readily done by agarose gel electrophoresis ( GENIE video of gel eletrophoresis). After digestion with the restriction
enzyme, the DNA sample is loaded into a slot in a slab of agarose gel containing ethidium bromide. An electric current
is passed through the gel and the DNA moves through the gel, small fragments moving most rapidly and larger
fragments migrating more slowly. After electrophoresis, DNA bands can be detected under UV light by the fluorescence
of ethidium bromide bound to the DNA. By running a mixture of DNA fragments of known size alongside an unknown
sample, a fragment size calibration curve can be constructed and the sizes of other fragments estimated with
reasonable accuracy.
OUTLINE OF THE EXERCISE
You are provided with photographs of gels that have been run. The exercise is best if divided into a number of stages:
Stage 1: Examine the two photographs provided. One is of single and double digestions of lambda DNA carried out
with the enzymes EcoRI, KpnI, XbaI and XhoI. The second photograph contains data with respect to a series of partial
digestions.
You need to determine the sizes of the fragments generated by restriction enzyme digestion, and begin to work out the
restriction map of phage lambda. You should also begin to think about the strategy for assembling the map.
Stage 2: The maps should now be completed as far as possible using the data from the first photograph. At this stage
there will be uncertainties in the map. Some of these uncertainties can be resolved using deletion mutants of lambda.
The second photograph depicting digests of wild-type λ DNA and two different deletion mutants of λ DNA will help you
to resolve the restriction map.
PROCEDURE
1. You will be provided with two sheets of photographs of electrophoresis gels. The first sheet is labelled ‗―Complete
Digests‖ — phage λ restriction mapping experiment‘ and contains two similar gel images side-by-side with a legend to
the left. The second sheet is labelled ‗―Partials & Deletions‖ — phage λ restriction mapping experiment‘ and
contains two dissimilar gel images with the legend running down the centre of the sheet between the gels.
2. Start with the “Complete Digests” sheet. The DNA samples electrophoresed on the two gels are identical — only
the electrophoresis conditions differ. The left gel has been run in a way that allows you to determine the sizes of the
small DNA fragments. The right gel is better suited to the determination of the sizes of larger DNA fragments. Neither
gel is ideal for the determination of the sizes of the very largest DNA fragments.
3. You MUST deal with each gel separately when determining the sizes of the DNA fragments and plot separate
standard curves for each. Measure the distance migrated by each marker DNA fragment. For each marker fragment,
plot its size against the distance that it has migrated on semi-logarithmic graph paper. Connect the points with a smooth
curve that actually passes through each of the points. The curve is not necessarily a parabola or a quadratic curve — it
may be a strange shape but it MUST pass through all of the points. If you find the task impossible, it‘s probable that you
have plotted one or more points in the wrong position.
The marker DNA in track 1 is λ DNA digested with the restriction enzyme HindIII (λ x HindIII) which produces a set of
fragments of which only the largest 7 are normally easily visible:
23.1 kb
9.4 kb
6.6 kb
4.4 kb
2.3 kb
2.0 kb
0.56 kb
Track 2 contains undigested λ DNA (48.5 kb) which, although more than double the size of the largest size marker
fragment in lane 1, migrates only marginally less slowly.
4. Once you have plotted the standard curve for the DNA marker fragments, measure the mobilities of fragments in the
experimental λ DNA digests (tracks 3–12) and use the standard curve to calculate their sizes. You will need to use the
data from both gels to obtain accurate sizes for the fragments. Keep in mind that each fragment has only one size
whatever the electrophoresis conditions. It will be difficult to determine the sizes of the very largest fragments with any
accuracy. It is best to add
together the sizes of all but the largest fragment and then subtract the sum from 48.5 kb to obtain the largest fragment
size.
5. Once you have made an attempt to do this, discuss your results with your tutor. You will then be supplied with a list
of correct fragment sizes. These correct sizes have been rounded to the nearest 100 bp (0.1 kb) and should be used for
the construction of the map.
6. Deduce the possible arrangement of cleavage sites in λ DNA. The following approach is suggested:-