Expt008 : Restriction map

Before technology made it easy to sequence a gene, ways had to be found to describe a gene or a fragment of DNA. A technique widely used was the production of restriction maps which show the positions of several restriction sites in a fragment of DNA. The relative positions of restriction sites on a map are determined by double-digest experiments.

Method

Load Experiment Expt008. It contains 4 samples of DNA.

Sample 1 is the size marker we have already used,
Samples 2 to 4 are fragments of DNA of approximately 9000 bp.

Use double-digests to draw a map of the fragment in Sample 2, marked with the positions of the following restriction sites: AluI, BamHI, EcoRI, HindIII, PstI, SmaI and XbaI.

All the practical aspects of this exercise have been covered earlier. However, with this kind of problem, it is important to be well organised, and to keep a good record of what you have done, and of the results of each digest. You can use the Sample table provided.

Hint

A good method would be to start with a single digest of the fragment with each of the enzymes in turn.

Then a small number of double-digests should enable you to draw the map.

Results of the analysis of Sample 2

Digestion of the fragment with each enzyme individually shows that

BamHI, EcoRI, KpnI and XbaI cut the fragment once.

AluI, HindIII, PstI, and SmaI do not cut the fragment at all.

The gel with all the digests is shown in diagram 1.

Diagram 2 shows the BamHI (B), EcoRI(E), KpnI (K) and XbaI (X) digests alongside the size markers. The size of each marker is written on the diagram, to make it easier to measure the band sizes.

In a research lab the size of the size markers would be used to draw a standard curve of log size vs distance migrated. This would then be used to estimate the size of unknown fragments. You can do it this way if you want to, or you can find the fragment sizes by clicking on the well above the track you are interested in. This gives you a detailed view of the bands in that track, which includes the size and distance migrated of each band.

This table shows the sizes, in bp, of the fragments obtained after digest.

Fragments	BamHI	EcoRI	KpnI	XbaI
1 (biggest)	8221	6764	7061	4811
2 (smallest)	749	2206	1909	4159

Let's deal with each enzyme one at a time.

Bam H I

There is one BamHI site (B): BamHI releases a 749 bp fragment and a 8221bp fragment. The site can be placed on the map in either of these two positions:

Position of B
or

We choose the first option arbitrarily.

Eco R I

There is one EcoRI site (E): EcoRI releases a 2206bp fragment and a 6764bp fragment. The site can therefore be placed on a map in either of these two positions: 1 and 2.

Position of E: hypothesis

In the case of hypothesis 1, a double-digest (EcoRI + BamHI) would generate three fragments with sizes of 749bp, 1457bp, and 6764bp. The gel would therefore show the banding pattern in diagram 3a. The biggest fragment from the EcoRI cut is not cut by BamHI.

Hypothesis 1

In the case of hypothesis 2, the double-digest (EcoRI + BamHI) would generate three fragments with sizes of 749bp, 2206bp, and 6.15bp. The gel would therefore show the banding pattern in diagram 3b. The smallest fragment from the EcoRI cut is not cut by BamHI.

Hypothesis 2

To decide which of these hypotheses is correct and place the EcoRI site (E) on the map, relative to the BamHI site (B), we need to do a double-digest EcoRI + BamHI.

The digest is done as described in Expt007 , and the result is shown in Diagram 3. [The coloring and the arrows help to visualise which fragments are cut once and which are cut twice. The yellow arrows show how the fragments are cut.]

The banding pattern we get after the double-digest experiment confirms that the second hypothesis is correct.

Gel real Bam+Eco

At this stage the restriction map looks like this:

KpnI

There is one KpnI site (K): KpnI releases a 1909bp fragment and a 7061bp fragment (Diagram 2 and Table) The site can therefore be placed on a map in either of these two positions: 1 and 2.

Position of K: hypothesis

In the case of hypothesis 1, a double-digest (KpnI + BamHI) would generate three fragments with sizes of 749bp, 1161bp, and 7061bp. The gel would therefore show the banding pattern in diagram 4a. The biggest fragment from the KpnI cut is not cut by BamHI.

Hypothesis 1

In the case of hypothesis 2, the double-digest (KpnI + BamHI) would generate three fragments with sizes of 749bp, 1909bp, and 6312bp. The gel would therefore show the banding pattern in diagram 3b. The smallest fragment from the EcoRI cut is not cut by BamHI.

To decide which of these hypotheses is correct and place the KpnI site (K) on the map, relative to the BamHI site (B), we need to do a double-digest KpnI + BamHI.

The digest is done as described in Expt007 , and the result is shown in Diagram 4. [The coloring and the arrows help to visualise which fragments are cut once and which are cut twice. The yellow arrows show how the fragments are cut.]

The banding pattern observed after the double-digest experiment confirms that hypothesis 1 is correct.

KpnI+BamHI

K can be placed relatively to B like this:

Position of K

and we can combine these two results to draw a map with BamHI, EcoRI and Kpn I.

B, E, K

XbaI

There is one XbaI site (X): XbaI releases a 5000bp fragment and a ~4200bp fragment (Diagram 2 and Table) The site can therefore be placed on a map in either of these two positions: 1 and 2.

Position of X: hypothesis

In the case of hypothesis 1, a double-digest (XbaI + BamHI) would generate three fragments with sizes of 7490bp, 3402bp, and 4811bp. The gel would therefore show the banding pattern in diagram 5a. The biggest fragment from the XbaI cut is not cut by BamHI.

Hypothesis 1

In the case of hypothesis 2, the double-digest (BamHI + XbaI) would generate three fragments with sizes of 748bp,4062bp, and 4159bp. The gel would therefore show the banding pattern in diagram 5b. The smallest fragment in the EcoRI cut is not cut by BamHI. Note that the 4300 and 4200bp fragments have sizes too similar to be differentiated by this kind of gel. We would observe a thicker band.

To decide which of these hypotheses is correct and place the XbaI site (X) on the map, relative to the BamHI site (B), we need to do a double-digest XbaI + BamHI.

The digest is done as described in Expt007 , and the result is shown in Diagram 5. [The coloring and the arrows help to visualise which fragments are cut once and which are cut twice. The yellow arrows show how the fragments are cut.]

The banding pattern we get after the double-digest experiment confirms that hypothesis 2 is correct.

B, X double-digest

Following the same principle, it is easy to place the XbaI site relatively to Bam HI.

When we combine all the previous results, we can draw the final map with the four restriction sites and the distances they are separated.
Map

Your turn

Expt008 contains more samples of DNA. Try to draw their restriction maps. In some cases you may find that you can't come to any conclusion...

You will find the results here