bs2009 practicals 2008

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Virtual Genetics Education Centre: http://www.le.ac.uk/ge/genie/vgec/index.html

VGEC: Student Notes

RESTRICTION ENZYME MAPPING OFTHE (lambda) PHAGE GENOME

This is a paper exercise where you will use images of restriction enzyme digestion data to construct aphysical map of the phage lambda genome.

INTRODUCTION

Restriction endonucleases are powerful tools for the molecular analysis of complex genomes such as thoseof mammals. These enzymes can be isolated from a wide variety of micro-organisms and have the propertyof cutting both strands of double-stranded DNA only at a specific nucleotide sequence, usually 4-6 basepairs long. A considerable number of different restriction endonucleases are now available, each onecapable of cleaving DNA at a different specific sequence.

This exercise will show how these enzymes can be used to cleaveDNA, a linear DNA molecule 48500 bplong, into various numbers of discrete fragments depending on the enzyme used. By using combinations ofdifferent restriction endonucleases, these fragments can be ordered into a physical map of cutting sitesalong DNA.

BACKGROUND INFORMATION

AGAROSE GEL ELECTROPHORESIS

The success of making maps of DNA using restriction endonucleases depends of course on having amethod for resolving DNA fragments of different sizes. For example, if an enzyme E cuts DNA at two sitesas below;

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 ismost readily done by agarose gel electrophoresis (GENIE video of gel eletrophoresis). After digestion withthe restriction enzyme, the DNA sample is loaded into a slot in a slab of agarose gel containing ethidiumbromide. An electric current is passed through the gel and the DNA moves through the gel, small fragmentsmoving most rapidly and larger fragments migrating more slowly. After electrophoresis, DNA bands can bedetected under UV light by the fluorescence of ethidium bromide bound to the DNA. By running a mixture ofDNA 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.
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OUTLINE OF THE EXERCISE

You are provided with photographs of gels that have been run. The exercise is best if divided into a numberof stages:

Stage 1:Examine the two photographs provided. One is of single and double digestions of lambda DNAcarried out with the enzymes EcoRI, KpnI, XbaI and XhoI. The second photograph contains data withrespect to a series of partial digestions.You need to determine the sizes of the fragments generated by restriction enzyme digestion, and begin towork out the restriction map of phage lambda. You should also begin to think about the strategy forassembling the map.Stage 2:The maps should now be completed as far as possible using the data from the first photograph. Atthis stage there will be uncertainties in the map. Some of these uncertainties can be resolved using deletionmutants of lambda. The second photograph depicting digests of wild-type DNA and two different deletionmutants of 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 Digestsphage restriction mapping experimentandcontains two similar gel images side-by-side with a legend to the left.

The second sheet is labelled Partials & Deletions phage restriction mapping experimentandcontains two dissimilar gel images with the legend running down the centre of the sheet between thegels.

2. Start with the Complete Digestssheet.The DNA samples electrophoresed on the two gels areidentical only the electrophoresis conditions differ. The left gel has been run in a way that allowsyou 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 ofthe sizes of the very largest DNA fragments.

3. You MUSTdeal with each gel separately when determining the sizes of the DNA fragments and plotseparate 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-logarithmicgraph 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 itMUSTpass through all of the points. If you find the task impossible, its probable that you have

plotted one or more points in the wrong position.

The marker DNA in track 1 is DNA digested with the restriction enzyme HindIII(x HindIII) which produces a set of fragments of which only the largest 7 are normally easily visible:

23.1 kb9.4 kb6.6 kb4.4 kb2.3 kb2.0 kb0.56 kb

Track 2 contains undigested DNA (48.5 kb) which, although more than double the size of the largestsize 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 DNA digests (tracks 312) and use the standard curve to calculatetheir 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 bedifficult to determine the sizes of the very largest fragments with any accuracy. It is best to add

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together the sizes of all but the largest fragment and then subtract the sum from 48.5 kb to obtain thelargest fragment size.

5. Once you have made an attempt to do this, discuss your results with your tutor. You will then besupplied with a list of correct fragment sizes. These correct sizes have been rounded to the nearest100 bp (0.1 kb) and should be used for the construction of the map.

6. Deduce the possible arrangement of cleavage sites in DNA. The following approach is suggested:-

(a)

How many DNA fragments are produced by KpnI?

Do their combined lengths equal that of DNA?

How many KpnI cleavage sites are there?

What are the 3 possible arrangements of KpnI sites within the map?

It should be evident from a comparison of tracks 9 and 10 that the KpnI sites in DNA are containedwithin a single EcoRI digest fragment. Only one of the EcoRI fragments seen in track 9 is affectedby digestion with KpnI in track 10.

The left-hand gel of the Partials & Deletionsphotographdepicts a series digests of DNA thatare complete with respect to digestion with EcoRI, but are partial with respect to KpnI (tracks 210).The DNA in track 2 of the partial-digest gel is completely digested with both enzymes and

corresponds to track 10 on the complete-digest gels.From the partial and complete digests of (x EcoRI) with KpnI, deduce the KpnI cleavage patternwithin the largest EcoRI fragment. There is no need to plot a standard curve you will be providedwith sizes of the fragments that are important for working out the map. This should give you the fullKpnI map plus the map position of the largest EcoRI fragment.

(b)

How many fragments are produced by XhoI?

How many XhoI sites are there?

From the KpnI + XhoI double digest, fit the XhoI site(s) on to the KpnI map.

(c)

How many XbaI cleavage sites are there?

Fit the XbaI site(s) on to the KpnI map.

(d)

How many EcoRI fragments are there?(CAUTION- two of the fragments run close together giving a single more-intense band under someelectrophoresis conditions)

How many EcoRI sites are there?

From double digests with EcoRI and another enzyme, try to locate as many EcoRI sites as possibleon the KpnI, XhoI, XbaI restriction map.

(e) Draw the map to scale to the extent that you can and describe any remaining uncertainties.

MAPPING THE DELETION MUTANT DNAs

To resolve the remaining uncertainties it is necessary to consider mapping data with respect to twonaturally-occurring mutants of b519 and b538 each carry a different single internal deletion. Theright hand panel of the second photographdepicts digestions of wild-type , b519 and b538 DNAseach with EcoRI and EcoRI + KpnI (tracks 12-22). Analyse the data and deduce the size and approximateposition of each deletion. Comparison of the EcoRI single digests of each of the DNAs (tracks 13, 15 and17) is particularly helpful in resolving any uncertainties with respect to the order of the EcoRI fragments.

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QUESTIONS1. Using the DNA fragment sizes for wild type that you were provided with, describe how the

restriction map is deduced and draw the map to scale, indicating the location of the cut sites foreach of the restriction enzymes.

2. Describe the remaining uncertainties in the map derived using the complete-digest data provided forwild-type .

3. Explain how the deletion data resolve the uncertainties.4. What can you say about the role of the DNA in the deleted regions of the genomes of the two

deletion mutants?

5. State any remaining uncertainties and devise an experiment to resolve them.

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