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LAB 4: DNA ANALYSIS Theory and Procedures

OBJECTIVES Upon completion of this lab you should be able to: 1. Define: restriction enzyme or restriction endonuclease and identify recognition sites on DNA for BamHI, EcoRI and HindIII. 2. Understand how fragments of DNA of different lengths are produced by each restriction endonuclease. 3. Understand how these fragments are separated according to size by forcing them to move through a gel along an electrical gradient. 4. Determine the size of the digested DNA fragments by comparison with a known digest and a 1 kb DNA ladder plotted on semi-log graph paper. HOMEWORK AND PRE-LAB PREPARATION Read BioSkills #9 found on pages B10-B13 in the blue appendix at the back of your text-book Read Using Restriction Endonucleases and Ligases to Cut and Paste DNA in section 19.1 of your textbook (page 368 - 373) Read through this entire lab. Prepare any tables that might be required in your lab notebook. Complete the Pre-Lab Assignment work sheet (included in this manual on page 11) Complete the Pre-Lab Quiz on AVENUE Bring at least two sheets of 3-cycle semi-log graph paper (available on AVENUE) to the lab.

INTRODUCTION Restriction endonucleases (or restriction enzymes) are produced by prokaryotic organisms to protect them against foreign DNA. One source of foreign DNA is the bacteriophage (also called just phage) virus which infects bacteria. These viruses inject their DNA into the bacterial cell and co-opt the cellular machinery to produce more phage particles. Many different types of bacteria produce restriction enzymes (so called because they restrict the virus ability to infect the cell) which are able to cut the viral DNA into pieces too small to carry on the viral infection. Each of these enzymes acts on very specific areas or base pair sequences in the viral DNA. These sequences are known as recognition or restriction sites. Recognition sites are symmetrical sequences. The restriction endonucleases attach to the DNA strand in dimers each cuts one strand- and they orient themselves in opposite directions so that the same bond between the same two bases is cut on each of the polynucleotides (see Figure 4.1)

Figure 4.1: Recognition sequence of a restriction enzyme. The restriction enzyme Hind III breaks the phosphodiester linkage between two adjacent adenine residues, but only when they are found in exactly the symmetrical sequence shown in the recognition site.

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Some types of restriction enzymes produce a blunt ended cut, but more often the cut is off-centre and fragments like the ones shown in Figure 4.1 are produced with single stranded or sticky ends. The ability of a restriction enzyme to cut DNA depends entirely on the presence of at least one of the recognition site sequences for that enzyme in the DNA. Different restriction enzymes have different recognition sites, as shown in Table 4.1. Table 4.1: Restriction recognition sites for EcoRI, BamHI, and HindIII.

The frequency of recognition sites such as these occurring in viral DNA varies from virus to virus. For example, EcoRI cuts the DNA of SV40 virus only once (the viral genome is 5243 base pairs) whereas HindIII recognizes and cuts the same DNA at five sites. The fragments produced by digesting SV40 viral DNA with the two restriction enzymes, then, are quite different in size and number. In the lab today, you will be digesting the DNA from a phage known as (lambda) which infects E. coli. Its entire genome is 48,502 base pairs (bp) in length. You will be digesting the DNA with three different restriction enzymes, EcoRI, BamHI and HindIII, each with 5 or more recognition sites in the genome. The digested fragments will be loaded into wells of a 0.8% agarose gel and an electrical current will be run through the gel. Because of the many negative charges on the phosphate groups of the DNA molecule, the fragments will tend to move towards the positive pole. The smaller fragments move faster through the pores of the gel than the larger ones, and with time the fragments become separated into distinct bands. Each restriction enzyme produces a characteristic pattern of bands which are made visible under UV light by staining with ethidium bromide. By convention, DNA gels are read from left to right with the sample wells at the top. The area below the well in which the bands of digested DNA will move is called a lane. All the bands in a lane represent the fragments produced by a single restriction enzyme. Reading across the lanes will identify fragments of similar size produced by different restriction enzymes. Fragments of DNA will migrate in the electrical field at rates which are inversely proportional to the log10 of their molecular weight (to simplify the analysis, base pair numbers are substituted for molecular weight). Plotting the distance a band has traveled from the sample well on semi-log graph paper will enable you to estimate its base pair length. Table 4.2 shows the fragment sizes for DNA digested with Hind III. You will use this information and that obtained from a 1 kb DNA ladder to determine the standard relationship between base pair lengths and distance migrated in the gel.

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Figure 4.2: Linear restriction maps of the genome for each restriction endonuclease you will be using today. These restriction fragments are generated when the phage genome is in linear form. In Figure 4.2, the first line shows the linear genome which is 48,502bp long. The second line shows the BamHI restriction sites. The numbers above the line represent the location of the cut, expressed as bp number beginning from 0 on the left. The numbers below the line show the size of the fragment which is produced when the DNA is cut at these positions. The third and fourth lines show the same information for EcoRI and HindIII. List all the fragments in the table below in the order they would appear on your gel, from largest to smallest. The phage DNA can exist as a circular, as well as a linear, molecule. At each end of the linear molecule is a single stranded sequence of 12 nucleotides called the COS site. The COS sites at either end of the DNA are complementary, thus, when they come together, they can base pair to form a circular molecule. When the phage DNA is circular, the two end fragments are actually found joined together. For example, in the HindIII digest, the 23,130 bp fragment from one end of the molecule will be joined to the 4361 bp fragment from the other end of the molecule (See Figure 4.3). Therefore, you would expect to see a fragment that is 27,491 bp in size instead of the 23,130 bp fragment and the 4361 bp fragment. The expected fragments resulting from the circular form of HindIII digested phage is shown in Table 4.2, below. Often, phage DNA exists in BOTH forms. The commercially prepared DNA you will use in lab today is a mixture of both the linear and circular forms. This will cause more bands to appear on your gel than you could predict with only linear DNA molecules and the partial loss of other predicted fragments.

Page 4 48.5 / 0 44.1

37.6 37.5 36.9

phage DNA (48.5 kb) digested with HindIII

27.5 25.2

23.1

Figure 4.3: Circular restriction map for phage DNA digested with HindIII. (Note; the base pair designations are expressed as kilobase (or 1000 base) pairs. This is a common way of expressing size in DNA.) Table 4.2: Expected Fragment Sizes for DNA (in linear form, circular form, and combination) digested with Hind III.

HindIII Fragments (from LINEAR form) Why is this box empty? 23, 130 bp 9, 416 bp 6, 557 bp 4,361 bp 2,322 bp 2,027 bp 564 bp 125 bp

HindIII Fragments (from CIRCULAR form) 27, 491 bp Why is this box empty? 9, 416 bp 6, 557 bp Why is this box empty? 2,322 bp 2,027 bp 564 bp 125 bp

HindIII Fragments (if both forms are present) 27, 491 bp 23, 130 bp 9, 416 bp 6, 557 bp 4,361 bp 2,322 bp 2,027 bp 564 bp 125 bp

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PART A: Setting up the Restriction Digests 1. 2. Work in groups of FOUR during the lab. You will be required to analyze your gels and write your report INDIVIDUALLY. Label four 1.5 ml tubes with your initials and as follows: B = BamHI E = EcoRI H = HindIII -- = no enzyme Refer to Table 4.4, below. Follow the instructions numbered 3-7 carefully.

3.

IMPORTANT: The success of your experiment depends upon your care at this point. DO NOT USE THE SAME TIP IN DIFFERENT REAGENTS! You must not cross-contaminate the enzymes by neglecting to use a fresh pipette tip for each step! Table 4.4: The required volumes of reagents for each of the four reactions. DNA (l) 4.0 4.0 4.0 4.0 Restriction Buffer (l) 5.0 5.0 5.0 5.0 BamHI (l) 2.0 ---EcoRI (l) -2.0 --HindIII l) --2.0 -H2O (l) ---2.0

Tube B E H -4.

Place a fresh tip on your micropipette and add 4 l of DNA to each of the reaction tubes. Touch the tip of the pipette to the side of the tube to dispense pull the solution. Discard your tip. Place a fresh tip on your micropipette and add 5 l of restriction buffer to each reaction tube. DO NOT let the tip touch any of the DNA solution that is already in your tube. If it does touch the DNA drop in your tube, discard your tip and use a new one! When you have added 5 l of restriction buffer to each of your 4 tubes, discard your tip. Place a fresh tip on your micropipette and add 2 l of EcoRI to the liquid in the E tube. Discard your tip. Keep the EcoRI tube on ice at all times. Place a fresh tip on your micropipette and add 2 l of BamHI to the liquid in the B tube. Discard your tip. Keep the BamHI tube on ice at all times. Place a fresh tip on your micropipette and add 2 l of HindIII to the liquid in the H tube. Discard your tip. Keep the HindIII tube on ice at all times. Place a fresh tip on your micropipette and add 2 l of distilled H2O to the liquid in the -tube. Discard your tip. Close the tube tops and spin the tubes for a moment in the benchtop centrifuge to combine the solutions. Place the reaction tubes in the 37C warm bath for 20 minutes. Record the start and end times of this incubation in your lab notebook. While you are waiting, proceed to Part B.

5.

6. 7. 8. 9. 10. 11.

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PART B: Casting an Agarose Gel 1. While the restriction digestion is incubating, assemble the electrophoresis apparatus according to the instructions below. DO NOT PLUG THE POWER PACK INTO THE APPARATUS UNLESS THE COVER IS IN PLACE. Position the gel tray in the gel casting stand. Keep all equipment for the gel and electrophoresis on the plastic tray provided. Place the comb into position. Your TA will melt a flask of agarose in the microwave and add a small volume of ethidium bromide solution to the molten agarose. Agarose melts when it boils and it will start to solidify when it drops to a temperature of about 50 degrees Celsius. Before pouring the agarose into your apparatus, it should be cooled enough so that you can hold the flask in your hands. Ethidium bromide is a known mutagen and suspected carcinogen, so you must wear gloves when handling the gel and any equipment that comes in contact with the gel. Wear double gloves for all subsequent steps. Pour approximately 35 ml of molten agarose into the casting tray. 35 ml will cover the bottom of the combs teeth (see Figure 4.4, below). There is no need to accurately measure this volume of agarose just approximate.

2. 3. 4.

5.

Level of agarose

Figure 4.4: Pour molten agarose into apparatus until it covers the bottom of the combs teeth. 6. Use a fresh pipette tip to move bubbles or slide debris to the sides of the tray while the agarose is still liquid. This tip must be discarded in the waste container for ethidium bromide contaminated items. It will take about 15-20 minutes for the agarose to solidify. Take care not to disturb the casting tray during this time. When it is set, you should notice that it looks translucent rather than transparent. After 15 minutes, you may test to see whether it has solidified by gently touching the surface of the gel with a gloved finger (somewhere far from where the comb is). After the gel has solidified, apply a small amount of 1X running buffer to the surface of the gel to act as a lubricant to remove the comb (5 10 ml). VERY GENTLY AND VERY CAREFULLY remove the comb by pulling it straight up and out of the set agarose. Do not rock or wiggle the comb. NOTE: If the gel is not completely hardened or if the comb is removed too quickly, the wells may be damaged or gel fragments may be left behind. This will result in the bands on the gel being distorted. 9. 10. 11. Holding the sides of the gel tray, remove it from the casting stand. It may be necessary to bend the sides inward slightly. Set the gel tray into the gel box with the end containing the wells closest to the power supply (anode, or positive electrode). Pour buffer into the gel box to a level 3 5 mm above the surface of the gel.

7.

8.

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PART C: Loading and Running the Gel 1. 2. After your digest tubes have incubated for a minimum of 20 minutes, remove the digest tubes from the 37C water bath. Are you hands still gloved?? They must be!! Add 2 l of loading dye to each reaction tube. Be careful not to touch your tip to any of the solution within your tube. If you do, discard that tip and get a fresh one. Spin the tubes in the bench top centrifuge for 5-10 seconds to mix the solutions. Using a fresh tip for each tube, load 10 l from each digest into a separate well in the gel as shown below. a) b) c) steady the pipette over the well using both hands. if there is a bubble in the end of the pipette tip, depress the plunger to move the solution to the end of the tip. centre the pipette tip over the well, dip the tip in only enough to pierce the surface of the buffer and gently expel the sample into the well. Glycerol has been added to the loading dye to increase the density of the sample and ensure it will sink and fill the well without damage.

3.

4.

In your lab notebook, record which digest when into which lane of your gel. (Note: use the center 4 wells to avoid problems at the edges of the gel.)

Figure 4.5: Loading the lanes of an agarose gel.

Figure 4.6: Migration of a 1 kb ladder used to size DNA fragments.

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5.

Into one of the outside wells, and using a fresh tip on the micropipette, load 10 l of the 1 kb ladder. This ladder contains fragments from a plasmid containing a 1018 base pair repeat. It consists of 12 fragments ranging from 1018 bp to 12,216 bp. In addition, it contains smaller fragments up to ~500 bp that will not resolve well on your gel, and one fragment at 1636 bp which will help you to identify the other bands (Figure 4.6) Place the cover on the gel box. With the power supply unplugged, connect it to the gel box. Plug in the power cord and turn on the power switch. The pilot light will come on. Make sure that the polarity selector is set to the (+ - -) position. This will ensure the migration of negatively charged molecules away from the power supply. If you see your bands begin to move in the wrong direction, change the polarity! Set the voltage to 100V. Bubbles should begin to rise from the electrodes if current is flowing properly. You should soon see the loading dye begin to move away from the power source. It quickly resolves into two bands of colour which move at different rates. The faster moving purple coloured band is bromothymol blue and the slower moving aqua band is xylene cyanol. Bromothymol blue moves through agarose at about the same rate as a DNA fragment of about 300 base pairs. Xylene cyanol migrates at about the same rate as a fragment of DNA with about 4000 base pairs. The best separation for the analysis of DNA is achieved when the bromothymol blue migrates ~70 mm from the origin. This should take about 30-40 minutes. You must stop the electrophoresis before the bromothymol blue band runs off the end of the gel. Turn off the power supply, unplug and disconnect it from the gel box. This will allow the lid to be lifted and the gel removed. Wearing double gloves carefully remove the gel tray from the box and slide the gel into a baggie labeled with your initials and lab section number. Be very careful not to break your gel. It is fragile and can easily break if you are not careful. Pour the used electrophoresis buffer into the bottle marked USED ELECTROPHORESIS BUFFER.

6. 7. 8. 9.

10. 11.

12. 13.

14.

PART D: Gel Staining Ethidium bromide is the most sensitive and reliable DNA stain available but it is a known mutagen and a suspected carcinogen (as are many other commonly used chemicals in research labs) so it must be used carefully, disposed of properly and the user must be protected with gloves while handling a stained gel. Because the ethidium bromide was added to the agarose before the gel was run the DNA will pick up the dye as it travels through the agarose, it will have intercalated into the DNA molecules so that the DNA will be visible when the gel is viewed under Ultra-Violet light.

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PART E: Gel Imaging 1. Take your stained gel, in its sealed bag, to the imaging equipment located at the end of one of the benches in your lab. 2. A TA will help you take one picture of the gel for each member of your group. 3. After the photos are taken under UV light, you can dispose of your gel. Discard the gel (in its sealed bag) in the yellow biohazard waste containers. 4. Label the lanes of your gel image appropriately. 5. Have your TA initial and date your photo. If this is not done, you will receive a zero on your lab. DATA ANALYSIS 1. On your photograph, carefully measure the distance that each HindIII fragment migrated from the sample well. Measure from the front edge of the well to the leading edge of each band. Enter the distance migrated by each fragment into the Distance Migrated column of the following table. You may cut and paste this table into your notebook OR draw out this table in your lab notebook.

Table 4.5: Comparing the Estimated Sizes of Fragments to determine which ladder is best to use Hind III
size estimate from restriction map (bp)

EcoRI
size estimate from HindIII Graph (bp) size estimate from 1kb ladder Graph (bp) size estimate from restriction map (bp)

BamHI
size estimate from HindIII Graph (bp) size estimate from 1kb ladder Graph (bp) size estimate from restriction map (bp)

1 kb ladder

Distance Migrated (mm)

Distance Migrated (mm)

Distance Migrated (mm)

Distance Migrated (mm)

K-1 B-4 K-2 K-3 B-8 K-4 K-5 K-6 K-7 K-8 K-9 K-10 K-11 K-12 K-13 K-14 K-15

27,491* 23,130*
H-1 E-1 E-2 E-3 E-4 B-1 B-2 B-3

9,416
H-2 E-5 E-6 E-7 E-8 B-5 B-6 B-7

6,557
H-3 E-9 E-10 E-11 E-12 B-9 B-10 B-11 B-12

4,361
H-4 E-13 E-14 E-15 E-16 B-13 B-14 B-15 B-16

2,322
H-5 E-17 E-18 E-19 E-20 B-17 B-18 B-19 B-20

2,027
H-6

564
H-7

12216 11198 10180 9162 8144 7126 6108 5090 4072 3054 2036 1636 1018 517* 506* 396

125
H-8

Any two fragments that are close in size (<10% difference) may travel together and appear as one band. Any fragments less than 500 bp may not be detectable by this method.

Size (bp) from Fig 4.6

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2. 3. 4.

Do the same for the 1 kb ladder fragments. Refer to Figure 4.6 to determine the sizes of the fragments. Measure the distances migrated by each EcoRI and BamHI fragment in the same way and enter these values in Table 4.5. Set up 3-cycle semi-log paper with Distance Migrated on the x (arithmetic) and Size of Fragment (bp) on the y (logarithmic) axis. The first cycle of the y axis should begin at 100 bp and end with 1000 bp, counting in 100 bp increments. The second cycle begins with 1000 bp and ends with 10,000 bp in 1000 bp increments. The third cycle begins with 10,000 bp and ends with 100,000 bp in 10,000 bp increments. Plot the distance migrated against the known size of the fragment for each band in the HindIII lane. (For the 2 largest fragments, plot their average size). Connect the data points. This is your standard curve. It can be used to help you determine the sizes of the fragments in the other bands on your gel. Make a photocopy of this graph. You will need one for your lab notebook and one to hand in. Locate each distance migrated for the other fragments on the x-axis of the graph and draw a line up to the HindIII data line. Extend a horizontal line across to the y-axis and read the bp length of the fragment. Enter this number in the appropriate columns size estimate from HindIII Graph (bp) for each fragment. They should approximate the sizes of the fragments you predicted in Table 4.5 although you will notice some differences. Create another standard curve using the 1 kb ladder fragment sizes instead of the HindIII data. Using this line, estimate the sizes of the EcoRI and BamHI restriction fragments. Enter these estimates into the Table 4.5 in the size estimate from 1kb ladder Graph (bp) column. Make a photocopy of this graph. You will need one for your lab notebook and one to hand in.

5.

6.

7.

Questions to answer in your lab notebook and on the informal report sheet: 1. 2. 3. 4. 5. What is the purpose of the -- tube and what did the results from this tube show? What is the function of the loading dye? How did your graphically determined fragment sizes from the HindIII graph compare to the predicted fragment sizes you determined from the known sizes given in the restriction maps? How did your graphically determined fragment sizes from the 1 kb graph compare to the known sizes given in the restriction maps? For which fragment sizes were your graphs most accurate? For which fragment sizes were they least accurate? Which standard would be most useful in future to identify unknown restriction fragment sizes? Why? Two similarly sized fragments of DNA will sometimes appear as a single dark band on the gel (e.g., the two largest HindIII fragments; 27,491 and 23,130 bp). These are referred to as a doublet. Did you find any other doublets on your gel? What would you need to do in order to resolve the two bands?

6.

MARKING SCHEME: Questions on Informal Report Semi-log Graph Circular DNA Map Gel Photo Total 10 marks 5 marks 4 marks 1 mark 20 marks

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PRE-LAB ASSIGNMENT LAB IV: DNA ANALYSIS (TO BE COMPLETED BEFORE ATTEMPTING THE PRE-LAB QUIZ)
1. Draw two circular maps of phage DNA (one digested with BamHI, one digested with EcoRI in your lab notebook. You will also be asked to submit a drawing of one of these maps (done on a separate sheet of paper) with your informal lab report for grading. Use Figure 4.3 as a guide, along with these instructions: i. Draw two circles (about 8-10 cm in diameter) to represent DNA in circular form. ii. Label the 12 oclock position 48.5/0 to represent the COS site where the ends of the molecule are joined. iii. Using data from the restriction maps in Figure 4.2, make a rough circular restriction map for each of BamHI-digested phage DNA and EcoRI-digested phage DNA. Examine the restriction map for HindIII in Figure 4.2 and Figure 4.3. In circular form, we expect fewer fragments since two of the fragments join together at the COS sites. Determine the expected fragments when phage DNA is found exclusively in linear form, exclusively in circular form, or in a combination of linear and circular forms for BamHIdigested phage DNA and EcoRI-digested phage DNA. Use Table 4.3 to record your expected fragment sizes. Organize your fragments in order from LARGEST to SMALLEST. Leave blanks in the table where a fragment will not exist. Your pre-lab quiz on ELM will ask you to input the values of certain boxes from this table. If there are any two fragments that are close in size (less than 10% difference in size or any two fragments that are greater than 20,000 bp), you should not expect to see them as two separate fragments on the gel. Which of the above fragments do you expect to see as a doublet (two fragments that appear as one band)? Circle them together in the combined forms column.

2.

3.

4.

Table 4.3 Restriction fragment sizes predicted from the restriction maps, LISTED FROM LARGEST TO SMALLEST. This table should be copied into your lab notebook. Expected HindIII fragments (bp) Linear Form
23, 130 9, 416 6, 557 4,361 2,322 2,027 564 125 2,322 2,027 564 125 9, 416 6, 557

Expected BamHI fragments (bp) Circular Form

BC-1 BC-2

Expected EcoRI fragments (bp) Circular Form

EC-1 EL-1 EL-2 EL-3 EL-4 EL-5 EL-6 EC-2 EC-3 EC-4 EC-5

Circular Form
27, 491

Combined Linear Forms Form

27, 491
BL-1

Combined Linear Forms Form

BB-1 BB-2 BB-3 BB-4

Combined Forms
EB-1 EB-2 EB-3 EB-4 EB-5 EB-6 EB-7

23, 130 9, 416

BL-2 BC-3

6, 557
BL-3

4,361
BL-4 BC-4 BC-5 BB-5 BB-6 BB-7

2,322
BL-5

2,027
BL-6

564 125

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INFORMAL REPORT LAB 4: DNA ANALYSIS

(submitted individually by 1pm on the day following your lab day) SUBMITTED BY: ___________________________ TA: ________________________________ STUDENT ID: _________________ LAB SECTION: ________________

NAMES OF OTHER GROUP MEMBERS WHO SHOULD HAVE THE SAME GEL IMAGE: ______________________________________________________________________________

What is the purpose of the -- tube and what did the results from this tube show?

How many doublets are present in your EcoRI digest? ________ Which fragments form these doublets?

Of the five samples that you ran on your gel, which one would be most useful in future to identify unknown restriction fragment sizes? Why?

What would you need to do in order to resolve two fragments that appear as a doublet?

Which band on the gel or from table 4.3 confirms that some of the phage DNA was circular? Why?

Items to attach to this report ONE Semi-log graph (your TA will tell you which one to submit) ONE circular DNA map (your TA will tell you which one to submit) Gel photo (or photocopy of gel photo) with lanes labeled.

Lab 4 DNA Analysis Questions Semi-log graph Circular DNA map Attached Gel Photo Total : /10 /5 /4 /1 /20

N.B. Your TA must initial and date your gel photo before you leave the lab.