Objective 2.1 Lesson B Restriction Enzyme Analysis and ...data.cteunt.org/content/files/stem/advanced-biotechnology/unit-2/2... · Objective 2.1 Lesson B Restriction Enzyme Analysis

Copyright © Texas Education Agency 2012. All rights reserved

MODULE 2

Objective 2.1 Lesson B Restriction Enzyme Analysis and Electrophoresis Extension: Restriction Mapping

Course Advanced Biotechnology

Unit DNA Technology

Essential Question How do restriction enzymes contribute to DNA analysis?

TEKS 130.364 1A-K, 2F, 2G, 2H, 7F, 7I

TAKS Science 1A, 2A-D, 6A

Prior Student Learning DNA structure, electrophoresis of dyes, agarose gel

Rationale Restriction enzymes have proved to be invaluable for the physical mapping of DNA. They offer unparalleled opportunities for diagnosing DNA sequence content and are used in fields as disparate as criminal forensics and basic research. In fact, without restriction enzymes, the biotechnology industry would certainly not have flourished as it has. All modern molecular biology methods involve the use of restriction enzymes or restriction endonucleases for cloning and the creation of recombinant DNA. Objectives Students will:

1. Explain how restriction enzymes cleave DNA. 2. Learn to read and interpret restriction maps. 3. Understand how DNA fragments are sorted by size with gel

electrophoresis. 4. Work together to construct and compare different DNA fingerprints. 5. Describe and explain the process of gel electrophoresis. 6. Predict electrophoresis results from a restriction digest. 7. Perform a restriction digest using lambda DNA. 8. Perform restriction mapping using sample data.

Restriction enzymes bind to and cuts at a specific DNA sequence called the recognition site.


Engage Project the image below and ask students:

Can you think of some reasons why we would need/want to cut DNA?

Would it be better if cuts were made randomly or in the same place every time?

What are sticky ends?

How can we use this technology in biotechnology? Show students an application of restriction enzymes by viewing:

"Genetic Engineering and Working with DNA." Teachers' Domain. 22 Sep. 2011. Web. 13 Oct. 2012. http://www.teachersdomain.org/asset/biot11_vid_genengdna/

Key Points See Power Point from Module 2 Lesson 1: DNA Analysis

Activity Pre-Lab Activities

Below are two activities that compliment the wet- lab procedure. Chose one or both of them depending on the amount of time you have and the level of your students.

o Paper Activity: Restriction Enzymes Sort and See (Dolan DNA Learning Center, Cold Spring Harbor)

o Virtual Activity: Restriction Digest of Plasmid pBR322 (attached at the end of this lesson)

http://www.teachersdomain.org/asset/biot11_vid_genengdna/

http://www.dnai.org/teacherguide/guide.html



Lab Procedure: Restriction Enzymes

Teaching Tip: Have students set up lab for a grade.

Ordering Information: Materials for this lab can be purchased from your district science vendor.

o Restriction enzyme: BamHI o Restriction enzyme: EcoRI o Restriction enzyme: HindiIII o Lambda DNA o TEA Buffer 1X o DNA loading dye o Gel Staining solution

Post Lab: Restriction Enzymes

This exercise requires Microsoft Excel.

Students may also hand draw graphs if computers are not available.

Optional Extension Exercises

Restriction Mapping

Assessment

Restriction Enzymes Sort and See Gel

Virtual Digest of pBR322 Worksheet

Lab Grade for setting up lab correctly

Lab Grading Criteria

Restriction Mapping Exercise

Materials

Restriction Enzymes Sort and See (Dolan DNA Learning Center, Cold Spring Harbor)

Virtual Digest of pBR322 Worksheet

Lab Procedure: Restriction Enzyme

Post Lab Restriction Enzymes Worksheet

Lab Grading Criteria Handout

Restriction Mapping Exercise

Accommodations for Learning Differences Visit the Special Populations section of the CTE Career and

Technical Education Website: http://cte.unt.edu/special-pops.

National and State Education Standards Texas College and Career Readiness Standards I. Nature of Science: Scientific Ways of Learning and Thinking C1, C2, C3, D1, D2, D3. E1, E2 II. Foundation Skills: Scientific Applications of Mathematics A1, A6, A7, F1, F2, F3 III. Foundation Skills: Scientific Applications of Communication A1, B1, B2, B3, C1 IV. Science, Technology, and Society

D3



http://cte.unt.edu/special-pops

http://www.thecb.state.tx.us/files/dmfile/CCRS081009FINALUTRevisions.pdf


Name:________________________________

Virtual Lab: Restriction Digest of Plasmid pBR322

Go to: http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/virgel.html.

1. Read through the background information on how to set up and run the gel

simulation.

2. Scroll down the page to the interactive section. On the left, “Choose DNA to cut” and select pBR322 plasmid.

a. What is the size of the pBR322 in base pairs (bp)?

3. A map of this plasmid will appear in the window, showing recognition sites for a

number of restriction enzymes. The more recognition sites for an enzyme are on the plasmid, the greater the number of fragments of DNA that will result from cutting with this enzyme. The sizes of the fragments will be a function of how many base pairs separate the sites.

4. Now use the pull-down menus to the right of the “Load Lane with” to load each of these enzymes into one of the lanes.

5. Click on the “Load Lane” button and you should see the corresponding well in

the gel at right turn blue.) Don’t forget your molecular weight markers!

6. On the virtual gel side, turn the power on to start the virtual electrophoresis. The bands of dye markers will begin to move up the gel. To achieve the best product:

a. Turn on the UV light to see the actual DNA bands. In research applications, DNA is typically labeled with a fluorescent dye called ethidium bromide.

b. Run the gel until you can verify the number of fragments for each restriction enzyme AND you can estimate the size of each.

c. If several fragments are close together run the gel longer to get a better If the fragments are large, this may require running smaller fragments off the gel; make sure you have their sizes first!

7. What are the sizes, in bp, of the molecular weight standards?

http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/virgel.html


8. List the sizes of the fragments next to the name of each enzyme in the table below. Do the sizes agree with the apparent distances between the sites on the plasmid map?

9. Add up the sizes of the fragments in each lane. What value does each lane come close to? Does this make sense? Why or why not?

10. The line below represents the plasmid as though it had been cut at the origin of replication, so the whole sequence of 4361 base pairs is linear. Show the positions of each restriction site on the now linear DNA – mark which enzyme is cutting at what base pair

1 ------------------------------------------------------------------------------------------------------- 4361

11. Now imagine that you want to do a double digest using EcoRI and Hindi III. This means that both enzymes will cut the plasmid at the same time. How many fragments should result from this digest?

12. How big should each of the fragments be?

13. How could restriction mapping be useful to a biotechnologist?

Enzymes How many times is the plasmid cut by each

enzyme?

How many fragments are formed by each enzyme?

Sizes of restricted digest pieces

EcoR1

BglI

Ple 1

Hind III


Lab Procedure: Restriction Enzyme Name:_________________

Day 1: Restriction Digests

1. We want to digest 1 µg of DNA in these reactions. Using the concentration of the lambda

DNA, calculate how many microliters need to be added to each tube to have 1 µg in each

tube and record in Table 1.

2. The final volume in each tube after every reagent is added should be 20 µL. Using the

concentration of the buffer (usually 10X), calculate how much should be added so that

when the final volume is 20 µL, the buffer concentration is 1X and record in Table 1.

3. Using the concentrations of the two enzymes, calculate how much should be added to

each to tube if you want one enzyme unit per tube. Enzyme concentrations are usually in

enzyme units (U) per volume (for example, 1 U/µL). Calculate the amount of water needed

in each tube, after accounting for the volumes of the other reagents, to make a final

volume of 10 µL. Record in Table 1.

TABLE 1

Lambda Digest

Volume to add

Component

Concentration Mass or

Conc. required

Tube

B*

Tube

E*

Tube

H*

Tube

C*

DNA

0.25 μg/μL

1 µg

Universal buffer

10X

1X

Water

Bam HI 0.5 units/μL

1 unit

EcoRI 0.5 units/μL

1 unit

HindIII 0.5 units/μL

1 unit

TOTAL 20 µL 20 µL 20 µL

*Tube B: Bam HI and lambda DNA *Tube E: Eco RI and lambda DNA *Tube H: Hind III and lambda DNA *Tube C: Control lambda DNA only

Copyright © Texas Education Agency 2012. All rights reserved. ;ll ‘l

4. Label four microcentrifuge tubes B, E, H and C. Place the tubes in ice.

5. Using a fresh tip for every aliquot, pipet the reagents into each tube according to the Table 1. Be sure to touch the pipette tip to the side of the tube and make sure reagent has been deposited. Keep DNA and enzymes on ice. Use a new tip for each solution. Read note below before starting.

Note: DNA should be added first to the tubes; you may keep the same tip on the micropipette without contaminating the stock solution of DNA. The buffer, which should be added second, maintains the proper pH for enzymatic activity. Water should be added next so that the buffer is the correct concentration when you add the enzyme (last). Pipet directly into the solution at the bottom of the microcentrifuge tube, and pipet up and down to mix. Avoid making bubbles, which can degrade the enzyme activity.

6. Pop spin all the tubes in a microcentrifuge to ensure all components are condensed in the bottom of the tube and able to react. Be sure to balance microcentrifuge!

7. Place all tubes, with lids closed, (in a floating tube rack) in a 37 ˚ C water bath to incubate for at least 50 minutes and up to 3 hours; the DNA should be specifically digested after 50 minutes and any longer than 3 hours may result in unspecific cutting of the DNA.

Stop Point: Following incubation, freeze reaction tubes until Day 2 of lab. Tubes can be kept for up to one month.

Copyright © Texas Education Agency 2012. All rights reserved.

Day 2: Agarose Gel Electrophoresis

1. While the tubes are incubating or thawing from the freezer, the class should prepare 1.0% agarose gels in 1X TAE. Use SOP developed in dye electrophoresis lab.

2. When the incubation of your reaction tubes is over, remove the tubes from the water bath, and add 2 µl gel loading solution to each. Pipette up and down to mix and spin the contents down by microcentrifuging for a few seconds.

3. Remove the bumpers and comb from a gel and place the casting tray with gel into the electrophoresis chamber. Wait until each group has put their tray in, then have one person add enough 1X TAE buffer to cover the gels. Make sure that every well is full of buffer and completely submerged. Draw a picture of the chamber and label the position of each group’s gel relative to the positive and negative terminals.

4. Load 10 ul each sample into a separate well of your gel. If you have enough wells, leave the first and last empty, as they tend to be more distorted then the other wells. Record in your notebook, which sample was loaded into which well (always number from left to right).

5. Your instructor will load your unknown DNA samples.

6. Connect the red (positive) patch cord to the red terminal on the power supply.

Similarly, connect the black cord to the black terminus.

7. Electrophorese at 80 volts for 1.5 hours or until the dye has migrated to within a few mm of the end of the gel (the end nearest the positive terminal). If the loading dye has more than one color, use the aqua color as a guide. Turn off the power supply and disconnect the leads before removing the cover of the chamber. Remove your gel in its casting tray.

Stop Point: Following electrophoresis, wrap the gel in plastic wrap or in a zip lock bag with buffer and refrigerate until Day 3. Make sure to wear gloves.

Day 3: Photographing Gels

1. Stain your gel according to your instructor’s directions.

2. Photograph your gel with a ruler beside it.

3. Dispose of your gel properly.


Post Lab: Restriction Enzymes

Results and Analysis: Restriction Analysis The size of the fragments produced by a specific endonuclease can be determined by using standard fragments of known size. When you plot the data on semilog graph paper, the size of the fragments is expressed in the log of the number of base pairs they contain. This allows data to be plotted on a straight line. The migration distance of the unknown fragments, plotted on the x-axis, will allow their size to be determined on the standard curve. Make a Standard Curve for HindIII

1. Measure the migration distance (in mm) for each HindIII band on your gel. Measure from the bottom of the sample well to the bottom of the band. Record these measurements in the table on your answer sheet.

2. Plot the measured migration distance for each band of the standard HindIII digest against the actual base pair (bp) fragment sizes given in Table 1 using a semi log graph using Microsoft Excel. Add a best fit line to your graph. This will serve as a standard curve. See an example below


Interpolated Calculations for EcoRI From your standard curve for HindIII, made from known fragment sizes, you can calculate fragment sizes resulting from a digest with EcoRI. The procedure is as follows.

Measure the migration distance in mm for each EcoRI band. Record the data in the table under measured distance.

Determine the sizes of fragments of lambda phage DNA digested with EcoRI.

Locate on the x axis the distance migrated by the first EcoRI fragment. Using a ruler, draw a vertical line from this point to its intersection with a best fit data line. Now extend a horizontal line from this point to the y axis. This point gives the base pair size for this EcoRI fragment. Highlight these lines in YELLOW. See example below.

Repeat this procedure and determine the remaining EcoRI fragments. Enter your interpolated data in table 1, in the interpolated bp column.

Your teacher has provided you with the actual bp data. Compare your results to these actual sizes. Note: This interpolation technique is not exact. You should expect as much as 10% to 15% error. Calculate the error and see if this is so. Show work on you answer sheet/


Interpolated Calculations for BamHI From your standard curve for HindiIII, made from known fragment sizes, you can calculate fragment sizes resulting from a digest with BamHI. The procedure is as follows

Measure the migration distance in mm for each BamHI band. Record the data in the table under measured distance.

Determine the sizes of fragments of lambda phage DNA digested with EcoRI.

Locate on the x axis the distance migrated by the first EcoRI fragment. Using a ruler, draw a vertical line from this point to its intersection with a best fit data line.

Now extend a horizontal line from this point to the y axis. This point gives the base pair size for this EcoRI fragment. Highlight these lines in any color besides BLUE.

Repeat this procedure and determine the remaining BamHI fragments. Enter your interpolated data in table 6.1, in the interpolated bp column.

Your teacher has NOT provided you with the actual bp data. You must get this info yourself from your pre-lab results. Compare your results to these actual sizes. Note: This interpolation technique is not exact. You should expect as much as 10% to 15% error. Calculate the error and see if this is so. Show work on you answer sheet. Interpolate Calculations for the UKNOWN (loaded by your teacher) Create a table next to the BamHI table and record your results.


NAME

Answer Sheet: Restriction Digest

Table 1

HindIII EcoR1

Distance Act. bp Distance Measured/ Interpolated

bp.

Actual bp

*27,491 *24,756

*23,130 *21,226

9416 7421

6648 *5804

4361 *5643

2322 4878

2027 3530

**564

**125

*Pair appears as a single band

** Does not appear on this gel

BamHI

Distance Measured/Interpolated bp.

Actual bp

** Does not appear on this gel

Percent error calculations:


Post Lab Questions

1. Look at the interpolated bp (base pairs) for EcoR1 and compare it to the actual bp. For

which fragment size was your graph most accurate? For which fragment size was it least accurate? What does this tell you about the resolving ability of agarose-gel electrophoresis?

2. Discuss how each of the following factors would affect the results of electrophoresis: Voltage used: Running Time: Reversal of Polarity (+/- electrode):

3. Compare your gel with the ideal gel and try to account for the DNA in each lane. How

can you account for differences in separation and band intensity (darkness/lightness) between your gel and ideal gel?

4. What are restriction enzymes? How do they work? What are recognition sites?

5. Describe the function of electricity and agarose gel in electrophoresis?

6. What are the functions of loading dye in electrophoresis?

7. Use the standard graph you prepared from your lab data to predict how far (in cm) a

fragment of 8,000 bp would migrate.

8. In gel electrophoresis DNA molecules migrate from to ends of the gel


Gel Electrophoresis: Restriction Enzymes Grading Criteria

Agarose Gel Made properly (10 points): (Teacher Initials)

Lab Procedure

ALL THE TIME

(5 pts)

MOST OF THE TIME (4pts)

RARELY (1pt)

NEVER (0 pt)

On TASK

Followed Directions

Proper use of equipment

Cleaned Lab Table

Post Lab

ALL (18 pts) MOST (14 pts) FEW (6 points) FEW if ANY (2pt)

Questions answered correctly

Data Table complete and accurate

Graph generated with criteria

Instructor Comments:

Total Points: _


Restriction Mapping Exercise Name:____________________

View Restriction Mapping Tutorials

http://www.youtube.com/watch?v=8FqMUF96cPE&feature=source_video

http://www.youtube.com/watch?v=v2T8Y3-8674&feature=related

1. You want to clone a fragment of DNA into the

plasmid to the left. The plasmid is 4.25 kb long. If

you digest the plasmid with BamHI, how many

linear fragments will result? _____

2. If you digest the plasmid with BamHI and

EcoR1, how many linear fragments will result?

3. You have made a DNA library by cloning your desired DNA fragments into the vector

above at the ECRR1 site and then transforming the plasmid into E. coli. You select one

bacterial colony (derived from a single individual) and extract plasmid DNA. You digest

the plasmid with BamHI. You get a single 5.25 kb fragment. Does this plasmid contain a

cloned gene? Explain.

4. How many kb is the desired gene?

5. If you digest the plasmid above with EcoR1, how many fragments will you get and how

many kilobases will each be?

6. A 700 bp fragment of DNA is digested with EcoRI. Two fragments result, a 300 bp and

a 400 bp fragment. When the same DNA is digested with HaeIII, two fragments result, a

200 bp and a 500 bp. A double digestion produces three fragments, 100 bp, 200 bp,

and 400 bp. Map the fragment. Is the HaeIII restriction site within the EcoRI 400 bp

fragment?

http://www.youtube.com/watch?v=8FqMUF96cPE&feature=source_video

http://www.youtube.com/watch?v=v2T8Y3-8674&feature=related


7. A restriction map for a fictitious 4000 base pair plasmid is shown in the figure below. Create a figure predicting the appearance of a gel showing digests with EcoRI, BamHI, and EcoRI and BamHI combined. Your figure should include a lane showing a 1kb ladder for reference.

8. An unknown plasmid is digested with EcoRI and HindIII, and the fragments are separated by gel electrophoresis. A diagram of the resulting gel is shown below. Use this information to create a restriction map of the plasmid.

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Objective 2.1 Lesson B Restriction Enzyme Analysis and ...data.cteunt.org/content/files/stem/advanced-biotechnology/unit-2/2... · Objective 2.1 Lesson B Restriction Enzyme Analysis