ap biology - r. wingerden · ap biology investigation 9: restriction enzyme analysis learning...

Pearson Education, Inc., publishing as Person Benjamin Cummings College Board, AP Biology Curriculum Framework 2012-2013

Copyright © Rebecca Rehder Wingerden

Investigation 9: Biotechnology:Restriction Enzyme Analysis of DNA

AP Biology Investigation 9: Restriction Enzyme Analysis

Learning Objectives

• In this investigation, you will learn how to use restriction enzymes and gel electrophoresis to create genetic profiles.

• You will use these profiles to help narrow the list of suspects in the hypothetical crime.

Copyright © 2012 Rebecca Rehder Wingerden

PreLab:

• Complete Bozeman Science- Molecular Biology Questions in CompBook (handout online) - http://www.bozemanscience.com/molecule-biology

Investigation 9: Restriction Enzyme Analysis

BozemanScience.com: AP Biology Lab 6 - Molecular Biology (9:00 min.) http://www.bozemanscience.com/ap-bio-lab-6-molecular-biology


PreLab: • Complete Activity - A Process To Dye For: Gel

Electrophoresis in CompBook (handout) - Read Introduction, Background & Experimental

Overview - Complete Pre-Lab Activity 1-5 (#5 is a Table 1) - Copy Gel Drawing Worksheet (Figure 1)



Lab: • Complete Procedure

steps 1-11 and collect data & draw gel

Post Lab: • Complete Post-Lab

Questions 1-10


BufferDyes

Power Supply

+

-

Agarose gel

Cathode

Anode

Electrophoresis Equipment

•Power supply and chamber- a source of negatively charged particles with a cathode and anode

•Buffer- a fluid mixture of water and ions •Agarose gel- a porous material the DNA

migrates through •DNA ladder- mixture of DNA fragments of

known lengths •Loading dye- contains a dense material and

allows visualization of DNA migration •DNA Stain- allows visualization of DNA

fragments after electrophoresis



Electrophoresis - Loading the Gel




Dye Name Dye Well #

Migration Distance

(mm)

Migration Direction

(+/-)Dye Molecules

“Speed” Ranking

Malachite Green 1Orange G 2Safranin O 3

Alizarin Red S 4m-Cresol Purple 5

Unknown Sample letter? NA NA NA

Table 1: Gel Electrophoresis

Analysis: • Collect data to complete Table 1: Gel Electrophorese • Complete Fig. 1 Gel Drawing

Post Lab: • Complete Post-Lab Questions 1-10



Figure 1: Gel Drawing-

- +

+1

2

3

4

5

?

Background

• DNA Restriction enzymes found naturally in bacteria, can be used to cut DNA fragments at specific sequences, while another enzyme, DNA ligase, can attach or rejoin DNA fragments with complementary (sticky) ends.

• More than 200 restriction enzymes now available commercially, they are named after the bacterium in which they were first identified: - EcoRI was the first enzyme isolated

from Escherichia coli - HindIII was the third enzyme isolated

for Haemophilus influenzae (restriction enzyme we will be using in DNA Forensic Lab)




GGCCTGCGAATTCCCGATCGAAGGCCCGAATTCTGGCCA CCGGACGCTTAAGGGCTAGCTTCCGGGCTTAAGACCGGTEco RI

GGCCTGCG AATTCCCGATCGAAGGCCCG AATTCTGGCCA CCGGACGCTTAA GGGCTAGCTTCCGGGCTTAA GACCGGT

GGCCTGCGAATTCCCGATCGAAGGCCCGAATTCTGGCCA CCGGACGCTTAAGGGCTAGCTTCCGGGCTTAAGACCGGTHae III

GG CCTGCGAATTCCCGATCGAAGG CCCGAATTCTGG CCA CC GGACGCTTAAGGGCTAGCTTCC GGGCTTAAGACC GGT

Sticky Ends - ligation is very specific

Blunt Ends - ligation is non-specific

How do restriction enzymes work?

• Each restriction enzyme digest (cuts) DNA at a specific sequence, called the restriction site.



How do we visualize the DNA?

• Agarose Gel Electrophoresis is a method of separating molecules in an electrical field based on their size and charge; DNA has an overall negative charge



• Agarose gel acts as a sieve for separating DNA fragments; smaller fragments travel faster than large fragments

1% agarose

2% agarose

• Concentration of the agarose gel affects DNA migration - Low Concentration = larger

pores -- better resolution of larger DNA fragments

- Higher Concentration = smaller pores -- better resolution of smaller DNA fragments



Agarose Gel

DNA Fragments


Molecules are separated based on their size and charge. DNA has an overall negative charge.

Forensic DNA Fingerprinting



Restriction Enzymes

A A G C T T

T T C G A A

HindIII (Hin D Three) restriction enzyme

Standard Lambda/HindIII

CS S1 S2 S3 S4 S5

Complete the following before conducting this investigation:

I. Read Investigation 9: Biotechnology: Restriction Enzyme Analysis of DNA

II. Answer the following PreLab questions in your Comp Book:

1. Summarize what you will be doing in this investigation.

2. What is the primary question you will be trying to answer in this investigation?


PreLab


Inv. 9: Biotechnology: Restriction Enzyme Analysis of DNA

PreLab - Getting Started:

• Define: Restriction Enzyme, PCR, RFLP, and Gel Electrophoresis

• Activity I: Restriction Enzyme 2Q p. S113-S114

• Activity II: DNA Mapping Using Restriction Enzymes 5Q p. S114-S115 (Note: There are a total of 5 questions in the 2 bullets.)

• Activity III: Basic Principle of Gel Electrophoresis 1Q p. S115



Forensic DNA Fingerprinting Procedure: Lesson 1 Restriction Digestion

• Steps 1-8



CS S1 S2 S3 S4 S5

Pipet 10 µl of each DNA sample from the stock tubes and transfer to corresponding colored micro centrifuge tubes. Make sure the samples transferred to the bottom of the tubes.

DNA


• Steps 1-8



ENZ

CS S1 S2 S3 S4 S5

HindIII (Hin D Three) restriction enzyme

Using a fresh tip for each DNA sample, pipet 10 µl of ENZ into the bottom of each tube.


• Steps 1-8



CS S1 S2 S3 S4 S5

Tightly cap the tubes and mix the components by gently flicking the tubes with your finger. Place tubes in your labeled floating micro-centrifuge tube rack and give to instructor.

Forensic DNA Fingerprinting Procedure: Lesson 2 Agarose Gel Electrophoresis

• Steps 7-9



CS S1 S2 S3 S4 S5S

CS S1 S2 S3 S4 S5S

Using a separate tip for each sample, load 10 µl of Standard and 20 µl of digested DNA samples in to the correct wells of gel.


• Loading Dye - DNA samples are loaded using the dry method. Samples are loading into the wells using a micro pipet.

• The presence of dyes in the DNA samples allows visualization while running the gel. The dyes must not be allowed to run off the gel.


Designing and Conducting Your Investigation: • The Disappearance of Ms. Mason: Your task is to design

and conduct a procedure based on DNA evidence to determine whose blood is spattered on the classroom floor. - Purpose: What is the goal of this investigation? - Hypothesis: If (rational for the investigation), then

(outcome that you would expect). - Procedure: Steps outlining the lab techniques that you

will complete to test your hypothesis (Include the following techniques in your procedure: PCR, RFLP, and gel electrophoresis.)

- Data: Table 1: Electrophoresis Data: DNA Fingerprints of Five Suspects

- Approval by Instructor



Lambda/HindIII Size Standard

Crime Scene Suspect 1 Ms. Mason

Suspect 2 Mr. Gladson

Suspect 3 Bobby

Suspect 4 Unknown A

Suspect 5 Unknown B

Band Distance (mm)

Actual size (bp)

Distance (mm)

Actual size (bp)

Distance (mm)

Actual size (bp)

Distance (mm)

Actual size (bp)

Distance (mm)

Actual size (bp)

Distance (mm)

Actual size (bp)

Distance (mm)

Actual size (bp)

1 23,130

2 9,416

3 6,557

4 4,361

5 2,322

6 2,027

Table 1: Electrophoresis Data: DNA Fingerprints of Five Suspects


Designing and Conducting Your Investigation:


- Evidence collected: • Crime Scene DNA- blood

spatter in classroom (SC) • Ms. Mason’s DNA- saliva

on her coffee cup (S1) • Mr. Gladson’s DNA- tissue

with which he wiped his nose (S2)

• Bobby’s DNA- bubble gum (S3)

• Unknown A DNA- blond hair (S4)

• Unknown B DNA- brown hair (S5)


Analyzing and Evaluating Results: • Complete Graph 1: Standard Curve ~ Lambda/

HindIII Size Standard • Complete Table 1: Electrophoresis Data: DNA

Fingerprints of Five Suspects • Conclusion: Write a conclusion which takes into

account the DNA results at the “crime scene.” Your conclusion should address “who-dun-it” by including motive, means, opportunity, and the DNA evidence found in Ms. Mason’s classroom.

• Evaluating Results: #1-2 (p. S122) • Thinking About Your Results: #1-4 (p. S123)



Analyzing Results: • Calculating the Sizes of Restriction Fragment Length Polymorphisms (p. S120) base pair length (bp) is substituted for molecular weight when determining the size of DNA fragments.



* For this “ideal” gel, assume that these two bands appear as a single band instead of resolving into separate bands.** These bands do not appear on the ideal gel and likely will not be seen.

• Creating the Standard Curve: - Graph 1: Standard Curve

- Table 1: DNA Fragment - Migration Distance

11.00 mm13.00 mm15.00 mm

1000900800

700

600

500

400

300

100

200

908070

60

50

40

30

10

20

987

6

5

4

3

1

2

10 2 3 4 5 6 7 8 9 10

Graph 1: Standard Curve ~ Lambda/HindIII Size Standard

DN

A Fr

agm

ent S

ize

(#bp

)

i.e. band #2 is 9416 bp and it migrated 13 mm


i.e. band #3 is 6557 bp and it migrated 15 mm

10 20 30 40 50 60 70 80 90 1000

Distance Migrated (mm)

x100

9 8 7 6

5

4

3

2

1

x1,0

00

9 8 7 6

5

4

3

2

1

x10,

000

5

4

3

2

1i.e. band #1 is 23,130 bp and it migrated 11 mm

NOTE: The first fragment is too large to migrate properly in agarose and will not fit within your line of best fit, and should be discarded.

Complete Activity- Restriction Enzyme Cleavage of DNA (EDVOTEK 112)


PreLab - • Read Background Information and Experimental

Procedure (p. 4-9) • Copy data tables:

• Table 1: DNA Marker Standard • Table 2: Lambda DNA cut with EcoRI • Table 3: Lambda DNA (UNcut) • Figure 1: Lambda DNA cut with EcoRI




• Complete Procedure steps 1-6 (p.9) • Collect Data:

• Table 1: DNA Marker Standard • Table 2: Lambda DNA cut with

EcoRI • Table 3: Lambda DNA (UNcut)

• Analysis: • Graph: Size Determination of

DNA Restriction Fragments (p.10) • Answer Study Questions #1-2 (p.

12) Lambda DNA cut with EcoRI

DNA Ladder - known quantities of DNALambda DNA

Lambda DNA


Fragment Distance Migrated (mm) Length (bp)

1 (top) 23109

2 9416

3 6557

4 4361

5 3000

6 2322

7 2027

8 725

9 570

Table 1: DNA Marker Standard - Lane 1




1

2

3

4

5

6

Table 2: Lambda DNA cut w/ Eco RI - Lane 2


Table 3: Lambda DNA (UNcut) - Lane 3


1

Use the “best fit line” on your graph (Determination of Unknown DNA Fragment Size) to determine the length of the DNA fragments in lanes 2 and 3.


Figure 1: Lambda DNA cut with EcoRI

#1 = 18mm#2 = ? mm

#3 = ? mm

1 2 3

Lane 1: DNA Marker Standard



1 (top) 18 23109

2 ? mm 9416

3 ? mm 6557

4 4361

5 3000

6 2322

7 2027

8 725

9 570

Table 1: DNA Marker Standard - Lane 1



Graph 1: Determination of Unknown DNA Fragment Size

DNA Fragment Size (#bp)

Distance Migrated (mm)

10 20 30 40 50 60 70 80

x100

x1,000

x10, 000

i.e. fragment #2 is 9416 bp and itmigrated 23 mm


#1 = ? mm#2 = ? mm

#3 = ? mm

1 2 3

Lane 2: Lambda DNA cut w/ Eco RI



1 ? mm

2 ? mm

3 ? mm

4

5

6

Table 2: Lambda DNA cut w/ Eco RI - Lane 2

#1 = ? mm

1 2 3

Lane 3: Lambda DNA (UNcut)


Table 3: Lambda DNA (UNcut) - Lane 3


1 ? mm


Use the “best fit line” on your graph (Determination of Unknown DNA

Fragment Size) to determine the length of the DNA fragments in lanes 2 and 3.



• Complete Procedure steps 1-6 (p.9) • Collect Data:

• Table 1: DNA Marker Standard • Table 2: Lambda DNA cut with

EcoRI • Table 3: Lambda DNA (UNcut)

Lambda DNA cut with EcoRI

DNA Ladder - known quantities of DNALambda DNA

Lambda DNA


• Analysis: • Graph: Size Determination of

DNA Restriction Fragments (p.10) • Answer Study Questions #1-2 (p.

12)

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