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The Biotechnology Education Company ®

EDVOTEK, Inc. • 1-800-EDVOTEK • www.edvotek.com

EVT 2012_04_26AM

EDVO-Kit #

337DrosophilaGenotyping Using PCR

Storage: See Page 3 for specifi c storage instructions

EXPERIMENT OBJECTIVE:

The objective of this experiment is to introduce students to Drosophila genotyping using the

Polymerase Chain Reaction.

This experiment is designed for DNA staining with InstaStain® Ethidium Bromide.

Updated

Revised

and

2The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

Drosophila Genotyping Using PCR337337Experiment

EVT 2012_04_26AM

EDVOTEK, The Biotechnology Education Company, and InstaStain are registered trademarks of EDVOTEK, Inc. UltraSpec-Agarose is a trademark of EDVOTEK, Inc.

Experiment Components 3

Experiment Requirements 4

Background Information 5

Experiment Procedures

Experiment Overview and General Instructions 9

Laboratory Safety 10

Module I: Isolation of Drosophila DNA 11

Module II: PCR Amplifi cation 12

Module III: Agarose Gel Electrophoresis 14

Study Questions 16

Instructor's Guidelines

Notes to the Instructor 18

Pre-Lab Preparations 21

Experiment Results and Analysis 23

Study Questions and Answers 24

Appendices

A PCR Experimental Success Guidelines 26

B Polymerase Chain Reaction Using Three Waterbaths 28

C Preparation and Handling of PCR Samples With Wax 29

D 1.0% Agarose Gel Preparation 30

E 1.0% Agarose Gels - Quantity Preparations 31

F Staining and Visualization of DNA with

InstaStain® Ethidium Bromide Cards 32

G InstaStain® Blue: One Step Staining

and Destaining 33

Material Safety Data Sheets 34

Table of Contents

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EDVOTEK - The Biotechnology Education Company® 1.800.EDVOTEK • www.edvotek.com

FAX: 202.370.1501 • email: [email protected] 2012_04_26AM

Storage

A. Tubes with PCR reaction pellets™ Room Temperature Each PCR reaction pellet™ contains • dNTP Mixture • Taq DNA Polymerase Buffer • Taq DNA Polymerase • MgCl2 B. Primer mix -20°C FreezerC. 200 base pair ladder -20°C FreezerD. Control DNA -20°C FreezerE. TE buffer Room temperatureF. Proteinase K Room temperatureG. Potassium Acetate Room temperatureH DNA extraction buffer Room temperature

Reagents & Supplies

(Store all components below at room temperature)

• Wild and White Drosophila*• UltraSpec-Agarose™• Electrophoresis Buffer (50x)• 10x Gel Loading Solution• InstaStain® Ethidium Bromide• InstaStain® Blue• Microcentrifuge Tubes• PCR tubes (0.2 ml - for thermal cyclers with 0.2 ml template)• Calibrated transfer pipets• Wax beads (for waterbath option or thermal cyclers without heated lid)

All components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor administered to or consumed by humans or animals.

THIS EXPERIMENT DOES NOT CONTAIN HUMAN DNA. None of the experiment components are derived from human sources.

This experiment is designed for 10 lab groups.

Sample volumes are very small. For liquid samples, it is important to quick spin the tube contents in a microcentrifuge to obtain suffi cient volume for pipeting. Spin samples for 10-20 seconds at maxi-mum speed.

Experiment Components

*Drosophila must be requested two weeks in advance.


Drosophila Genotyping Using PCR337337Experiment

EVT 2012_04_26AM

Requirements

• Thermal cycler (EDVOTEK Cat. # 541 highly recommended)

or three waterbaths*

• Horizontal gel electrophoresis apparatus

• D.C. power supply

• Balance

• Microcentrifuge

• Water bath (70° C)

• UV Transilluminator or UV Photodocumentation system

• UV safety goggles

• Automatic micropipets (5-50 µl) with tips

• Microwave, hot plate or burner

• Pipet pump

• 250 ml fl asks or beakers

• Hot gloves

• Disposable vinyl or latex laboratory gloves

• Ice buckets and ice

• Distilled or deionized water

• Isopropanol

*If you do not have a thermal cycler, PCR experiments can be conducted, with proper care, using three wa-terbaths. However, a thermal cycler assures a signifi cantly higher rate of success.

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Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 2002-2012 EDVOTEK, Inc., all rights reserved. EVT 2012_04_26AM

The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

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Drosophila melanogaster

For almost a century, Drosophila melanogaster (the fruit fl y) has been a valuable research tool in genetics and developmental biology. Drosophila was one of the fi rst animals used to illustrate principles of modern genetics, such as gene encoding of pro-teins, that make up the phenotype of the gene. Drosophila is inexpensive to maintain; its entire genome,165 million bases, an estimated 14,000 genes, has been completely sequenced, and several thousand genetic mutants are available for study. The sequence and mutant availabilities allow one to correlate changes at the DNA level with changes in phenotype.

Another advantage of Drosophila is their short life cycle, facilitating studies of genetic crossing. Embryos of Drosophila develop in 24 hours; the insects molt 1, 2, 4, and 6 days after hatching. The fi rst three molts give rise to larval forms while the last molt results in an inactive pupal stage. After 5 days, the fl y completes its metamorphosis and emerges from the pupa as a winged adult. Within one day, the adults are fertile and may mate to begin a new life cycle. Thus, one generation is complete within two weeks.

Another laboratory advantage for studying Drosophila is the ease of visualization of nuclear material. Salivary gland cells do not divide in adulthood, DNA is replicated but does not segregate into daughter cells and the newly formed strands remain attached. This results in chromosomes that contain hundreds of copies attached in perfect register. This duplicated state is called polytene. Polytene chromosomes, when stained, divide the Drosophila genome into 102 numbered bands, that are further subdivided into let-ter bands of A-F. The letter bands are further subdivided into 1-13 numbered divisions. Using this banding, the gene studied in this experiment, known as white, is located at position 3B6, located on the X chromosome. In addition to the banding system, local-ized expansions, known as “puffs”, may be observed on polytene chromosomes. Puffs unfold chromatin due to RNA synthesis and thus may be used to demarcate active genes. Drosophila chromosome puffs have been used in research on chromatin struc-ture, transcription, and developmental gene regulation.

In this experiment, the eye color of fruit fl ies will be studied. Drosophila eye color is determined by enzymes encoded by genes that produce pigments. Two types of eye pigments, pteridines and ommochromes, combine to impart the dull red eye color of the fruit fl y. Five different genes for eye color exist on the Drosophila X chromosome. The white gene encodes an enzyme that transports pigment precursors (used by subsequent enzymes) in both the pterididine and ommochrome pathways. The white-1 mutation re-sults from an insertion of a 4.4 kb mobile genetic element, known as the Doc retrotrans-poson, into the promoter region of the white gene. As a result, transcription of the gene is repressed and no mRNA is synthesized and no transport of the enzyme occurs. This results in neither the pteridine nor ommochrome pigments to be synthesized. The fi nal result is that the eye of the mutant insect is a bleach-white color.

The Doc retrotransposon is a mobile genetic element that uses an RNA intermediate. The mobility of retrotransposons is believed to occur through an RNA intermediate. This RNA is then reverse transcribed, by an enzyme called reverse transcriptase, to double stranded DNA. This DNA intermediate can then be integrated into relatively random sites within the Drosophila genome. Thus the original copy of the retrotransposon is maintained, but a new copy results at another location in the genomic DNA.



337337 Experiment

Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc.

Copyright © 2002-2012 EDVOTEK, Inc., all rights reserved. EVT 2012_04_26AM

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Figure 1: F1 Phenotype 2 white male 2 white female

Figure 2: F2 Phenotype 2 red male (wild) 2 white female

white males can only produce white offspring of both sexes (Figure 1). On the other hand, crossing white females with wild-type males will yield fe-male offspring that all have wild-type eyes and males that are all white-eyed (Figure 2).

To detect genetic differences at the DNA level (rather than the phenotype level), the polymerase chain reaction (PCR) is often used. PCR, invented in 1984, has gained such widespread use that its inventor, Kary Mullis, was awarded a Nobel Prize in 1994. The enormous utility of the PCR method is based on its ease of use and its ability to allow the amplifi cation of small DNA fragments. PCR amplifi cation (Figure 3, page 8) uses Taq DNA poly-merase. This enzyme is purifi ed from a bacterium isolated from hot springs and is stable at very high temperatures.

The Doc retrotransposon is a member of a group of retrotransposons known as long interspersed nuclear elements (LINEs). LINEs are found in many different organisms, are all about 5 kb in length, and (like other retrotransposons) are believed to encode their own reverse transcriptase. Although some retrotranspo-sons can occasionally excise from their site of inte-gration, most always retain an original copy of the element as well as the newly integrated copy. There-fore, the number of LINEs continually amplify so that there are now many thousand copies of LINEs in many animals. LINEs that have recently duplicated prob-ably retain the ability to transpose to new locations, while those that are older are probably fi xed at one locus. The white-1 Doc insertion is probably unable to excise from its location but has been shown to encode a reverse transcriptase, and is therefore probably able to replicate horizontally (to another genome location) as well as vertically (generation to generation).

Since the white-1 mutation results in the suppres-sion of the enzyme that transports pigment precur-sors for the ommochrome and pteridine pathways, it is a recessive mutation. Furthermore, it is located on the X chromosome and is therefore also a sex-linked mutation. Similar to other sex-linked recessive mutations, female white fl ies must carry two copies of the mutant allele, while males carry only one copy (as males only have one X chromosome and one Y chromosome). Matings between white females and


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In the fi rst step of PCR, known as denaturation, the DNA complementary strands are separated at 94 degrees, while the polymerase remains stable. In the second step, known as annealing, the sample is cooled to a temperature from 42° C to 65° C to allow hybridization of small (15-30 nucleotide) synthetic oligonucleotides, known as “prim-ers”, to the DNA to be amplifi ed, also known as the “target”. In this experiment the target is the white gene locus. In the third step, known as extension, the temperature is raised to 72° C and the polymerase then adds nucleotides to the primers to complete each new complementary strand of the target. These three steps constitute one “cycle”. This process is typically repeated from 25-30 cycles, amplifying the target exponentially (Figure 3). PCR is performed in a thermal cycler, which is programmed to heat the sample at the designated temperature for each of the three steps.

In this experiment, PCR will be used to detect the presence of the Doc insertion in white mutant fl ies. Since the white-1 allele differs from the wild-type allele by the insertion of the 4.4 kb Doc element, the use of primers that span the insertion site can differentiate between the wild-type and white-1 alleles. The primers were chosen to yield a 220 base pair product for the wild-type allele, and a 4.6 kb product in the white fl ies. However, large PCR products are diffi cult to amplify and are not usually seen under normal PCR conditions. Therefore, the wild-type allele will show a PCR product of 220 bp, while the white-1 fl ies will show no PCR product. As a positive control, an unrelated locus (related to a wing protein) is amplifi ed; this PCR product is 1000 bp. Thus, the wild type fl ies will display both the 220 bp and 1000 bp PCR products, while the mutant fl ies will show only the 1000 bp product.




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Drosophila Melanogaster

Figure 3: Polymerase Chain Reaction

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BEFORE YOU START THE EXPERIMENT

1. Read all instructions before starting the experiment.

2. If you will be conducting PCR using a thermal cycler without a heated lid, also read the Appendix entitled "Preparation and Handling PCR Samples with Wax ". If you will be using three waterbaths to conduct PCR, read the two appendices entitled "Polymerase Chain Reaction Using Three Waterbaths" and "Handling samples with wax overlays".

3. Write a hypothesis that refl ects the experiment and predict experimental outcomes.

EXPERIMENT OBJECTIVE:

The objective of this experiment is to introduce students to Drosophila genotyping using the Polymerase Chain Reaction.

BRIEF DESCRIPTION OF EXPERIMENT:

In this experiment, PCR is used to detect the presence of the Doc insertion in white mutant fl ies. Since the white-1 allele differs from the wild-type allele by the insertion of the 4.4 kb Doc element, the use of primers that span the insertion site can differentiate between the wild-type and white-1 alleles. The primers were chosen to yield a 220 base pair product for the wild-type allele, and a 4.6 kb product in the white fl ies. However, PCR products of the larger size are not usually seen under normal conditions. Therefore, the wild-type allele will show a PCR product of 220 bp, while the white-1 fl ies will show no PCR product. As a positive control, however, an unrelated locus (related to a wing protein) is amplifi ed; this PCR product is 1000 bp. Thus, the wild type fl ies will display both the 220 bp and 1000 bp PCR products, while the mutant fl ies will show only the 1000 bp product. This experiment has three modules:

I. Isolation of Drosophila DNA II. PCR Amplifi cation III. Separation of PCR Reactions by Agarose Gel Electrophoresis

GEL SPECIFICATIONS

This experiment requires a gel with the following specifi cations:

• Recommended gel size 7 x 14 cm (long tray) • Number of sample wells required 4 • Placement of well-former template fi rst set of notches • Gel concentration required 1.0%

Experiment Overview and General Instructions



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Laboratory Safety

1. Gloves and goggles should be worn routinely as good laboratory practice.

2. Exercise extreme caution when working with equip-ment that is used in conjunction with the heating and/or melting of reagents.

3. DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS.

4. Exercise caution when using any electrical equipment in the laboratory.

• Although electrical current from the power source is automatically disrupted when the cover is removed from the apparatus, fi rst turn off the power, then unplug the power source before disconnecting the leads and removing the cover.

• Turn off power and unplug the equipment when not in use.

5. EDVOTEK injection-molded electrophoresis units do not have glued junctions that can develop potential leaks. However, in the unlikely event that a leak develops in any electrophoresis apparatus you are using, IMMEDIATELY SHUT OFF POWER. Do not use the apparatus.

6. Always wash hands thoroughly with soap and water after handling reagents or biological materials in the laboratory.

Wear gloves and safety goggles

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WARNING!Use only screw-cap tubes when incubating in the waterbath for DNA isolation. Do not use snap-top tubes.

OPTIONAL STOPPING POINT

The supernatant may be stored at -20°C until the experiment is continued.

Module I: Isolation of Drosophila DNA

1. Transfer one "wild type" fl y to a 0.5 ml microcentrifuge tube labeled "wild". Place the tube in the freezer for a minute to anesthetize the fl y.

NOTE: Dead fl ies should also work for the experiment. Positive PCR results are not compromised even if fl ies are dead.

2. Use a clean pipet tip and draw up 100 µl of the DNA extraction buffer (contain-ing Proteinase K).

3. Without dispensing the 100 µl of buffer into the tube, insert the pipet tip into the tube containing the fl y and mash the fl y with the tip for 10 seconds (a small amount of buffer will release from the tip). Expel the remaining buffer and mix the solution by pipetting up and down. Place the tube on ice.

4. Using a clean tube and tip, repeat steps 1-3 with a "white type" fl y. Place the tube on ice.

5. Float both tubes in a 70°C waterbath for 15 minutes.

6. Add 14 µl of potassium acetate to each tube and mix for 5 seconds. Leave on ice for 5 minutes.

7. Spin the tubes in a microcentrifuge at maximum speed for 5 minutes.

8. Carefully transfer just the supernatant into a fresh, labeled 0.5 ml microcentri-fuge tube, being careful not to disturb the pellet. (Discard the tube with the pellet after the supernatant is saved).

9. Precipitate the DNA in the supernatant by adding 45 µl of room temperature isopropanol to each tube (wild and white). Use a marker to draw a circle at the bottoms of the tubes so you can locate the DNA pellet after centrifugation (see drawing at left). Spin at maximum speed for 5 minutes.

10. Carefully remove and discard all of the supernatant (avoid losing the DNA pel-let). A very small DNA pellet should be visible at the bottom of the tube where it was marked in the previous step.

WARNING: The DNA pellet is very small. Take care not to lose it!

11. Wash the pellet carefully with 20 µl of 70% ETOH. Spin the tube(s) at maximum speed for 2 minutes.

12. Discard the supernatant and allow the pellet to completely dry. Resuspend the DNA pellet in 25 µl of TE buffer.

13. Store the tubes on ice until you are ready to proceed with Module II, or freeze the tubes for future use.

Protocol Hint:Use compressed air to quickly dry the DNA pellet.

After centrifugation, mark location of the pellet



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The PCR reaction pel-let™ contains Taq DNA polymerase, the four deoxytriphosphates, Mg+2

and buffer.

Sample volumes are very small. For liquid samples, it is important to quick spin the tube contents in a microcentrifuge to ob-tain suffi cient volume for pipeting. Spin samples for 10-20 seconds at maxi-mum speed.

Module II: PCR Amplifi cation

PCR REACTION:

1. Transfer the PCR bead to the appropriate sized tube (e.g. 0.5 ml or 0.2 ml) for your thermal cycler.

2. Label two tubes, each containing one PCR reaction pellet™, with your initials. Label one of the tubes "wild" and the other "white".

3. Tap the reaction tube to assure the reaction pellet is at the bottom of the tube. There are also materials to perform two control sample PCR reactions.

4. To prepare the PCR reaction mix tube, add the following to the correct tube making sure that the liquid comes into contact and dissolves the PCR reaction pellet™:

Primer solution 20 µl "white", "wild", or "control DNA" 5.0 µl

5. Gently mix the individual PCR reaction tubes (separately) and quick spin in a microcentrifuge to collect all the sample at the bottom of the tube.

6. If your thermal cycler is equipped with a heated lid, proceed directly to polymerase chain reaction cycling.

If your thermal cycler does not have a heated lid, or if you are cycling manually with three water baths, add one wax bead to the tube before proceeding to polymerase chain reaction cycling.

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OPTIONAL STOPPING POINT

Samples can be frozen after addition of 5 μl of 10x Gel Loading Solution until ready for electrophoresis.

Module II: PCR Amplifi cation

POLYMERASE CHAIN REACTION CYCLING

8. Put the PCR tubes in a thermal cycler programmed as follows:

Initial Denaturation 35 cycles @ Final Extension

94°C for 2 min. 94°C for 45 seconds 72°C for 5 min. 67°C for 45 seconds. 72°C for 90 seconds

NOTE: The thermal cycler can be programmed to hold the samples at 4°C overnight.

9. Add 5 µl of 10x Gel Loading Solution to the sample and store on ice until ready for electrophoresis. Alternatively, samples can be frozen for electrophoresis at a later time.

10. Proceed to instructions for preparing a 1.0% agarose gel (7 x 14 cm) and separating the PCR products by electrophoresis.



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Module III: Separation of PCR Products by Agarose Gel Electrophoresis

AGAROSE GEL REQUIREMENTS

• Recommended gel size: 7 x 14 cm

7 x 14 cm gels are recommended to achieve better resolution of the PCR products. Each gel can be shared by several students or groups.

• Placement of well-former template: fi rst set of notches

• Agarose gel concentration: 1.0%

If you are unfamiliar with agarose gel preparation and

electrophoresis, detailed instructions and helpful

resources are available at www.edvotek.com

PREPARING THE AGAROSE GEL

1. Close off the open ends of a clean and dry gel bed (casting tray) by using rubber dams or tape.

2. Place a well-former template (comb) in the fi rst set of notches at the end of the bed. Make sure the comb sits fi rmly and evenly across the bed.

3. To a 250 ml fl ask or beaker, add agarose powder and buffer as indicated in the Refer-ence Tables (Appendix A) provided by your instructor. Swirl the mixture to disperse clumps of agarose powder.

4. With a marking pen, indicate the level of the solution volume on the outside of the fl ask.

5. Heat the mixture using a microwave oven or burner to dissolve the agarose powder.

IMPORTANT NOTE: Continue heating until the fi nal solution appears clear (like water) without any undissolved particles. Check the solution carefully. If you see “crystal” particles, the agarose is not completely dissolved.

6. Cool the agarose solution to 60°C with careful swirling to promote even dissipation of heat. If detectable evaporation has occurred, add distilled water to bring the solution up to the original volume marked in step 4.

After the gel is cooled to 60°C:

7. Place the bed on a level surface and pour the cooled agarose solution into the bed.

8. Allow the gel to completely solidify. It will become fi rm and cool to the touch after ap-proximately 20 minutes.

9. After the gel is solidifi ed, be careful not to damage or tear the wells while removing the rubber dams or tape and comb(s) from the gel bed.

10. Place the gel (on its bed) into the electrophoresis chamber, properly oriented, centered and level on the platform.

11. Fill the electrophoresis apparatus chamber with the appropriate amount of diluted (1x) electrophoresis buffer (refer to Table B on the instruction Appendix provided by your instructor).

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Module III: Separation of PCR Products by Agarose Gel Electrophoresis

BEFORE LOADING THE SAMPLES

This experiment requires a 1.0% agarose gel and is designed for staining with InstaStain® Ethidium Bromide.

LOADING DNA SAMPLES

1. (Optional Step) Heat the 200 bp DNA ladder and PCR samples for two min-utes at 50° C. Allow the samples to cool for a few minutes.

2. Make sure the gel is completely submerged under buffer before loading the samples. Load the entire volume (30 µl) of the samples in the following sequence.

Lane 1 200 bp DNA ladder 2 Control DNA PCR Product 3 White DNA PCR Product 4 Wild DNA PCR Product

3. Record the position of your sample in the gel for easy identifi cation after staining.

RUNNING THE GEL

4. After the DNA samples are loaded, properly orient the cover and carefully snap it onto the electrode terminals.

5. Insert the plugs of the black and red leads into the corresponding inputs of the power source.

6. Set the power source at the required voltage and conduct electrophoresis for the length of time determined by your instructor.

7. Check to see that current is fl owing properly - you should see bubbles forming on the two electrodes.

8. After the electrophoresis is completed, disconnect the power and remove the gel from the bed for staining.

STAINING AND VISUALIZATION OF DNA After electrophoresis, agarose gels require staining to visualize the separated DNA samples. Your instructor will provide instructions for DNA staining with InstaStain® Ethidium Bromide.

Reminder:

Before loading the samples, make sure the gel is properly oriented in the apparatus chamber.

+Black Red

Sample wells

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