daphnia 9-12 - nslc · 2005. 9. 11. · title: microsoft word - daphnia 9-12.doc author: tom elgin...

SEEING RED: Daphnia and Hemoglobin Genes A HIGH SCHOOL LABORATORY UNIT USING DNA SCIENCE FOR ECOLOGICAL INVESTIGATION Cheryl Stephens Washington High School Washington, Missouri 2005 Summer Research Fellowships for Science Teachers Sponsored by Howard Hughes Medical Institute Washington University Science Outreach

Seeing Red: Daphnia and Hemoglobin Genes – Teacher Manual 1

SEEING RED: DAPHNIA AND HEMOGLOBIN GENES Teacher Manual

LESSON OVERVIEW

This unit is designed to introduce students to laboratory techniques that distinguish differences between species using current genomic practices. DNA isolation, gel electrophoresis, and PCR are basic lab methods.

Students will need an understanding of the following: 1. DNA structure and function 2. Gene theory – definition, sequencing 3. Laboratory techniques of gel electrophoresis and DNA amplification by

PCR This teacher manual is more detailed than the student manual, with additional

comments and hints in the appropriate sections. Materials and resources are listed at the end of the manual. FIT TO STANDARDS The NSF Science Content Standards (9-12) are aligned as follows for this lesson:

1. Science as Inquiry Content Standard A: As a result of activities in grades 9-12, all students should develop

o Abilities necessary to do scientific inquiry o Understandings about scientific inquiry

2. Life Science Content Standard C: As a result of their activities in grades 9-12, all students should develop understanding of

o The cell o Molecular basis of heredity

3. Science and Technology Content Standard E: As a result of activities in grades 9-12, all students should develop

o Abilities of technological design o Understandings about science and technology

4. History and Nature of Science Content Standard G: As a result of activities in grades 9-12, all students should develop understanding of

o Science as a human endeavor o Nature of scientific knowledge o Historical perspectives


TIMELINE

This lab activity will take a full 5-day week to complete. The first day should cover introduction to the lab and pre-planning of activities. The second, third, and fourth days will be hands-on lab activities (Procedure Part I, Part II, and Part III). The last day will close with observations and conclusions. Suggested time schedule is:

1 month (or more) before: order materials for lab 1 week before: mix solutions/reagents/agarose 1 day before: gather materials, ice, set water bath, set up lab equipment

BACKGROUND INFORMATION DAPHNIA Daphnia are freshwater zooplankton found in ponds and lakes all over the world. Daphnia are commonly called ‘water flea’ due to their jerky swimming motions. Some types of Daphnia can be seen with the human eye, while others must be identified with a microscope. Depending on the species they can range in size from 0.5mm to 1cm. Their outer covering, or carapace (the thick shell that covers the back of animals and serves to protect or isolate from external influence), is transparent, so many internal organs can be seen, especially the beating heart. On the head there is a compound eye and a pair of antennae, which are used for swimming. Females are usually larger than males and have a brood chamber under their outer carapace where eggs are carried.

Daphnia exhibit a number of natural responses to environmental stress and are often used as a bio-indicator (changes in organisms that can be reliably used to indicate a change in the environment; these changes may be physiological, chemical, or behavioral) of watershed health. Daphnia can detect the presence of kairomone chemicals (a subclass of pheromone, defined as an interspecific secretion which benefits the receiver) released by predators and in response can grow larger head and tail spines or develop through a shorter gestation period. They are extremely sensitive to changes in water toxicity, which can be monitored through changes in heartbeat or Daphnia death. When Daphnia are exposed to hypoxic (a reduced concentration of dissolved oxygen in a water body leading to stress or even death in aquatic organisms) conditions (low oxygen) the reaction is to increase hemoglobin production. Due to their clear outer carapace, they will appear red when hemoglobin production has increased. Daphnia are also an extremely important part of aquatic food chains. They eat primary producers such as algae, yeast, and bacteria but are the prey of tadpoles, salamanders, newts, aquatic insects, and many types of small fish. Fluctuations in Daphnia populations can cause algae overgrowth, a drastic drop in fish populations, and can even affect larger animals caught or eaten by humans.

The most common bioindicator species of Daphnia are D. pulex and D. magna (the larger of the two). At present the only method available to assess Daphnia populations in the wild is microscopic identification after aquatic sampling. This method is time consuming when large samples must be processed. Recently Daphnia lumholtzi, an invasive species originally from Africa and Asia, began invading watersheds in the United


States. Efficient and accurate analysis of large numbers of water samplings with a wide variety of organisms could be developed through DNA profiling, using genetic databases to identify species differences. Note the enlarged head and tails spines on D. lumholtzi, which inhibits predation by fish.

Daphnia magna Daphnia pulex Daphnia lumholtzi

HEMOGLOBIN

Hemoglobin (Hb) is the most common transport molecule in almost all higher organisms. Within closed circulatory systems it serves as an oxygen carrier, removes carbon dioxide produced by metabolism, and regulates the pH of the blood.

Invertebrate Hb is extra-cellular but is dissolved in the hemolymph. (This is the blood analogue that is used by all arthropods and most mollusks that have an open circulatory system. In these animals there is no distinction between blood and interstitial fluid. The liquid fills the entire interior (hemocoel) of the body and surrounds all cells.)

Invertebrates, such as Daphnia, obtain oxygen for respiration through diffusion. These animals’ hearts, generally found in the upper central portion of the back, pump blood forward to the animal's brain through thin-walled vessels and somatically (referring or relating to the body) through channels or spaces in the tissues. Oxygen molecules along with water and other dissolved macromolecules are carried throughout the Daphnia. Under stressful environmental conditions Daphnia can increase hemoglobin production allowing it to survive in hypoxic conditions. If water conditions return to normal, Daphnia can lower hemoglobin production.

Daphnia hemoglobin (Hb) is a multi-subunit, multi-domain (compactly folded structures with their own hydrophobic core and which serve a variety of functions for a protein) macromolecule coded for by at least four Hb genes in Daphnia species. The genetic sequence for Daphnia hemoglobin has been identified and has many genetic and ecological applications. When analyzed it is found that there are several differences between nucleotide sequences for the same Hb subunits found in different Daphnia species. DNA from even a single Daphnia amplified by PCR with a specific hemoglobin primer and visualized through staining of an electrophoresis gel can discriminate and confirm the identification of different species. Once the DNA for invading species of


Daphnia is sequenced it will be possible to identify them genetically instead of microscopically. ELECTROPHORESIS

Gel electrophoresis is a technique that uses the properties of charge, mass, and shape to separate macromolecules, such as proteins and nucleic acids. An agarose or polyacrylamide gel is poured into the electrophoresis apparatus and electrodes create positive and negative ends toward which the macromolecules travel. Nucleic acids, which are negatively charged, are repelled by the anode and attracted towards the cathode.

The gel provides a matrix through which macromolecules travel and impedes their progress differentially. The larger, more massive molecules travel slower and remain closer to the point of origin. A DNA size marker, or ladder of known weight and size is used to provide a reference point. (A DNA ladder is a solution of known molecular weight markers that is used for electrophoresis gel calibration used to analyze weights of samples.) Various dyes are mixed with the samples before they are applied to individual lanes, which when properly stained and visualized, will result in bands that can be compared to the ladder. The sizes of the DNA segments can then be estimated. For instance, whole genome DNA is very large and remains closer to the wells than a Hb gene segment of DNA. PCR (POLYMERASE CHAIN REACTION) AMPLIFICATION OF DNA

PCR is a laboratory technique that creates multiple exact copies of selected pieces of DNA. Small amounts of DNA template (for example, crime scene trace evidence or the whole genome of a small creature like Daphnia) can be amplified enough times to provide workable amounts of genetic material. A single copy of a DNA sequence (a segment of DNA, a gene, or a whole chromosome) multiplied through 25 cycles results in over 33 million exact copies of the original DNA template in just a few hours.

DNA is macromolecule made up of units of a phosphate, deoxyribose sugar and one of four nucleotides. Hydrogen bonds hold the nucleotides together, A to T and C to G, as base pairs. Codons, groups of three base pairs, code for amino acids, which ultimately are joined to form proteins.

A PCR reaction mixture is created using the targeted template DNA, primers, dNTPs, DNA polymerase, and buffer components. The targeted DNA must have known nucleotide sequences on either end of it, but can be unknown itself. In a thermal cycler, polymerase chain reactions (PCR) repeat a three-step cycle, alternately heating and cooling the mixture to precise temperatures. The first step, denaturation, separates the double helix coil of DNA into individual, single strands by heating the DNA and breaking the hydrogen bonds between the nucleotide base pairs. The much cooler second step, annealing, allows the primers to anneal to complementary sequences on the template DNA. Finally, in the warmer third step, extension, the DNA polymerase causes the dNTPs to attach to the primer ends and synthesize exact duplicates of the template DNA. Then the process repeats as many at 35 times.


http://users.ugent.be/~avierstr/principles/pcrsteps.gif

The 300 million base-pair Daphnia genome currently being sequenced comprises 10 to 12 chromosome pairs found in the known Daphnia species. Four of the more than 3000 genes code for different subunits of Daphnia hemoglobin and those hemoglobin genes have been sequenced for D. pulex and D. magna. The differences between the sequences of their hemoglobin genes suggest that distinctions can be made between species.

PCR primers are short series of DNA base pairs that flank both ends of a desired template sequence. Primers are paired; the forward primer amplifies from the 5′ end and the reverse primer from the 3′ end of the DNA template. Both must be supplied to a PCR reaction mixture. Using the sequences of Daphnia Hb genes, three primers were selected:

Daphniarev- amplifies segment common to both species, reverse primer Dpulex1- amplifies hemoglobin precursor in D. pulex, forward primer Dmagna1- amplifies hemoglobin DHB1 subunit in D. magna, forward primer Each primer for Daphnia Hb will amplify a segment of approximately 300 bp. D.

pulex DNA does not have a complimentary sequence to match the D, magna primer (and vice versa) so amplification of DNA will not occur if the wrong primer is used. Samples of unknowns would require separate PCR set-ups with each primer to distinguish between each species.

In addition to the template DNA and primers, a PCR reaction mixture must include Taq polymerase and dNTPs. The polymerase enzyme initiates the reaction. The dNTPs, the subunits with which to build new DNA, need to be supplied in excess. The buffers and other components are supplied to provide optimal conditions for the amplification of DNA.

Gel electrophoresis, with the appropriate ladder, will confirm the amplification of the selected template DNA. Bands of amplified DNA can then be compared to those of


known species. Even the presence of a single organism can be established through a PCR-based DNA analysis of an unknown sample. PURPOSE

To perform a qualitative test of a water sample that distinguishes different species of the water flea, Daphnia using DNA analysis. MATERIALS / EQUIPMENT Water bath Microcentrifuge (8000 rpm-14,000 rpm) Electrophoresis Gel box Micropipettors (2-1000� L) Eppendorf tube racks PCR tubes PCR machine Vortexer Fluorescent light box CONSUMABLES QIAGEN DNeasy Tissue Kit 100% ethyl alcohol 1X TAE buffer 10X PBS (phosphate buffered saline) LE Agarose 10X KTLA buffer KTLA Taq polymerase dNTPs (1.25 � M each) Orange G dye Kontes Pellet Pestles Eppendorf tubes (1.5-2.0 mL) 1 Kb DNA Ladder 100bp DNA Ladder 4 M Betaine Ethidium bromide Daphniarev primer Dmagna primer Dpulex primer Distilled DNase/RNase free water Daphnia magna (live) Daphnia pulex (live)


ADVANCE PREPARATION

1. Order Daphnia in time for delivery before lab. Live specimens must be used. 2. Order primers for lab. Order or secure all other consumables. 3. Mix solutions. Dilutions and mixtures will be found in Materials and Methods

section below. 4. Preheat water bath on day of Part 1 – DNA Isolation.

DILUTION OF STOCK SOLUTIONS

1. dNTPs: 10.0 mM stock solution diluted to 200µL of 1.25mM concentration. Add 25µL dNTP stock solution to 175µL dH2O. Keep frozen.

2. 1Kb DNA ladder: Add 5µL Orange G dye to 25µL ladder stock. Keep frozen. 3. 1X TAE Buffer: 100mL 10X TAE stock to 900 mL dH20 4. Ethidium bromide stain: Add 15µL of 10 mg/mL EtBr solution to 150 mL dH20.

Harmful – use caution. 5. 0.8% LE Agarose Gel: Add 2.0 g Agarose to 250mL TAE Buffer. Can make large

amount and melt for pouring gel at a later time. 6. 2.0% LE Agarose Gel: Add 5.0 g Agarose to 250mL TAE Buffer. Can make large

amount and melt for pouring gel at a later time. MASTER PCR MIX FOR 10 STUDENTS (5 PAIRS)

In Eppendorf tube mix the following: 1) 22.275µL dH2O 2) 27.5µL 10X Buffer KTLA 3) 89.375µL 4M Betaine 4) 55.0µL dNTPs (1.25 µM) 5) 3.85µL Taq DNA polymerase Add immediately before the lab.

198µL total volume

Have students aliquot 36µL of Master PCR Mix into PCR tube. Then add to each tube:

2µL Daphniarev primer 2µL Dpulex (or Dmagna) primer 10µL D. pulex (or D. magna) DNA

Be sure students add the correct primers to the correct DNA samples.


REFERENCES AND ADDITIONAL RESOURCES QIAGEN DNeasy Tissue Kit: QIAGEN Inc. Cat # 69504

27220 Turnberry Lane, Valencia, CA 91355. Phone: 800 426-8157. Fax: 800 718-2056. Web: http://www1.qiagen.com

100% ethyl alcohol: Flinn Scientific, Inc.

PO Box 219, Batavia, IL 60510. Phone: 800 452-1261. Fax: 866 452-1436. E-mail: [email protected]. Web: http://www.flinnsci.com/

1X TAE buffer: SIGMA-ALDRICH, CO. 10X Concentrate Cat #T-9650

PO Box 14508, St. Louis, MO 63178. Phone 314 771-5750. Web: http://www.sigmaaldrich.com

Orange G dye: Fisher Scientific. Orange G dye (10 g) Cat # AC41655-0100

Phone: 800 766-7000. Fax: 800 926-1166. Web: http://www.fishersci.com/

10X PBS (phosphate buffered saline): SIGMA-ALDRICH, CO. Cat # P5493-AL

PO Box 14508, St. Louis, MO 63178. Phone 314 771-5750. Web: http://www.sigmaaldrich.com

LE Agarose: ISC BioExpress. GenePure LE Agarose (25g) Cat # E-3120-25

420 North Kays Drive, Kaysville, UT 84037. Phone: 800 999-2901. Fax: 800 574-7890. Web: http://www.bioexpress.com

100bp DNA ladder: ISC BioExpress. GeneMate Quanti-Marker Cat # C-5088-200

420 North Kays Drive, Kaysville, UT 84037. Phone: 800 999-2901 Fax: 800 574-7890. Web: http://www.bioexpress.com Store in freezer. Keep on ice while using.

10X KTLA buffer: SIGMA-ALDRICH, CO.

PO Box 14508, St. Louis, MO 63178. Phone 314 771-5750. Web: http://www.sigmaaldrich.com Free with KlenTaq LA Taq Polymerase

KTLA Taq polymerase: SIGMA-ALDRICH, CO. Cat # D5062-125UN

PO Box 14508, St. Louis, MO 63178. Phone 314 771-5750. Web: http://www.sigmaaldrich.com Store in freezer. Keep on ice while using.

dNTPs: Fisher Scientific. Promega dNTP Mix 10mM Cat # PR-U1511

Phone: 800 766-7000. Fax: 800 926-1166. Web: http://www.fishersci.com/ Store in freezer. Keep on ice while using.


4M Betaine: SIGMA-ALDRICH, CO. Cat # E8751-1G PO Box 14508, St. Louis, MO 63178. Phone 314 771-5750. Web: http://www.sigmaaldrich.com

Distilled DNase/RNase free water: Invitrogen Corporation. Cat # 10977-015

Web: http://www.invitrogen.com Daphnia magna (live): Carolina Biological. Cat # HT-14-2330

2700 York Road, Burlington, NC 27215. Phone: 800 334-5551. Web: http://www.carolina.com/

Daphnia pulex (live): Carolina Biological. Cat # HT-14-2314

2700 York Road, Burlington, NC 27215. Phone: 800 334-5551. Web: http://www.carolina.com/

Kontes Pellet Pestle: Fisher Scientific. Cat # K749520-0090

Phone: 800 766-7000, Fax: 800 926-1166. Web: http://www.fishersci.com/ Microcentrifuge tubes (1.5 mL): Fisher Scientific. Cat # 05-406-22

Phone: 800 766-7000, Fax: 800 926-1166. Web: http://www.fishersci.com/ Primers: Integrated DNA Technologies, Inc. 1710 Commercial Park, Coralville, IA 52241-2760. Phone: 800 328-2661.

Fax: 319 626-8444. Web: http://www.idtdna.com PRIMER SEQUENCES: Order 25 nmol each. Store in freezer. Keep on ice while using. Dpulex = 5′- GCA GTT CCT CAA GAT CGC TCT TTT CTT TG- 3′ (29 bases) Dmagna = 5′-ATG GCT TCT TTT AAG ATT GCC CTT CTC CTC-3′ (30 bases) Daphniarev = 5′-GAT GTT TTC CCA GCT TCT CTG GAC ATC-3′ (27 bases)


PROCEDURE Part I – Isolate Daphnia DNA using the QIAGEN DNeasy Tissue Kit Remind students to check step when completed. Have students pre-label tubes. Check temperature of water bath before lab. Materials and tubes can be places into container of water/bleach before disposal. The teacher should make the DNA unknowns for use in Part III. Unknowns can be D. pulex, D. magna, or a combination of both. ___1. Grind 7-10 live D. magna and D. pulex in separate Eppendorf tubes using the

appropriate pellet pestles (labeled DmD and DpD). Follow the next steps for each tube.

___2. Add 180 µL 10X PBS. Mix. ___3. Add 20 µL Proteinase K. ___4. Add 200 µL Buffer AL. ___5. Vortex mixture and incubate 10 minutes in 70°C water bath. ___6. Add 200µl 100% ethanol and vortex to mix. ___7. Pipette the mixture from step 6 (including any precipitate) into the DNeasy Mini

spin column placed in a 2 mL collection tube (provided). Centrifuge at 8000 rpm for 1 min. Discard flow-through and collection tube.

___8. Place the DNeasy Mini spin column in a new 2 ml collection tube (provided), add 500µL Buffer AW1, and centrifuge for 1 min at 8000 rpm. Discard flow-through and collection tube.

___9. Place the DNeasy Mini spin column in a new 2 ml collection tube (provided), add 500µL Buffer AW2, and centrifuge for 3 min at 14,000 rpm to dry the DNeasy membrane. Discard flow-through and collection tube.

___10. Place the DNeasy Mini spin column in a clean 1.5 ml or 2 ml microcentrifuge tube (not provided), and pipette 100µL Buffer AE directly onto the DNeasy membrane. Incubate at room temperature for 1 min, and then centrifuge for 1 min at 8000 rpm to elute.

___11. Repeat elution step 11, using the same microcentrifuge tube, resulting in 200µL total.


Image and text from QIAGEN DNeasy Tissue Kit Handbook

Use about 7 live Daphnia. Do not use frozen/preserved specimen. Grind up animals with pellet pestle.

The Buffer ATL will help lyse the individual cells, while the Proteinase K breaks up the proteins within the cell and nuclear membranes.

The DNeasy Mini spin column has a special coating that binds the DNA to it. Two washes (with Buffers AW1 And AW2) clean up the DNA.

The elution steps remove the DNA from the spin column and dissolve it in a buffer solution.

DNA in the Buffer AE can now be used in a PCR reaction or frozen for later use.

The ethanol precipitates the DNA.


Part II – Gel Electrophoresis Confirmation of Genomic DNA Remind students to check each step. Ethidium bromide is harmful; gloves must be worn when handling and solution can be disposed of down the sink with sufficient running water. Several students can use the same gel to run their samples. UV light is harmful; goggles must be worn. ___1. Set up 0.8% LE Agarose gel. ___2. When gel is set, remove comb carefully and add 1X TAE Buffer to cover. ___3. Load wells as follows:

Lane #1 1kb DNA Ladder (with dye) Lane #2 Daphnia pulex DNA (DpD) – (3µL dye + 12µL DpD) Lane #3 Daphnia magna DNA (DmD) – (3µL dye + 12µL DmD)

___4. Run gel at approximately 90V until dye is 2-3 cm from end of gel. ___5. Carefully transfer gel from tray to staining container. Add about 150 mL

EtBr/water. ___6. Gently agitate for 10 minutes. Rinse in dH20 for 10 minutes. ___7. Visualize bands under UV light ___8. Mark the location of DNA bands on Data/Observations Sheet. ___9. If bands confirm presence of Daphnia DNA proceed to Part III. If not, repeat Part I

and II to obtain DNA template material for Part III.


Part III – PCR Amplification of Daphnia DNA Remind students to check each step when completed. Label all tubes before beginning the lab. Students should be provided one unknown for use in PCR tubes DuDp and DuDm. ___1. Label 4 small PCR tubes prior to mixing materials for the reaction. ___2. In 4 labeled PCR tubes mix the following: Tube: DpD PCR 1) 36.0 µL of Master PCR mixture,

containing a- 4.05µL dH2O b- 5.0µL 10X Buffer KTLA c- 16.25µL 4M Betaine d- 10.0µL dNTPs (1.25 µM) e- 0.7µL Taq DNA polymerase

2) 2.0µL Daphnia reverse primer (Drp) 3) 2.0µL Daphnia pulex primer (Dpp) 4) 10.0µL Daphnia pulex DNA (DpD)

50.0µL total PCR reaction

Tube: DmD PCR 2) 36.0 µL of Master PCR mixture,

containing a- 4.05µL dH2O b- 5.0µL 10X Buffer KTLA c- 16.25µL 4M Betaine d. 10.0µL dNTPs (1.25 µM) e. 0.7µL Taq DNA polymerase

2) 2.0µL Daphnia reverse primer (Drp) 3) 2.0µL Daphnia magna primer (Dmp) 4) 10.0µL Daphnia magna DNA (DmD)


Tube: DuDp PCR 1) 36.0 µL of Master PCR mixture,


2) 2.0µL Daphnia reverse primer (Drp) 3) 2.0µL Daphnia pulex primer (Dpp) 4) 10.0µL Daphnia unknown DNA


Tube: DuDm PCR 1) 36.0 µL of Master PCR mixture,


2) 2.0µL Daphnia reverse primer (Drp) 3) 2.0µL Daphnia magna primer (Dmp) 4) 10.0µL Daphnia unknown DNA


___3. Set the “Daphnia” PCR program on the thermocycler. Run the PCR reaction as soon as possible after mixing the components.

68˚C 5 min Initial denaturation 93˚C 10 sec Denaturation 55˚C 30 sec Annealing 35 cycles 68˚C 4 min Extension

___4. When the PCR is finished, reaction products can be refrigerated overnight.

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Part IV – Gel Electrophoresis of PCR Products Remind students to check each step. Ethidium bromide is harmful; gloves must be worn when handling and solution can be disposed of down the sink with sufficient running water. Several students can use the same gel to run their samples. UV light is harmful; goggles must be worn. ___1. Set up 2% LE Agarose gel. ___2. When gel is set remove comb carefully and add 1X TAE Buffer to cover. ___3. Load lanes as follows:

Lane 1- 10µL 100bp DNA ladder (with dye) Lane 2- 15 µL D. pulex DNA plus dye (3 µL Orange G dye/12µL PCR product) Lane 3- 5µL D. magna DNA plus dye (3 µL Orange G dye/12µL PCR product) Lane 4- 15µL Unknown DNA plus dye (optional) (3 µL Orange G dye/12µL PCR product)

___4. Run gel at about 90V until dye is near end of gel. Disconnect power source. ___5. Carefully transfer gel from tray to staining container with 150 mL EtBr/water

solution. ___6. Gently agitate for 10 minutes. Rinse in dH20 for 10 minutes. ___7. Visualize bands under UV light. ___8. Mark the location of DNA bands on Data/Observations Sheet.


DATA / OBSERVATIONS Part II – Gel Electrophoresis Confirmation of Genomic DNA

1. Using the 1 KB ladder as a guide, draw DNA bands in the appropriate lanes as seen on the gel.

The Daphnia genome is approximately 3.4 x 108 bp. The bands will be slightly past the wells. 2. Is the presence of genomic DNA confirmed? What size is indicated?

a. D. pulex _____________________________ b. D. magna ____________________________

D. pulex (DpD) D. magna (DmD)


Part III and IV – PCR

Using the 100 bp ladder as a guide, draw PCR product DNA bands in the appropriate lanes as seen on the gel.

Bands will be present at the same place for all positive results. Students given D. pulex DNA for the Unknown will not have a band in the DuDm lane because D. pulex DNA can not be amplified by the D. magna forward primer. Students given D. magna DNA for the Unknown will not have a band in the DuDp lane because D. magna DNA can not be amplified by the D. pulex forward primer.

DpD DmD DuDp DuDm


ANALYSIS / CONCLUSIONS / DISCUSSION

1. Daphnia are often used as model species. Give several reasons why they make good bioindicators. Daphnia are very common to fresh water environments, easy to catch and identify, and very susceptible to changes in water quality and populations differences.

2. Daphnia lumholtzi is an invading species that now threatens North American fresh water environments. Why would a PCR-DNA analysis of a water source be useful for ecological testing? When surveying water sources for the presence of an invading species it is likely that a large number of samples will be taken. PCR-DNA analysis would be quicker than microscopic examination and can detect the presence of even one organism.

3. In Daphnia hemoglobin is not produced unless needed, describe some causes of hypoxic conditions in the water. Causes of hypoxia could include overpopulation, overgrowth of algae, plankton or duckweed, and contamination of the water supply.

4. How could differences in the nucleotide sequences of Hb genes in different species of Daphnia still result in a functional hemoglobin protein? The differences in the nucleotide sequences could be in non-coding regions of the Hb gene.

5. In Part I, the mixture is heated to 70º C after Proteinase K and Buffer AL are added. Why is it not incubated at room temperature instead? Proteinase K cleaves peptide bonds and is used to remove DNases and RNases when isolating DNA and RNA from tissues or cell lines. The optimum temperature is 65°C. Rapid denaturation of Proteinase K occurs above 65°C.

6. Buffers AW1 andAW2 wash away what kinds of substances? The DNA binds to the resin in the column so the washes get rid of anything that is not DNA.

7. If PCR amplifies DNA, why did the lab protocol call for grinding 7-10 Daphnia instead of only one? Or a hundred? One Daphnia ground up might be so diluted that careless mixing and pipeting would result in not enough DNA for amplification.

8. What is the purpose of each ingredient in the PCR mix? a) dH2O – Dilutes chemicals to correct concentrations b) 10X Buffer KTLA – Provides optimal conditions for polymerase activity c) 4M Betaine – Suppresses polymerase pauses and stops during amplification d) dNTPs – Provides free-floating single nucleotide bases for DNA synthesis e) Daphniarev reverse primer – Recognizes specific sequence site and acts as

starting point for DNA synthesis f) Dpulex or Dmagna forward primer – Recognizes specific sequence site and

acts as starting point for DNA synthesis g) Taq polymerase – Enzyme that catalyzes DNA synthesis

9. Separate tubes are used in the PCR set up for each forward primer. Why can’t you use both forward primers with a sample? Because the different forward primers amplify DNA segments of approximately the same length, there would be no way to distinguish which forward primer resulted


in the PCR product. Using the different forward primers separately will allow the band to appear with the corresponding primer and not with the other.

10. Describe the bands you might see on a gel if a pond is inhabited by Daphnia pulex only. Daphnia magna only? Both Daphnia pulex and Daphnia magna? Neither Daphnia pulex nor Daphnia magna? D. pulex only would result in a band in the DuDp lane only. D. magna only would result in a band in the DuDm lane only. Both types of Daphnia would result in bands in all lanes. No Daphnia would result in no bands in any lanes.

SEEING RED: Daphnia and Hemoglobin Genes Student Manual Name(s) _________________________________________________________________ Class Hour _____________ Project Start Date ________________________________

Seeing Red: Daphnia and Hemoglobin Genes – Student Manual 1

BACKGROUND INFORMATION DAPHNIA Daphnia are freshwater zooplankton found in ponds and lakes all over the world. Daphnia are commonly called ‘water flea’ due to their jerky swimming motions. Some types of Daphnia can be seen with the human eye, while others must be identified with a microscope. Depending on the species they can range in size from 0.5mm to 1cm. Their outer covering, or carapace, is transparent, so many internal organs can be seen, especially the beating heart. On the head there is a compound eye and a pair of antennae, which are used for swimming. Females are usually larger than males and have a brood chamber under their outer carapace where eggs are carried.

Daphnia exhibit a number of natural responses to environmental stress and are often used as a bio-indicator of watershed health. Daphnia can detect the presence of kairomone chemicals released by predators and in response can grow larger head and tail spines or develop through a shorter gestation period. They are extremely sensitive to changes in water toxicity, which can be monitored through changes in heartbeat or Daphnia death. When Daphnia are exposed to hypoxic conditions (low oxygen) the reaction is to increase hemoglobin production. Due to their clear outer carapace, they will appear red when hemoglobin production has increased. Daphnia are also an extremely important part of aquatic food chains. They eat primary producers such as algae, yeast, and bacteria but are the prey of tadpoles, salamanders, newts, aquatic insects, and many types of small fish. Fluctuations in Daphnia populations can cause algae overgrowth, a drastic drop in fish populations, and can even affect larger animals caught or eaten by humans.

The most common bioindicator species of Daphnia are D. pulex and D. magna (the larger of the two). At present the only method available to assess Daphnia populations in the wild is microscopic identification after aquatic sampling. This method is time consuming when large samples must be processed. Recently Daphnia lumholtzi, an invasive species originally from Africa and Asia, began invading watersheds in the United States. Efficient and accurate analysis of large numbers of water samplings with a wide variety of organisms could be developed through DNA profiling, using genetic databases to identify species differences.

Daphnia magna Daphnia pulex Daphnia lumholtzi


HEMOGLOBIN Hemoglobin (Hb) is the most common transport molecule in almost all higher

organisms. Within closed circulatory systems it serves as an oxygen carrier, removes carbon dioxide produced by metabolism, and regulates the pH of the blood.

Invertebrate Hb is extra-cellular but is dissolved in the hemolymph. Invertebrates, such as Daphnia, obtain oxygen for respiration through diffusion. These animals’ hearts, generally found in the upper central portion of the back, pump blood forward to the animal's brain through thin-walled vessels and somatically through channels or spaces in the tissues. Oxygen molecules along with water and other dissolved macromolecules are carried throughout the Daphnia. Under stressful environmental conditions Daphnia can increase hemoglobin production allowing it to survive in hypoxic conditions. If water conditions return to normal, Daphnia can lower hemoglobin production.

Daphnia hemoglobin (Hb) is a multi-subunit, multi-domain macromolecule coded for by at least four Hb genes in Daphnia species. The genetic sequence for Daphnia hemoglobin has been identified and has many genetic and ecological applications. When analyzed it is found that there are several differences between nucleotide sequences for the same Hb subunits found in different Daphnia species. DNA from even a single Daphnia amplified by PCR with a specific hemoglobin primer and visualized through staining of an electrophoresis gel can discriminate and confirm the identification of different species. Once the DNA for invading species of Daphnia is sequenced it will be possible to identify them genetically instead of microscopically. ELECTROPHORESIS

Gel electrophoresis is a technique that uses the properties of charge, mass, and shape to separate macromolecules, such as proteins and nucleic acids. An agarose or polyacrylamide gel is poured into the electrophoresis apparatus and electrodes create positive and negative ends toward which the macromolecules travel. Nucleic acids, which are negatively charged, are repelled by the anode and attracted towards the cathode.

The gel provides a matrix through which macromolecules travel and impedes their progress differentially. The larger, more massive molecules travel slower and remain closer to the point of origin. A DNA size marker, or ladder of known weight and size is used to provide a reference point. Various dyes are mixed with the samples before they are applied to individual lanes, which when properly stained and visualized, will result in bands that can be compared to the ladder. The sizes of the DNA segments can then be estimated. For instance, whole genome DNA is very large and remains closer to the wells than a Hb gene segment of DNA. PCR (POLYMERASE CHAIN REACTION) AMPLIFICATION OF DNA

PCR is a laboratory technique that creates multiple exact copies of selected pieces of DNA. Small amounts of DNA template (for example, crime scene trace evidence or the whole genome of a small creature like Daphnia) can be amplified enough times to provide workable amounts of genetic material. A single copy of a DNA sequence (a segment of DNA, a gene, or a whole chromosome) multiplied through 25 cycles results in over 33 million exact copies of the original DNA template in just a few hours.


DNA is macromolecule made up of units of a phosphate, deoxyribose sugar and one of four nucleotides. Hydrogen bonds hold the nucleotides together, A to T and C to G, as base pairs. Codons, groups of three base pairs, code for amino acids, which ultimately are joined to form proteins.

A PCR reaction mixture is created using the targeted template DNA, primers, dNTPs, DNA polymerase, and buffer components. The targeted DNA must have known nucleotide sequences on either end of it, but can be unknown itself. In a thermal cycler, polymerase chain reactions (PCR) repeat a three-step cycle, alternately heating and cooling the mixture to precise temperatures. The first step, denaturation, separates the double helix coil of DNA into individual, single strands by heating the DNA and breaking the hydrogen bonds between the nucleotide base pairs. The much cooler second step, annealing, allows the primers to anneal to complementary sequences on the template DNA. Finally, in the warmer third step, extension, the DNA polymerase causes the dNTPs to attach to the primer ends and synthesize exact duplicates of the template DNA. Then the process repeats as many at 35 times.

http://users.ugent.be/~avierstr/principles/pcrsteps.gif

The 300 million base-pair Daphnia genome currently being sequenced comprises 10 to 12 chromosome pairs found in the known Daphnia species. Four of the more than 3000 genes code for different subunits of Daphnia hemoglobin and those hemoglobin genes have been sequenced for D. pulex and D. magna. The differences between the sequences of their hemoglobin genes suggest that distinctions can be made between species.

PCR primers are short series of DNA base pairs that flank both ends of a desired template sequence. Primers are paired; the forward primer amplifies from the 5′ end and


the reverse primer from the 3′ end of the DNA template. Both must be supplied to a PCR reaction mixture. Using the sequences of Daphnia Hb genes, three primers were selected:

Daphniarev- amplifies segment common to both species, reverse primer Dpulex1- amplifies hemoglobin precursor in D. pulex, forward primer Dmagna1- amplifies hemoglobin DHB1 subunit in D. magna, forward primer Each primer for Daphnia Hb will amplify a segment of approximately 300 bp. D.

pulex DNA does not have a complimentary sequence to match the D, magna primer (and vice versa) so amplification of DNA will not occur if the wrong primer is used. Samples of unknowns would require separate PCR set-ups with each primer to distinguish between each species.

In addition to the template DNA and primers, a PCR reaction mixture must include Taq polymerase and dNTPs. The polymerase enzyme initiates the reaction. The dNTPs, the subunits with which to build new DNA, need to be supplied in excess. The buffers and other components are supplied to provide optimal conditions for the amplification of DNA.

Gel electrophoresis, with the appropriate ladder, will confirm the amplification of the selected template DNA. Bands of amplified DNA can then be compared to those of known species. Even the presence of a single organism can be established through a PCR-based DNA analysis of an unknown sample.


PURPOSE To perform a qualitative test of a water sample that distinguishes different species

of the water flea, Daphnia using DNA analysis. MATERIALS / EQUIPMENT Water bath Microcentrifuge (8000 rpm-14,000 rpm) Electrophoresis Gel box Micropipettors (2-1000� L) Eppendorf tube racks PCR tubes PCR machine Vortexer Fluorescent light box CONSUMABLES QIAGEN DNeasy Tissue Kit 100% ethyl alcohol 1X TAE buffer 10X PBS (phosphate buffered saline) LE Agarose 10X KTLA buffer KTLA Taq polymerase dNTPs (1.25 � M each) Orange G dye Kontes Pellet Pestles Eppendorf tubes (1.5-2.0 mL) 1 Kb DNA Ladder 100bp DNA Ladder 4 M Betaine Ethidium bromide Daphniarev primer Dmagna primer Dpulex primer Distilled DNase/RNase free water Daphnia magna (live) Daphnia pulex (live)


PROCEDURE Part I – Isolate Daphnia DNA using the QIAGEN DNeasy Tissue Kit Pre-label all tubes and check off each step as it is completed. ___1. Grind 7-10 live D. magna and D. pulex in separate Eppendorf tubes using the

appropriate pellet pestles (labeled DmD and DpD). Follow the next steps for each tube.

___2. Add 180 µL 10X PBS. Mix. ___3. Add 20 µL Proteinase K. ___4. Add 200 µL Buffer AL. ___5. Vortex mixture and incubate 10 minutes in 70°C water bath. ___6. Add 200µl 100% ethanol and vortex to mix. ___7. Pipette the mixture from step 6 (including any precipitate) into the DNeasy Mini

spin column placed in a 2 mL collection tube (provided). Centrifuge at 8000 rpm for 1 min. Discard flow-through and collection tube.

___8. Place the DNeasy Mini spin column in a new 2 ml collection tube (provided), add 500µL Buffer AW1, and centrifuge for 1 min at 8000 rpm. Discard flow-through and collection tube.

___9. Place the DNeasy Mini spin column in a new 2 ml collection tube (provided), add 500µL Buffer AW2, and centrifuge for 3 min at 14,000 rpm to dry the DNeasy membrane. Discard flow-through and collection tube.

___10. Place the DNeasy Mini spin column in a clean 1.5 ml or 2 ml microcentrifuge tube (not provided), and pipette 100µL Buffer AE directly onto the DNeasy membrane. Incubate at room temperature for 1 min, and then centrifuge for 1 min at 8000 rpm to elute.

___11. Repeat elution step 11, using the same microcentrifuge tube, resulting in 200µL total.


Image and text from QIAGEN DNeasy Tissue Kit Handbook

Use about 7 live Daphnia. Do not use frozen/preserved specimen. Grind up animals with pellet pestle.

The Buffer ATL will help lyse the individual cells, while the Proteinase K breaks up the proteins within the cell and nuclear membranes.

The DNeasy Mini spin column has a special coating that binds the DNA to it. Two washes (with Buffers AW1 And AW2) clean up the DNA.

The elution steps remove the DNA from the spin column and dissolve it in a buffer solution.

DNA in the Buffer AE can now be used in a PCR reaction or frozen for later use.

The ethanol precipitates the DNA.


Part II – Gel Electrophoresis Confirmation of Genomic DNA Check off each step as it is completed. NOTE: Ethidium bromide is harmful; gloves must be worn when handling.

UV light is harmful; goggles must be worn. ___1. Set up 0.8% LE Agarose gel. ___2. When gel is set, remove comb carefully and add 1X TAE Buffer to cover. ___3. Load wells as follows:

Lane #1 1kb DNA Ladder (with dye) Lane #2 Daphnia pulex DNA (DpD) – (3µL dye + 12µL DpD) Lane #3 Daphnia magna DNA (DmD) – (3µL dye + 12µL DmD)

___4. Run gel at approximately 90V until dye is 2-3 cm from end of gel. ___5. Carefully transfer gel from tray to staining container. Add about 150 mL

EtBr/water. ___6. Gently agitate for 10 minutes. Rinse in dH20 for 10 minutes. ___7. Visualize bands under UV light ___8. Mark the location of DNA bands on Data/Observations Sheet. ___9. If bands confirm presence of Daphnia DNA proceed to Part III. If not, repeat Part I

and II to obtain DNA template material for Part III.


Part III – PCR Amplification of Daphnia DNA Check off each step as it is completed. Label all tubes before beginning the lab. The teacher will give you one unknown DNA sample for use in PCR tubes DuDp and DuDm. ___1. Label 4 small PCR tubes prior to mixing materials for the reaction. ___2. In 4 labeled PCR tubes mix the following: Tube: DpD PCR 1) 36.0 µL of Master PCR mixture,

containing a- 4.05µL dH2O b- 5.0µL 10X Buffer KTLA c- 16.25µL 4M Betaine f- 10.0µL dNTPs (1.25 µM) g- 0.7µL Taq DNA polymerase

2) 2.0µL Daphnia reverse primer (Drp) 3) 2.0µL Daphnia pulex primer (Dpp) 4) 10.0µL Daphnia pulex DNA (DpD)


Tube: DmD PCR 1) 36.0 µL of Master PCR mixture,

containing a- 4.05µL dH2O b- 5.0µL 10X Buffer KTLA c- 16.25µL 4M Betaine f. 10.0µL dNTPs (1.25 µM) g. 0.7µL Taq DNA polymerase

2) 2.0µL Daphnia reverse primer (Drp) 3) 2.0µL Daphnia magna primer (Dmp) 4) 10.0µL Daphnia magna DNA (DmD)


Tube: DuDp PCR 1) 36.0 µL of Master PCR mixture,

containing a- 4.05µL dH2O b- 5.0µL 10X Buffer KTLA c- 16.25µL 4M Betaine d- 10.0µL dNTPs (1.25 µM) f- 0.7µL Taq DNA polymerase

2) 2.0µL Daphnia reverse primer (Drp) 2) 2.0µL Daphnia pulex primer (Dpp) 4) 10.0µL Daphnia unknown DNA


Tube: DuDm PCR 1) 36.0 µL of Master PCR mixture,

containing a- 4.05µL dH2O b- 5.0µL 10X Buffer KTLA c- 16.25µL 4M Betaine f- 10.0µL dNTPs (1.25 µM) g- 0.7µL Taq DNA polymerase

2) 2.0µL Daphnia reverse primer (Drp) 3) 2.0µL Daphnia magna primer (Dmp) 4) 10.0µL Daphnia unknown DNA


___3. Set the “Daphnia” PCR program on the thermocycler. Run the PCR reaction as soon as possible after mixing the components.

68˚C 5 min Initial denaturation 93˚C 10 sec Denaturation 55˚C 30 sec Annealing 35 cycles 68˚C 4 min Extension

___4. When the PCR is finished, reaction products can be refrigerated overnight.

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Part IV – Gel Electrophoresis of PCR Products Check off each step as it is completed. NOTE: Ethidium bromide is harmful; gloves must be worn when handling.

UV light is harmful; goggles must be worn. ___1. Set up 2% LE Agarose gel. ___2. When gel is set remove comb carefully and add 1X TAE Buffer to cover. ___3. Load lanes as follows:

Lane 1- 10µL 100bp DNA ladder (with dye) Lane 2- 15 µL D. pulex DNA plus dye (3 µL Orange G dye/12µL PCR product) Lane 3- 5µL D. magna DNA plus dye (3 µL Orange G dye/12µL PCR product) Lane 4- 15µL Unknown DNA plus dye (optional) (3 µL Orange G dye/12µL PCR product)

___4. Run gel at about 90V until dye is near end of gel. Disconnect power source. ___5. Carefully transfer gel from tray to staining container with 150 mL EtBr/water

solution. ___6. Gently agitate for 10 minutes. Rinse in dH20 for 10 minutes. ___7. Visualize bands under UV light. ___8. Mark the location of DNA bands on Data/Observations Sheet.


DATA / OBSERVATIONS Part II – Gel Electrophoresis Confirmation of Genomic DNA

1. Using the 1 KB ladder as a guide, draw DNA bands in the appropriate lanes as seen on the gel.

2. Is the presence of genomic DNA confirmed? What size is indicated?

c. D. pulex _____________________________ d. D. magna ____________________________

D. pulex (DpD) D. magna (DmD)


Part III and IV – PCR

Using the 100 bp ladder as a guide, draw PCR product DNA bands in the appropriate lanes as seen on the gel.

DpD DmD DuDp DuDm


ANALYSIS / CONCLUSIONS / DISCUSSION

1. Daphnia are often used as model species. Give several reasons why they make good bioindicators.

2. Daphnia lumholtzi is an invading species that now threatens North American fresh

water environments. Why would a PCR-DNA analysis of a water source be useful for ecological testing?

3. In Daphnia hemoglobin is not produced unless needed, describe some causes of

hypoxic conditions in the water. 4. How could differences in the nucleotide sequences of Hb genes in different species

of Daphnia still result in a functional hemoglobin protein?

5. In Part I, the mixture is heated to 70º C after Proteinase K and Buffer AL are

added. Why is it not incubated at room temperature instead?


6. Buffers AW1 andAW2 wash away what kinds of substances? 7. If PCR amplifies DNA, why did the lab protocol call for grinding 7-10 Daphnia

instead of only one? Or a hundred? 8. What is the purpose of each ingredient in the PCR mix?

a) dH2O b) 10X Buffer KTLA

c) 4M Betaine

d) dNTPs

e) Daphniarev reverse primer

f) Dpulex or Dmagna forward primer

g) Taq polymerase

9. Separate tubes are used in the PCR set up for each forward primer. Why can’t you use both forward primers with a sample?


10. Describe the bands you might see on a gel if a pond is inhabited by Daphnia pulex only. Daphnia magna only? Both Daphnia pulex and Daphnia magna? Neither Daphnia pulex nor Daphnia magna?

daphnia 9-12 - nslc · 2005. 9. 11. · title: microsoft word - daphnia 9-12.doc author: tom elgin...

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