introduction to biotechnology methods

BIOLOGY

Introduction to Biotechnology Methods

Investigation Manual

INTRODUCTION TO BIOTECHNOLOGY METHODS

Overview DNA gel electrophoresis was developed in the 1970s and has been vital in bringing about many advances in biotechnology. This technique has a variety of applications in medicine, genetics, forensics, biochemistry, and more. This lab investigation introduces fundamental techniques used in biotechnology. In the first activity, you will explore restriction enzyme digestion, polymerase chain reaction, and gel electrophoresis through a simulation investigating the presence or absence of the sickle cell mutation. In the second activity, you will use a mini gel setup to electrophorese dyes across an agarose medium. This procedure is very similar to that used in genetic testing.

Outcomes• List and describe the steps of the polymerase chain reaction

(PCR).• Describe how restriction enzymes work and their uses

in biotechnology.• Explain the principle behind the separation of biomolecules by

gel electrophoresis.• Identify and describe the function of the equipment involved in

electrophoresis.• Perform an electrophoresis reaction, and analyze the results.

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Table of Contents

2 Overview 2 Outcomes3 Time Requirements3 Background8 Materials9 Safety10 Preparation11 Activity 112 Activity 214 Disposal and Cleanup

KeyPersonal protective equipment (PPE)

goggles gloves apronfollow link to video

photograph results and

submit

stopwatch required

warning corrosion flammable toxic environment health hazard

KeyPersonal protective equipment (PPE)

goggles gloves apronfollow link to video

photograph results and

submit

stopwatch required

warning corrosion flammable toxic environment health hazard

Made ADA compliant by NetCentric Technologies using the CommonLook® software

BackgroundScientists can analyze genetic variation to predict relationships between groups or individuals. This practice, referred to as DNA profiling or DNA fingerprinting, is often used to establish kinship or make genetic matches between an individual and a found DNA sample. In Activity 1 of this investigation, you will use DNA typing to determine the presence or absence of the sickle cell mutation.

Sickle Cell Mutation Sickle cell anemia is a genetic disease caused by a point mutation in the beta-globin gene. The point mutation is a simple switch of one adenine nucleotide to a thymine, but this single change has significant results. This mutation changes a glutamic acid to valine, altering the shape of the hemoglobin in such a way that blood cells become curved and misshapen. The rigid, sticky, sickle-shaped cells block small blood vessels and cause pain, organ damage, and other serious complications.

Some people suffering from sickle cell anemia have this single base-pair mutation in both alleles (genotype SS) of the beta-globin gene, while those without the disease carry two wild-type alleles of the beta-globin gene (genotype AA). Carriers of the trait have a

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single sickle cell allele and a wild-type allele (genotype AS). The health risk for people who have only one mutated allele is minor. They may experience some sickling of red blood cells in low-oxygen environments and some problems with their kidneys. Perhaps the greatest risk carriers face is that of passing on the allele to their offspring.

Polymerase Chain Reaction (PCR) Because the location and sequence of the sickle cell point mutation is known, DNA typing is a useful tool for diagnosing individuals. DNA typing can determine whether an individual is homozygous wild-type, heterozygous, or homozygous for the sickle cell mutation.

To perform DNA typing, a sample is first collected from the individual. Because DNA is in the nucleus of every human cell, cells from skin, hair, saliva, blood, or other bodily fluids can serve as the source of a DNA sample. Each sample, prepared in solution, contains the entire human genome. However, only a small region of the beta-globin gene is of interest for testing for sickle cell anemia. The entire human genome contains 3.4 billion base pairs. The beta-globin gene is on chromosome 11, which includes over 134 million base pairs. Because it is not

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Time RequirementsPreparation ..................................................................... 30 minutesActivity 1: Sickle Cell Anemia Detection Simulation ....... 30 minutesActivity 2: Mini Gel Electrophoresis of Dyes ................... 60 minutesNote: Activity 1, which should be performed before Activity 2, can be conducted while the gel for Activity 2 is setting. The amount of time required for running the electrophoresis gel may vary with the number of batteries used for the activity.



temperature is lowered (typically to 50–65 °C, depending on the primer sequence). One primer binds to each DNA strand, and each primer is the reverse complement of the DNA strand to which it binds. The region between the two primers is the sequence of DNA that will be amplified.

3. Extension: During the extension step, the temperature is raised again (typically to 68 °C). DNA synthesis occurs as the DNA polymerase adds nucleotides to the 3' end of each primer. DNA polymerase is an enzyme that catalyzes the DNA synthesis reaction by adding free nucleotides to the DNA strand. During the synthesis reaction, the primers are extended to become the complement strands of the templates resulting in double-stranded DNA products. The DNA polymerases used in PCR are produced by bacteria that live in very hot environments, such as hot springs and thermal vents on the seafloor. These bacteria evolved to produce proteins that are resistant to denaturation when heated. Thermostable DNA polymerases, such as Taq polymerase, are essential to current PCR protocols. DNA polymerases from other sources would be destroyed during the first heating step.

4. Amplification: Once the extension step is complete, the process of denaturation, annealing, and extension is repeated several times. The second and subsequent rounds of synthesis occur using both the original DNA strands and the newly synthesized strands of DNA. The three-step cycle can be repeated until the reactants are depleted, with each cycle doubling the number of DNA molecules.

possible to detect the mutation from a single copy of DNA, the desired region must be copied over and over in a process called amplification to determine whether the mutation is present. The polymerase chain reaction (PCR) method can be used to amplify the region of interest (the region known to contain the mutation site). After the PCR is complete, millions of copies of the region of DNA containing the mutation site will be present.

PCR is a powerful technique that generates large quantities of a specific DNA sequence from a very small amount of starting DNA, such as the amount isolated from a drop of blood or a single hair follicle. The method leads to exponential amplification of the DNA region of interest by doubling the number of copies of the region during every cycle. For example, an initial sample containing two copies of a DNA sequence amplified through 35 cycles of PCR would yield 235 or 34,359,738,368 (3.4 × 1010) copies of the DNA sequence.

A PCR reaction (see Figure 1) requires template DNA (the starting sample), free nucleotides (dNTPs), sequence-specific DNA primers, DNA polymerase, water, and salts for buffering.

Steps of Each PCR Cycle1. Denaturation: DNA is heated to near boiling

(typically 95 °C) so that the two strands of DNA separate.

2. Annealing: DNA primers (generally 18–22 bases long) are designed and synthesized in a laboratory based on known sequences at the beginning and end of the region of interest. During annealing, primers bind to their complementary sequences as the

Background continued




Figure 1.

In the diagram, the primers are depicted by a four-nucleotide sequence. However, for actual PCR reactions, primers are generally 18–22 bases long. DNAP = DNA polymerase (Taq polymerase)



on the same gel. The band with DNA fragments that match the size of the region of interest can be extracted from the gel for further testing.

Restriction Enzymes In this simulation, the amplified fragments of DNA contain the region known to have the sickle cell mutation, but it is unknown if the mutation is present or absent. Remember, the nucleotides, and thus the mutation, are much too small to visualize. Therefore, molecular tools are needed to determine if the mutation is present. Among these tools are restriction endonucleases, also called restriction enzymes. Restriction endonucleases are enzymes that act like tiny scissors that can cut DNA at specific nucleotide sequences. Restriction endonucleases occur naturally in bacteria. Bacteria use these enzymes to cut nucleotide strands of invading viruses. Scientists collect the enzymes from bacteria and use them for DNA profiling because the enzymes digest animal DNA just as well as they digest viral DNA. Not all restriction endonucleases cleave in the same way; some examples are listed in Table 1. Some restriction enzymes cleave specific DNA sequences so that all the nucleotides are still bound to their complementary bases. When this is the case, the ends of the DNA strands are called “blunt ends.” Other restriction enzymes cut in such a way that uneven ends remain, where some nucleotides are not bound to their complementary bases. The resulting uneven DNA strands are called “sticky ends.” Depending on the goals of the experiment, scientists select an enzyme that cuts at the desired site or sites.

Verifying PCR Products The next step in DNA profiling is to visualize the PCR products (the amplified DNA sequence) using electrophoresis. The sequence of the region of interest for beta globin is known. The DNA fragments in the amplified sample should be of a similar length. However, it is possible that a different region or more than one region was amplified. Running the PCR products on a gel verifies that the desired region of DNA was, first, amplified and, second, the only region of DNA amplified. If the researcher is confident, usually through experience, that the primers and PCR protocol only amplified the desired region, this gel electrophoresis step may be skipped.

Each DNA sample is pipeted into a small well in an agarose gel. Because DNA has a negative electrical charge, when an electric current is passed through the gel, DNA migrates toward the anode (the positive pole). DNA fragments travel through gels at speeds inversely related to their size. When the current is stopped, fragments of DNA remain at various places within the gel. DNA fragments of the same size appear as bands in the rectangular shape of the well to which they were loaded. Larger DNA fragments remain closer to the well, while smaller fragments travel farther through the gel.

Once the DNA fragments are separated by size on a gel, they can be visualized by staining. DNA can be stained with a fluorescent dye that requires UV light for visualization, such as ethidium bromide, or with a nonfluorescent dye. The approximate size of the DNA fragments in each band can be determined by comparing the distance traveled by the band to a DNA ladder composed of DNA fragments of known sizes run

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restriction enzyme used made a single cut. If there is only one band on the gel, then there were either no restriction sites within the specific DNA sequence or the restriction enzyme did not function properly.

For example, to analyze a DNA sample for the presence or absence of the nucleotide sequence AGCT, one might use the enzyme Alu1, which cleaves the bonds between guanine and cytosine on both strands when it recognizes an AGCT sequence. If the sequence, AGCT, does not occur in the DNA sample, Alu1 will not recognize any sites for digestion, and the DNA will remain intact.

Electrophoresis The next step in DNA typing is visualizing the results of the nucleotide restriction of the PCR-amplified DNA sequence by gel electrophoresis. The number of bands and their sizes indicate the presence or absence of restriction sites within the amplified DNA. If there are two distinct bands, then we can assume there was one restriction site and the

Table 1.

Restriction Endonuclease

Recognition Site

Cleavage Pattern

End Description

Alu1 AGCT AG/CT TC/GA blunt end

Cla1 ATCGAT AT/CGAT TACC/TA

5' CG overhang sticky end

Dde1 CTNAG C/TNAG GANT/C

5' TNA overhang sticky end

EcoRV GATATC GAT/ATC CTA/TAG blunt end

Mal1 GATA GA/TC CT/AG blunt end

“N” denotes any nucleotide. “/” represents the locations where the enzyme cleaves the DNA.




MaterialsIncluded in the materials kit:

Six-well comb

Carbon fiber sheet

SyringeGel box (gel electrophoresis chamber)

Red electrode (wire lead with clips)

Black electrode (wire lead with clips)

1% Melt-N-Pour Agarose, 30 mL

6 Yellow pipet tips

Tubing (to make a syringe adapter)

Methyl orangeBromophenol blue

1× TBE buffer solution, 75 mL

Explorer IXylene cyanolPonceau G

Explorer II


Safety

Metric ruler

Needed from the equipment kit: Wear your goggles, gloves, and lab apron at all times while conducting this investigation.

Read all the instructions for the laboratory activ-ities before beginning. Follow the instructions closely, and observe established laboratory safety practices, including the use of appropriate personal protective equipment (PPE).

When melting the agarose, use an oven mitt or several layered paper towels to handle the bottle. Loosen the bottle cap before heating the bottle; otherwise pressure may build up, causing the top to pop off or the bottle to explode.

The gel runs at a low voltage. To avoid electric shock, do not touch the gel or buffer when there is current running through it (that is, when the electrodes are connected to the batteries). Do not use more than five 9-volt batteries to run the gel.

The dyes provided in this kit may stain skin or clothing. Avoid contact.

Do not eat, drink, or chew gum while performing the activities. Wash your hands with soap and water before and after performing each activity. Clean up the work area with soap and water after completing the investigation. Keep pets and children away from lab materials and equipment.

Reorder Information: Replacement supplies for the Introduction to Biotechnology Methods investigation can be ordered from Carolina Biological Supply Company, kit 580150.

Call: 800.334.5551 to order.

Needed but not supplied:• 3–5 batteries, 9 volt• Microwave oven• Scissors• Camera or mobile device capable of taking

digital photos• Printout of Figure 4• Pen or pencil• Paper towels or oven mitt• Stopwatch or other timing device

Preparationor an oven mitt around the bottle and gently swirl the agarose. If parts of the agarose are not yet melted, place the bottle back in the microwave and heat it until the agarose is completely melted. The agarose will have no lumps and should pour easily.

c. Position the 6-well comb at one end of the gel box, also called a gel electrophoresis chamber (see Figure 3). Make sure the comb is pushed down as far as possible, leaving the teeth of the comb approximately 1 mm from the floor of the gel box.

d. Let the melted agarose cool for approximately 1 minute. Be careful; the agarose will be hot.

7. Place the gel box on a flat surface, and pour approximately 10 mL of the melted agarose


1. Read the investigation manual thoroughly, and become familiar with the kit materials and activities before beginning.

2. Obtain all materials.3. Clean and sanitize the work area.4. From the carbon fiber sheet provided,

measure and cut two pieces, each approximately 22 × 42 mm. These pieces will be used as electrodes. Set them aside for later use.

5. Preparation of the agarose gel is described in Steps 6–8. View the following video before

preparing the gel.

Pouring a Gel http://players.brightcove.net/17907428001/HJ2y9UNi_default/index.html?videoId=4573409701001

6. Completely and thoroughly melt the agarose. a. Loosen, but do not remove, the agarose

bottle cap. b. Using a microwave oven, heat the agarose for 30 seconds. During

these 30 seconds, check the agarose. As soon as it starts to boil, place a paper towel that has been folded several times

Figure 3.


Figure 2.


http://players.brightcove.net/17907428001/HJ2y9UNi_default/index.html?videoId=4573409701001


into the center of the box without allowing the agarose to flow over the lips and the ends of the box (see Figure 3). Make sure to use a paper towel or an oven mitt to hold the bottle.

8. Allow the agarose to harden in the gel box for approximately 20–30 minutes without moving or jostling it. Proceed to Activity 1 while the gel hardens.



ACTIVITY 1 A Sickle Cell Anemia Detection

Simulation: PCR, Restriction Enzyme Digest, and Electrophoresis

In this activity, the genotyping of a newborn baby will be simulated with respect to the sickle cell mutation. Does the baby have a wild-type beta-globin gene, a mutation on one allele (carrier), or a mutation on both alleles (homozygous)? Known samples of wild-type and homozygous mutant beta globin will be used for comparison.

1. Write down the known partial DNA sequence for the wild-type beta-globin gene as seen below. This sequence represents the portion of the beta-globin gene that has been amplified using PCR. Typically, amplified sequences will be longer than this example, and some restriction enzymes need longer sequences to function. However, for ease in this activity, the sequences are short.

Wild-type: 5' CTG ACT CCT GAG 3' 3' GAC TGA GGA CTC 5'

2. Beside the wild-type sequence, write the DNA sequence for the known mutation associated with sickle cell anemia.

Homozygous mutant: 5' CTG ACT CCT GTG 3' 3' GAC TGA GGA CAC 5'

3. Determine the recognition site and cleavage pattern of Dde1 by referring to the restriction enzyme table provided in the background section (see Table 1). Remember that the “N” notation represents any of the four nucleotides.

ACTIVITY

If the gel is prepared more than 30 minutes in advance, cover the hardened gel with a small amount of buffer to minimize evaporation. Do not prepare the gel more than a few hours in advance because the agarose will dry out.

-

Figure 4.

ACTIVITY



ACTIVITY 2

A Mini Gel Electrophoresis of DyesIn this activity, DNA fingerprinting is simulated, substituting dye samples for the DNA samples used in professional laboratories. The following video provides an overview of Steps 1–5.

Loading a Gel http://players.brightcove.

net/17907428001/HJ2y9UNi_default/index.html?videoId=4573412123001

1. Assemble the syringe (see Figure 5). a. Fit the piece of tubing on the end of the

syringe to create a syringe adapter.

4. Using the information in Table 1, find the recognition site for Dde1 in your written DNA sequences. Draw a circle around the Dde1 recognition site(s) in your DNA sequence, and draw a bold line where Dde1 would cleave. Remember, Dde1 will only cut where there is an exact match in sequence.

5. In this step, you will draw bands on the gel diagram in Figure 4 representing DNA samples from individuals with different sets of beta-globin alleles.

a. Begin by counting the number of DNA fragments that were produced by treatment with Dde1 for the wild-type and homozygous mutant samples. The number of fragments after Dde1 treatment tells you the number of bands you will draw for each sample. Recalling that a heterozygote will have one of each allele, determine the number of bands that will be present in the heterozygous sample.

b. Count the number of base pairs for each fragment. Compare your counts to the size of the DNA markers shown on the far-left side of the gel diagram. The size of each DNA fragment, expressed in nucleotide base pairs, determines its position in the gel at the end of electrophoresis. Draw the bands for each sample: wild-type, heterozygous mutant, and homozygous mutant.

c. Compare the band patterns drawn on the gel diagram to the sample DNA fragment from the baby, on the far-right side of the gel. This will enable you to determine the baby's genotype.

d. Take a photo of your completed gel diagram to document your work.

ACTIVITY 1 continued



b. Trim the tubing so that it is flush with the end of the syringe tip.

c. Fit one yellow pipet tip on the syringe over the tubing. The tubing should remain on the syringe when the yellow pipet tip is replaced.

2. Gently remove the well comb from the hardened gel by slowly wiggling it upward.

3. Pour enough 1× TBE buffer solution into the gel box to completely cover the agarose gel. The buffer level should be 2–3 mm above the gel.

4. Draw a diagram to indicate which dye slution you will load into each well.


Figure 5.


Figure 6.

5. You will use the syringe with the adapter and pipet tip to load dye into the wells of the gel.

a. Very slowly and carefully, draw up a small amount of the dye from the first tube into a yellow pipet tip, avoiding any air bubbles (see Figure 6).

b. Pipet the dye mixture into the first well. Hold the syringe with only the very tip in the upper portion of the well. Work carefully, and avoid piercing the bottom of the well with the syringe tip. Eject only the dye into the well because air bubbles will push the dye out of the well and into the buffer above.

During shipping, the dye solutions may become scattered inside their tubes. To collect the dye solution back to the bottom of the tube, tap the tube bottom repeatedly on a hard surface.

Note that the Explorer I and Explorer II tubes may contain any of the dyes in the other tubes, or even a mixture of dyes. At the end of the electrophoresis procedure, you should be able to determine which dyes were present in these two tubes.

ACTIVITY 2 continued

ACTIVITY


should be a current flowing. Small bubbles may be forming on the carbon fiber.

11. Observe the gel as it runs. Using three batteries, it may take up to 3.5 hours for the dye to travel through the gel. Using five batteries will reduce the run time to approximately an hour.

12. Allow the gel to run until the fastest dye is 1 cm from the end of the gel nearest the positive electrode.

13. Stop the electrophoresis by unclamping one of the alligator clips from the carbon fiber sheet.

14. Immediately take a photo of the gel and dye bands for reference. Once the gel is disconnected from the power

source, the dye will begin to diffuse through the gel and the bands will become blurred and difficult to observe.

Disposal and Cleanup1. Disconnect the batteries from the red and

black electrodes to stop the current through the gel.

2. The gel and yellow tips should be disposed of in the household trash.

3. Dispose of solutions down the drain with the water running. Allow the faucet to run a few minutes to dilute the solutions.

4. Rinse and dry the lab equipment, and return the materials to your equipment kit.

5. Sanitize the work space.6. Wash your hands.


6. Load the remaining five dye solutions into the remaining five wells. Use a new yellow pipet tip for each dye.

7. Get the carbon fiber sheets you cut during preparation.

8. Connect three to five 9-volt batteries (see Figure 7). The gel electrophoresis will run more quickly with five 9-volt batteries. Do not use more than five batteries.

9. Connect the black wire to the negative electrode (carbon fiber sheet) at the end of the gel box nearest the wells and the red wire to the positive electrode at the opposite end by folding each carbon fiber sheet over one edge of the box and clamping the appropriate alligator clip onto the sheet to hold it in place. Make sure the carbon fiber sheet makes contact with the buffer as shown in Figure 7.10. Connect the black wire to the negative

terminal of the battery and the red wire to the positive terminal of the battery. There

Figure 7.


NOTES

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BIOLOGY Introduction to Biotechnology Methods

Investigation Manual

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