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3.1

Pre-lab Assignment Sheet for PRACTICAL 3

Family Name: First name: Bench No: Stream No: Mark: Date:

This laboratory session is designed to give you an overview of the technical steps necessary to detect a gene within an organism's DNA. Specifically you need to understand the processes of DNA extraction, PCR and electrophoresis.
1. What does the Bt gene produce and how does it affect insects that eat plants that express the gene? (1 marks)

2.

The isolation of DNA involves three steps. Complete the table below indicating what chemicals or solutions are used and the purpose of each step (2 marks)

Steps 1. Break up tissue

Chemicals / solutions
Grind up tissue with a buffer and detergent

Purpose

2. DNA extraction

3. DNA precipitation

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3.

The DNA test designed by GeneSleuth Inc to detect the genetically engineered corn (MON810) will amplify two different alleles in hybrid Bt corn. Describe what element(s) makeup each allele and give their approximate size in base pairs (bp). (1 mark)

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LABORATORY 3 Detecting an inserted gene

At the end of this practical you should be able to: Summarise the processes and techniques used in inserting a foreign DNA sequence into a host organism Describe the process of DNA extraction Understand the basis for the polymerase chain reaction (PCR) Describe the process of electrophoresis Interpret an electrophoresis gel and justify the need for positive and negative controls in a PCR experiment Suggested reading: Campbell Biology 9th Ed. Chapter 20 pp. 448-451, 467-468 Campbell & Reece, Biology 8th Ed. Chapter 20 pp. 403-405, 421-422

Maize kernels

From Fig. 38.19b Campbell & Reece, Biology 6th Edn

Corn Borer

Insertion of genes into corn A Scenario

Last week Sunshine Bumblebee Organic Foods LTD became suspicious that some plants on one of its farms had been cross-pollinated with pollen from the corn variety MON810. MON810 was being grown on a neighbouring farm. MON810 is a genetically engineered (GE) variety of Zea mays (maize/corn) engineered to contain the Cry gene from the bacterium Bacillus thuringiensis (Bt). Concerned over public perception, Sunshine Bumblebee employed GeneSleuth (Inc) - a company that provides a commercial service for gene detection. One complication facing GeneSleuth was that Sunshine Bumblebee spray their corn with Bacillus thuringiensis bacteria (which is organically accredited) and so the Cry gene and the Cry protein would be present on all plants regardless of whether they were GE or not. To overcome the problem, GeneSleuth designed a DNA-based test in which the PCR primers bound to the maize genome sequences flanking the engineered Cry gene. These sequences are not present in the naturally occurring Cry gene. GeneSleuth was given samples of 1000 individual plants from the organic farm plus samples of wild type corn and MON810. DNA was extracted from each sample. The DNA was PCR amplified with the two PCR primers. Zea mays is diploid and so plants that have hybridised with GE plants will have some kernels that are heterozygous for the Cry gene.

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Maize/corn flowers Maize/corn flowers occur in distinct clusters (called inflorescences). The clusters (called tassels) at the top of the plant, contain only male flowers. The other clusters, on the lateral shoots lower down, contain only female flowers. The corn cob that we are familiar with is a cluster of (several hundred) individual seeds (kernels) each having developed from ovules in a female flower. The many ovaries in female flowers were fertilised independently by many (male) pollen grain. The pollen grains were transferred by wind currents from male flowers to the female flower. Remember - each ovule was fertilised separately by a pollen grain that could have come from a totally different tassel to the pollen of its neighbours

Female Flower

Male Flower

Silk

Tassel

The silk strands (styles) each lead to individual maize kernels.

Each tassel has numerous pollen containing anthers.

From Fig. 38.19b Campbell & Reece, Biology, 6th Edn. 2002
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3.5

(Source: Vivian Ward, SBS)

Bt and its action

Bacillus thuringiensis (Bt) is a naturally occurring bacterium that has the ability to produce a crystalline protein that is toxic to certain insect larvae. Upon ingestion by insect larvae, Bt toxins cause gut cells to swell and lyse (burst), leading to larval death. Commercial sprays making use of this bacterial property has been used for decades by gardeners and organic farmers. Different subspecies of Bt are selectively toxic to different insect orders. Different strains selectively target Lepidoptera (butterflies and moths); Diptera (flies and mosquitoes) and Coleoptera (beetles). The Bt gene (Cry) has been sequenced and inserted into crops (e.g. corn) to provide insect resistance. In general when inserting the Bt gene into a crop, researchers choose the subspecies of toxin that affects the main pest of that crop. The main target of the Bt gene in MON810 corn is the corn borer.

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DNA extraction - obtaining a sample of DNA from plants

The process of isolating DNA from the rest of the cellular material is called DNA extraction. DNA is contained in the cell nucleus and is packaged in chromosomes. DNA is extracted by grinding the tissue (e.g. leaves or kernels) together with a buffer that has a detergent in it, to release cellular contents. Organic solvents such as phenol and chloroform are used to remove the proteins, lipids and carbohydrates then the DNA is precipitated using ethanol, this serves to concentrate the DNA.

The Polymerase Chain Reaction (PCR)

The polymerase chain reaction (PCR) is a method for making many copies of a specific segment of DNA. The starting material is a solution of DNA containing the nucleotide sequence targeted for copying. The scientist adds a heat resistant DNA polymerase (usually TAQ polymerase, isolated from the thermophyllic bacterium Thermus aquaticus), a supply of all four nucleotides, and primers. The primers are needed because the DNA polymerase can only add nucleotides to a pre-existing DNA chain. Primers are generally designed to be very specific and the primers used in your experiment will only amplify the maize/corn DNA.
1 Denaturation - the DNA is briefly heated to separate its strand. 2 Annealing - cooled to allow the primers to bind by hydrogen bonding to the ends of the target sequence, one primer on each strand. 3 Amplification - then the DNA polymerase extends the primers, using the target DNA strands as templates. Within about five minutes the target DNA sequence has been copied. The solution is then heated again, starting another cycle of strand separation, primer binding and DNA synthesis. The cycle runs again and again, duplicating the target sequence many times.

PCR amplifies short target DNA sequences more readily than long ones, so in practice target sequences are typically less than 2000 base pairs (bp) long, but with care, lengths of over 10 kilobases (kb) can be amplified.

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3.7

The Polymerase Chain Reaction (PCR) continued

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Electrophoresis of DNA
Electrophoresis means, literally, to carry with electricity. DNA, as an organic acid, is negatively charged. Hence when placed in an electric field, DNA molecules are attracted to the positive pole and repelled from the negative pole. We can separate differently sized DNA fragments by electrophoresis. 1 An agarose gel acts as a molecular sieve through which smaller fragments can move more quickly than larger ones. 2 Thus, over a given time, smaller fragments migrate relatively further from the origin than do the larger fragments. 3 Melted agarose (a polysaccharide derived from seaweed) is poured into a casting tray in which a plastic comb has been inserted. As it cools the agarose solidifies to a gelatinous substance consisting of a dense network of crosslinked molecules. The solidified gel slab is immersed in a chamber filled with buffer solution containing ions needed to conduct electricity. Removal of the comb leaves behind a series of wells into which the DNA samples are loaded. Prior to loading the DNA is mixed with a loading dye consisting of sucrose and one or more dyes. The movement of the dye allows us to monitor the migration of the unseen DNA fragments. Current is applied through electrodes at either end of the gel chamber. Following electrophoresis the gel is removed and exposed to medium wavelength ultraviolet light. A stain in the gel binds to the DNA band and fluoresces. It is important to remember that a band of DNA seen in a gel does not represent a single molecule of DNA, but rather the total fluorescence from millions of DNA fragments of the same base-pair length.

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Interpreting the Electrophoresis Gel

In diploid wild type corn, the PCR primers amplify a 500 bp control region of DNA on both chromosomes. When the amplified control region is run on an electrophoresis gel the DNA migrates rapidly down the gel resulting in a single band. In hybrid Bt type corn both a 500 bp control region and a 2500 bp region of DNA are amplified. The 2500 bp region is a combination of the Bt gene and the control region. When run on an electrophoresis gel the 2500 bp region migrates more slowly than the 500 bp control region, resulting in two clearly defined bands. Note: If the PCR fails, for example if the DNA polymerase is inhibited, no bands will be present. A positive (+ve) control is necessary in all PCR tests and contains a mix of DNA from a wild type plant and a MON810 plant. A negative (-ve) control is also necessary. The PCR reaction is run containing water in the place of DNA.

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The Scenario...
Source: Vivian Ward, SBS

Scenario: Sunshine Bumblebee Organic Farms Ltd crop growing next to GE MON810 crop. Corn seed produced by organic farm.

DNA extraction.

This seed and next generation plants: (source of plant DNA used in extraction).

DNA amplification by PCR. Target sequence is amplified.

PCR products (target DNA sequence) are separated using gel electrophoresis.

Bands of different sized DNA fragments are visualised using UV light.

During the laboratory, you will complete steps 6 and 7 of this process.
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Your task is to analyse the samples of amplified DNA from the corn and determine if there is any hybrid corn growing on the organic farm (essentially part six and seven of this scenario)

The Task: Electrophoresis of PCR products

Part A: Pouring an agarose gel
For this exercise, work in pairs You will separate the PCR products using gel electrophoresis But FIRST do you have everything you need? Tick this checklist for your group of two

No. 1 1

Items Gel chamber including comb Plastic bulb pipette Gloves

Tick

1 1

500 ml Schott bottle of buffer 50 ml Schott bottle of agarose (in water bath)

1. Put on gloves before starting. 2. Place the gel base in the chamber - see image and diagram comb in the gel base in the end position - see image and diagram 2 . 3. When you pour the gel, the gel base should be positioned with the seals (red ends) hard up against the green sides of the gel chamber.
1

. Then place the

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4. Get a Schott bottle of agarose dissolved in buffer from the water bath at the side of the lab (careful its hot!) and gently swirl the contents before removing the lid. Pour all the liquid into the gel base. 5. Allow 15 minutes for the agarose gel to set. Now realign the base containing the gel with red ends facing the white ends of the gel chamber. The wells should be positioned over the black stripe - see diagram 3 . 6. Pour the entire volume of buffer (in the labelled Schott bottle) into the electrophoresis chamber - see digram 4 . Note that the gel should be covered by buffer. This is why this type of gel can be called a submarine gel. 7. Now remove the comb by gently pulling it directly upwards - see diagram 4 . The holes (known as "wells") that the comb leaves in the gel are where you will load your samples. 8. The gel is now ready for loading of samples. Before loading your samples wash wells using a plastic bulb pipette, this helps your samples load. Your demonstrator will show you how.

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Part B: Loading PCR reactions onto the agarose gel

But first do you have everything you need? Tick this checklist for your group of two. No. 8 1 1 Items PCR tubes, one set for each group Loading dye (orange colour) in an eppendorf tube (orange tube) P20 Micropipette & Yellow Tips Tick

1. Take a DNA sample. Each microfuge tube contains 10 l of the PCR sample. 2. Using the micropipette, add 5 l of the loading dye to the microfuge tube (= 15 l total volume) and flick the tube with your finger to properly mix the two solutions. Tap the microfuge tube on the bench so the sample will collect at the bottom. 3. Repeat step 2 for each of the eight samples. From now on use a new tip for

each addition!

4. Using the pipette, take the first sample (all 15 l of it) and insert tip through the buffer and expel the contents into the first well in your agarose gel.

Start with sample 1 in well 1. The positive (+ve) control should thus be in well 7 and the negative (ve) control in well 8. To improve your laboratory skills each person should load four of the eight samples. Also ensure that, for each sample you record the well number (always important to be absolutely certain where you placed your samples).

5. When all samples are loaded, put the lid on the gel chamber. Then see your demonstrator who will connect the power supply. 6. Set the timer for 30 minutes and press start.

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Part C: Detecting the DNA, viewing the gel, and interpreting results
1. Once your gel has run for 30 minutes, see your demonstrator and they will turn off the power supply and make sure that it is safe to remove your gel. 2. Take your gel in the tray provided and get the demonstrator to take a digital picture of your gel. The demonstrator will print you a copy of your results which you can now interpret. 3. Tutors and demonstrators will assist you to interpret results. Refer to page 3.7 to guide you.

Glossary
Base pairs (bp): a set of two bonded nucleotides on opposite strands of DNA. There are two possible base pairs: C-G and A-T. DNA polymerase: Generates a second strand of DNA from single stranded template. DNA polymerase can only extend existing double stranded regions and therefore requires a primer. Maize/corn: In general use these two words are synonymous and refer to the plant Zea mays, or the grain produced by that plant. Nucleotide: a purine or pyrimidine ribose or deoxyribose sugar and a phosphate base Primer: short (usually 18 30 nucleotides long) synthetic molecules of single stranded DNA complementary to the ends of the targeted DNA.

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Hazardous Substances
CHEMICAL Agarose SYBR*safe DNA gel stain FORMULA HAZARD ADDITIONAL INFORMATION Low risk
Flush with plenty of cold water, if eye or skin contact occurs

Tris

H2NC(CH2OH)3

Harmful

Harmful if swallowed or inhaled. Causes irritation to skin, eyes and respiratory tract May be harmful if swallowed/ inhaled. Avoid contact with skin Ethylenediaminetetraethanoic acid. Skin, eye and respiratory irritant Respiratory irritant in highly concentrated mist May be harmful if swallowed, inhaled or absorbed through skin. TOXICOLOGY NOT FULLY INVESTIGATED Avoid contact with eyes or skin

Sodium Ethanoate (Sodium Acetate) EDTA

CH3COONa.3H2O

Harmful

C10H16N2O8

Irritant

Glycerol Orange g

C3H8O3 C6H5N: NC10H4(OH)(CO3Na)2

Flammable Irritant

Ethanoic (acetic) acid

CH3COOH

Corrosive

CLEANING UP INSTRUCTIONS
1. Leave buffer in gel boxes. DO NOT EMPTY. 2. Place perspex wells in container on side bench 3. Place a) microfuge tubes (i.e. gel sample tubes) b) yellow tips (RETURN empty beaker to your bench). c) gels IN YELLOW BIOHAZARD BINS.

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