2011 Lab 3 Genetics Lab manual.pdf

8/14/2019 2011 Lab 3 Genetics Lab manual.pdf

1/18


2/18


3/18

3.1LABORATORY 3

LABORATORY GUIDE BIOSCI 101

Pre-lab Assignment Sheet for

PRACTICAL 3

Family Name:

First name:

Bench No:

Stream No:

Mark:Date:

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 (2marks)

This laboratory session is designed to give you an overview of the technical steps

necessary to detect a gene within an organism's DNA. Specifcally 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? (1marks)

Steps Chemicals / solutions Purpose

1. Break up tissue Grind up tissue with abuffer and detergent

2. DNA extraction

3. DNA precipitation


4/18

3.2 LABORATORY 3

BIOSCI 101 LABORATORY GUIDE

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 al-

lele and give their approximate size in base pairs (bp). (1mark)


5/18

3.3LABORATORY 3


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 Crygenefrom 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 thuringiensisbacteria (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 anking 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

amplied with the two PCR primers. Zeamaysis diploid and so plants that have hybridisedwith GE plants will have some kernels that are heterozygous for the Cry gene.

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, Biology8th Ed. Chapter 20 pp. 403-405, 421-422

From Fig. 38.19b

Campbell & Reece,

Biology 6th Edn

Maize kernels

Corn Borer


6/18

3.4 LABORATORY 3


Maize/corn owers

Maize/corn owers occur in distinct clusters (called inorescences). The clusters (called

tassels) at the top of the plant, contain only male owers. The other clusters, on the

lateral shoots lower down, contain only female owers.

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 ower. The many ovaries

in female owers were fertilised independently by many (male) pollen grain. The pollen

grains were transferred by wind currents from male owers to the female ower.

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

The silk strands (styles) each lead to indi-

vidual maize kernels.

Each tassel has numerous pollen containing

anthers.

Female Flower Male Flower

SilkTassel

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


7/18

3.5LABORATORY 3


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 (butteries and moths); Diptera (ies 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.

(Source: Vivian Ward, SBS)


8/18

3.6 LABORATORY 3


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 init, 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 specic

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 specic and the primers used in your experiment will only amplify the maize/corn

DNA.

Denaturation - the DNA is briey heated to separate its strand.

Annealing - cooled to allow the primers to bind by hydrogen bonding to the ends of

the target sequence, one primer on each strand.

Amplication - then the DNA polymerase extends the primers, using the target

DNA strands as templates. Within about ve 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 amplies 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 over10 kilobases(kb) can be amplied.

1

2

3


9/18

3.7LABORATORY 3


The Polymerase Chain Reaction (PCR) continued


10/18

3.8 LABORATORY 3


Electrophoresis of DNA

Electrophoresis means, literally, to carry with electricity. DNA, as an organic acid, is negativelycharged. Hence when placed in an electric eld, DNA molecules are attracted to the positive

pole and repelled from the negative pole.

We can separate differently sized DNA fragments by electrophoresis. An agarose gel acts as

a molecular sieve through which smaller fragments can move more quickly than larger ones.

Thus, over a given time, smaller fragments migrate relatively further from the origin than

do the larger fragments.

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 solidies to a gelatinous substance

consisting of a dense network of crosslinked molecules. The solidied gel slab is immersed in

a chamber lled 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 uoresces.

It is important to remember that a band of DNA seen in a gel does not represent a singlemolecule of DNA, but rather the total uorescence from millions of DNA fragments of the

same base-pair length.

2

3

1


11/18

3.9LABORATORY 3


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 amplied 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

amplied. 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 the500 bp control region, resulting in two clearly dened 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.

1

2

3

4


12/18

3.10 LABORATORY 3


The Scenario...

During the laboratory, you will complete steps 6 and 7 of this process.

Source: Vivian Ward, SBS

Scenario: Sunshine Bumblebee Organic

Farms Ltd crop growing next to GE

MON810 crop.

This seed and next

generation plants:

(source of plant

DNA used in extrac-

tion).

Corn seed produced

by organic farm.

DNA extraction.

DNA amplication by

PCR. Target sequenceis amplied.

PCR products

(target DNA

sequence) are

separated using gel

electrophoresis.

Bands of different sized

DNA fragments are visu-

alised using UV light.


13/18

3.11LABORATORY 3


Part A: Pouring an agarose gel

For this exercise, work in pairsYou 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. Items Tick

1 Gel chamber including comb

1 Plastic bulb pipette

Gloves

1 500 ml Schott bottle of buffer

1 50 ml Schottbottle of agarose (in water bath)

1. Put on gloves before starting.

2. Place the gel base in the chamber - see image and diagram . Then place thecomb in the gel base in the end posi-tion - see image and diagram .

3. When you pour the gel, the gel baseshould be positioned with the seals(red ends) hard up against the greensides of the gel chamber.

Your task is to analyse the samples of amplied

The Task: Electrophoresis of PCR products

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)

1

2


14/18

3.12 LABORATORY 3


4. Get a Schott bottle of agarose dissolved in buffer from the water bath at theside of the lab (careful its hot!) and gently swirl the contents before removingthe lid. Pour all the liquid into the gel base.

5. Allow 15 minutes for the agarose gel to set. Now realign the base containing thegel with red ends facing the white ends of the gel chamber. The wells should bepositioned over the black stripe - see diagram .

6. Pour the entire volume of buffer (in the labelled Schott bottle) into theelectrophoresis chamber - see digram . Note that the gel should be coveredby 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 . Theholes (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 demonstratorwill show you how.

4

3

4


15/18

3.13LABORATORY 3


Part B: Loading PCR reactions onto the agarose gel

But frst do you have everything you need?

Tick this checklist for your group of two.

No. Items Tick

8 PCR tubes, one set for each group

1 Loading dye (orange colour) in an eppen-dorf tube (orange tube)

1 P20 Micropipette & Yellow Tips

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 ick the tube with your nger to properly mix the twosolutions. Tap the microfuge tube on the bench so the sample will collect at thebottom.

3. Repeat step 2 for each of the eight samples. From now on use a new tip foreach addition!

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

Start with sample 1 in well 1. The positive (+ve) control should thus be inwell 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 importantto be absolutely certain where you placed your samples).

5. When all samples are loaded, put the lid on the gel chamber. Then see yourdemonstrator who will connect the power supply.

6. Set the timer for 30 minutes and press start.


16/18

3.14 LABORATORY 3


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 re-

quires 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.

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 turnoff 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 digitalpicture of your gel. The demonstrator will print you a copy of your resultswhich you can now interpret.

3. Tutors and demonstrators will assist you to interpret results. Refer to page3.7 to guide you.


17/18

3.15LABORATORY 3


Hazardous Substances

CHEMICAL FORMULA HAZARD ADDITIONAL INFOR-

MATION

Agarose Low risk

SYBR*safe DNA gel stain Flush with plenty of coldwater, if eye or skin contact

occurs

Tris H2

NC(CH2

OH)3

Harmful Harmful if swallowed orinhaled. Causes irritation to

skin, eyes and respiratory

tract

Sodium Ethanoate

(Sodium Acetate)

CH3COONa.3H

2O Harmful May be harmful if swallowed/

inhaled. Avoid contact with

skin

EDTA C10

H16

N2O

8Irritant Ethylenediaminetetraethano-

ic acid. Skin, eye and respira-

tory irritant

Glycerol C3H8O3 Flammable Respiratory irritant in highlyconcentrated mist

Orange g C6H

5N:

NC10

H4(OH)(CO

3Na)

2

Irritant May be harmful if swallowed,inhaled or absorbed through

skin. TOXICOLOGY NOT

FULLY INVESTIGATED

Ethanoic (acetic)

acid

CH3COOH Corrosive Avoid contact with eyes or

skin

CLEANINGUP INSTRUCTIONS

1. Leave buffer in gel boxes. DO NOT EMPTY.2. Place perspex wells in container on side bench3. Place a) microfuge tubes (i.e. gel sample tubes) b) yellow tips (RETURN empty beaker to your bench). c) gels

IN YELLOW BIOHAZARD BINS.


18/18

3.16 LABORATORY 3

Documents

2011 Lab 3 Genetics Lab manual.pdf