What to do before Practical 1

DNA, a Gene, an Auxotroph

Before this practical you will need to:
& Read Knox et al. (2014) pp. 35-36, pp. 199-203
& Read Appendices A2: The Dissecting Microscope and A4: Procedures for Genetic Analysis
Watch TechTip: Using a micropipette.
Watch BioByte: The Chi Square Test
6 Print out and read Practical 1 notes
" Complete the flow chart of DNA extraction page P1-2

" Complete the Pre-practical test (worth 1 mark out of the 10 marks available for the
practical).
All references for pre reading are to your textbook:
Knox, B, Ladiges, P, Evans, B & Saint, R 2014, Biology, an Australian focus, 5th edn, McGraw-Hill
Education (Australia), North Ryde.

Bring to practical:
• Printed copy of Practical 1 notes
• Lab coat, safety glasses, sharp HB pencil ruler, eraser, pen, calculator
• Permanent marker pen for labelling
• Student card for scanning

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 Practical 1
DNA, a Gene, an Auxotroph
Purpose:

In this class you will observe the DNA material isolated from bacteria. You will investigate
the phenotypic changes caused by a single gene in Arabidopsis thaliana, propose a
genetic hypothesis to explain your observations and test this with statistical analysis.
You will observe the growth, or absence of growth, of auxotrophic bacteria in the presence
of supplements.

After completing this practical you should be able to:

• Extract and visualise DNA from E.coli.

• Analyse data from a genetic cross involving one autosomal gene locus.
• Observe bacterial growth on minimal media to determine which supplements are required
for growth of different auxotrophs.

Assessment

• Pre-practical Test
• In-practical assessment
• Integrated Post-practical questions in Module Test 2

Time Management
• Begin practical with the DNA extraction.
• During Steps 1 and 3 incubations (each 15 minutes) start the analysis of the Arabidopsis plates.
• DNA extraction should be completed at the end of one hour and shown to your demonstrator for
assessment.
• Complete the observations of the auxotroph.

Practical Timeline Estimated Time

Introduction 10 mins
Activity 1: DNA extraction from E. coli 50 mins
Activity 2: An Autosomal gene (Arabidopsis) 20 mins
Activity 3: Characterisation of Auxotrophs Part 1 15 mins
Clean up 5 mins

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BIOL10005: 2018 Practical 1: DNA, a Gene, an Auxotroph P1 – 3
Your safety in the laboratory is very important:
• At all times wear a lab coat and suitable shoes with enclosed heel and toe.
• Safety glasses should be worn whenever there is a risk of a splash from your activities
or from those around you.
• Always work to ensure your safety and the safety of those around you.
• Use caution when using sodium perchlorate. It is a strong oxidising agent.
• Open microfuge tubes with care. Do not hold near your face.
• Immediately report any injuries or spills to a demonstrator.
• Microorganisms will be used in this class, avoid touching things to your mouth and wash
hands carefully after the class.

A risk assessment has been carried out for the practical classes and identified risks minimised.
Please observe the safety signs displayed and ask your demonstrator if you would like to know
more, MSDS (Material Safety Data Sheets) are available in the laboratory. Further information
can be found at: http://safety.unimelb.edu.au

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P1- 4 Practical 1: DNA, a Gene, an Auxotroph BIOL10005: 2018

Activity 1: DNA Extraction – Isolating DNA from E. coli Cells

Escherichia coli (E. coli) is a rod-shaped bacterium about 1-2 μm in length. Some strains can be
found in the human digestive tract, and one of these will be used in this activity. It is not harmful.
We will extract the DNA from a suspension containing approximately 1000 million cells. Escherichia
coli has a semi-rigid cell wall of peptidoglycan which supports the cell and determines its shape.
The wall surrounds the plasma membrane. Each cell contains a single chromosome, which is a
DNA molecule tightly coiled inside the cell, forming the nucleoid (Figure 1). The absence of a
nuclear envelope is one of the differences between prokaryotes and eukaryotes, as taught in
semester one. The chromosome is “circular” (forms a loop) and comprises approximately 4.7
million base pairs and 1500 genes. Cells commonly contain small circular extra-chromosomal DNA
called plasmids (Knox et al. 2014 (5th edition) p. 319; Knox et al. 2010 (4th edition) p. 292).
If you are unsure of what a base or base pair in DNA is, consult Knox et al. 2014 (5th edition) p. 35;
Knox et al. 2010 (4th edition) p. 50).

Figure 1. Diagram of an E.coli cell.

The steps involved in extracting and isolating DNA are:

1. Rupturing the cell wall, and liberating the contents of the cell into solution. This is called lysis.
2. Separating the DNA from the protein.
3. Inactivating any enzymes released from the cell, which may break up the DNA when in contact
with it.

Prior to the practical complete a flow chart showing steps 1 to 7 of the DNA extraction.

Flow chart:

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BIOL10005: 2018 Practical 1: DNA, a Gene, an Auxotroph P1 – 5

Equipment
• A plastic tube containing E. coli suspension
• Pipettes
• Microfuge tubes containing lysozyme, protease, sodium dodecyl sulphate
• Conical flask containing sodium perchlorate
• Squeeze bottle containing 100% Ethanol

Procedure
The cells you will use were grown in nutrient broth and have been resuspended in a lysis buffer to
a volume of 2.5 ml. They are in a screw-top tube on your bench.
1. Using a pipette, add 0.5 ml of the enzyme lysozyme (10 mg/ml) to the cell suspension. With
a permanent marker, label the lid of the tube with your seat number and screw the lid on,
leaving it a little loose. Incubate at 37˚C for 15 minutes. Lysozyme digests the cell wall.
2. Add 200 μL of the 20% ionic detergent named sodium dodecyl sulphate (SDS). Screw the
lid on tightly then gently mix by inverting the tube about five times. Sodium dodecyl sulphate
causes the plasma membrane of the cells to rupture.
3. Loosen the lid of the tube slightly and incubate at 55˚C for 15 minutes. The solution will
become slightly viscous, but this won’t be obvious.
4. Add 0.3 ml of protease (20 mg/ml) to the suspension (now called a lysate). Screw the lid on
loosely and incubate at 37˚C for 20 minutes.
5. Cool the tube and its contents on ice. The tube should feel cool to the touch before
proceeding.
6. Add 0.8 ml of sodium perchlorate (5 M). Invert gently about five times. Take care when
mixing (see Safety Note). The sodium perchlorate will assist in separating the protein from
the DNA and stabilising the DNA.
7. Tilt the tube at an angle and using a measuring cylinder carefully and slowly add 5 ml of
cold ethanol down the side of the tube so that it forms a layer above the DNA solution. It is at
the interface of the ethanol and lysate that the DNA will precipitate out of solution.
8. Identify the DNA as strands that form at the interface immediately after the addition of the
ethanol. There will be bubbles associated with these strands. Keep your tube vertical. Take
your tube and this page to your demonstrator for assessment.

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P1- 6 Practical 1: DNA, a Gene, an Auxotroph BIOL10005: 2018

Question 1: The solution may sometimes become viscous after the cell wall and cell
membrane are ruptured. Why might this be?

Question 2: Protease is an enzyme. What is an enzyme? Name another enzyme.

Question 3: What function does the protease perform in this experiment?

In a laboratory setting, the DNA you have extracted from E. coli would be purified and used in
other procedures such as gel electrophoresis (see Practical 3)

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BIOL10005: 2018 Practical 1: DNA, a Gene, an Auxotroph P1 – 7

Activity 2: An Autosomal Gene in Arabidopsis thaliana

In this section of the practical you will investigate the inheritance of a gene which is involved in the
plant’s response to the hormone gibberellin. Gibberellins (GAs) are plant hormones that are
necessary for many developmental processes, including seed germination, stem elongation, leaf
expansion, trichome (hair) development, pollen maturation and the induction of flowering. Hence,
mutant plants that are deficient in GA exhibit a dwarf and late-flowering phenotype. For most
mutants, normal growth will occur with the application of gibberellin. The mutant you are
investigating is insensitive to gibberellin, and application of the hormone does not change the dwarf
phenotype.

Can you suggest what the role of this gene might be?

Two pure breeding (homozygous) varieties of plants have been crossed (P),
and their heterozygous progeny were allowed to self fertilise to produce F2:
Parents (P1 X P2) = purebreeding à F1 = heterozygous à F1 X F1 à F2

Equipment
• Demonstration containers of parents and F1
• Container of F2 offspring

Procedure
1. Look at the demonstration pots showing these two varieties in the
parents (P1 and P2) and their offspring (F1).

Question 4: What is the phenotype of the F1 plants?

Figure 2. Wild type plant of Arabidopsis thaliana

Question 5: What does the observation about the F1 plants indicate?

Figure 3. A. thaliana dwarf plant

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P1- 8 Practical 1: DNA, a Gene, an Auxotroph BIOL10005: 2018

Plants of the F1 generation were allowed to self-fertilise and set seed (Figure 4). The F2 seeds were
germinated. You have been supplied with pots containing young F2 plants.

Score the phenotype of the plants and record your own data and the data from your group in Table 1.

Table 1: Data for F2

Phenotype

Number of wild type (tall)

Number of mutant (dwarf) plants Total
plants

Own data

Group
data

Figure 4. A. thaliana in fruit. The plants are staked to support the fruits for seed harvesting. The fruit is a siliqua 5–20 mm
long, containing 20–30 seeds.

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BIOL10005: 2018 Practical 1: DNA, a Gene, an Auxotroph P1 – 9
2. Using the conventional allelic symbols for this gene GAI1 (wild type allele) and gai1 (dwarf
allele), propose a genetic hypothesis to account for these observations by filling in the gaps
below.

Tall Dwarf
Parental
×
genotypes

F1 genotypes

F2 genotypes meiosis

Question 6: The expected F2 phenotypic ratio is :

Question 7: State the genetic hypothesis by completing the following:

1. There is/ are ______ gene locus/i being considered in this cross with ______ allele/s. The
dominant phenotype is _________________________. The F2 of this _____________ cross will
produce a phenotypic ratio of ____________________.

2. Using the appropriate statistical test (c2) compare the predictions of your genetic hypothesis with
the observed results. Use the class data. Apart from the information in the TechTip, you can
also refer to the Appendix pages at the end of this practical P1-14 to P1-17.
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P1- 10 Practical 1: DNA, a Gene, an Auxotroph BIOL10005: 2018

Watch the BioByte: The Chi-square Test: testing an hypothesis

3. State the null hypothesis.
4. Calculate the Chi-square (c2) value for this comparison by completing Table 2 (for your
calculations in genetic problems correct to 2 decimal places).

Table 2: c2 Analysis

Phenotype
TOTAL
Tall Dwarf

Observed
(O)

Expected
(E)

(O-E)2
E

c2 = å (O-E)2
E

Use the Chi-square table (see Table A1 in Appendix 1, P1 - 17).

Number of degrees of freedom

Probability

Conclusion: Circle the appropriate alternatives:

The null hypothesis is rejected / not rejected. Therefore, we accept / reject, the underlying genetic
hypothesis that gave rise to the prediction. That is, the observed results are consistent / not
consistent with the genetic hypothesis.

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BIOL10005: 2018 Practical 1: DNA, a Gene, an Auxotroph P1 – 11

Modelling meiosis in the Arabidopsis cross

The gibberellin insensitive gene is located on chromosome 1 in Arabidopsis thaliana. On the
diagram below, track chromosome 1 marked with its appropriate alleles through meiosis in the two
parents to the F1. Draw the chromosomes in the parents as they would be after DNA replication.

Tall parent Dwarf parent

X
end of meiosis 1

end of
meiosis II

Show the chromosomes immediately

F1 following fertilization

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P1- 12 Practical 1: DNA, a Gene, an Auxotroph BIOL10005: 2018

This page is deliberately blank.

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BIOL10005: 2018 Practical 1: DNA, a Gene, an Auxotroph P1 – 13

Activity 3: Analysis of Auxotrophs in E. coli

Collect your plates from the container.
In the last practical class you prepared these plates each spread with one of the mutant strains of
E. coli (Strain A, B, C or D) onto minimal media.
There is a demonstration plate of the wild type E. coli spread on minimal media.
Work as a group to see all 4 strains.
Hold your plate up to the light or against a dark background to look for a halo of growth around the
discs.

Question 8: Describe what you see on these 5 plates (hold the plate up to the ceiling to see
whether there is growth or not).

a. Wild type E. coli on Minimal Media

___________________________________________________________________________

b. Strain A on Minimal Media

___________________________________________________________________________

c. Strain B on Minimal Media

___________________________________________________________________________

d. Strain C on Minimal Media

____________________________________________________________________________

e. Strain D on Minimal Media

____________________________________________________________________________
Question 9: What can you conclude from these observations?

Question 10:
I worked with Strain _________________
Draw the pattern of growth you see, on the
template.

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P1- 14 Practical 1: DNA, a Gene, an Auxotroph BIOL10005: 2018

Question 11: Collect data for the 4 strains and enter the results in Table 5. Indicate when
growth occurred around the disc with a + and – for no growth

Table 5: Results of auxotroph growth on minimal media

Amino
Strain A Strain B Strain C Strain D
Acid
Arg
Leu

Meth
Tyr
Phe

Water

Note: you will not be able to draw a single biochemical pathway for these results.

Question 12:

a. Why is one disc impregnated with just water?

b. Had there been growth of E. coli around the disc impregnated with water, how would this
influence the conclusions you could draw from your experiment?

Question 13: Describe in what amino acid pathway there was a mutation for each of the strains.

Strain A

Strain B

Strain C

Strain D

Question 14: What was unusual about strain D? How can you explain this result?

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BIOL10005: 2018 Practical 1: DNA, a Gene, an Auxotroph P1 – 15

Appendix 1: Procedures for Genetic Analysis

The basic steps in genetic analysis are as follows:
1. Genetic Hypothesis (Genetic Model)
2. Statistical model (Null hypothesis)
3. Statistical test
4. Conclusion

Each of these steps will be examined in turn and then worked examples of genetic analysis will be
provided.

1. Genetic Hypothesis (Genetic Model)

We commence the analysis of genetic data by formulating a genetic hypothesis. This is done by
examining the phenotypes of parents and offspring. In particular, the ratio of phenotypes among
offspring is considered to see if they fit recognisable genetic ratios (e.g. 3:1). Always start with the
simplest ratios; only move on to consider more complex explanations if the simple ones can be
excluded.
In any case our genetic hypothesis should include the following:
1. Is the inheritance autosomal or sex linked?
2. Number of loci
3. Number of alleles (per locus)
4. Relationship between phenotypes (dominance, epistasis etc....)
5. The cross(es) showing genotypes and phenotypes
6. Prediction, that is expected phenotypic ratios in the offspring.
It is good practice to write out the genetic hypothesis in full.

2. Statistical model (Null Hypothesis)

On the basis of the genetic hypothesis a prediction is made e.g. we may predict a 3:1 ratio. Our
observed results may deviate from the expected (predicted) for two reasons:
a. Our genetic hypothesis is in error
b. Sampling error
In the real world we seldom get ideal figures. For example, in a case where we predict a 3:1 ratio,
we may see 93:27 rather than 90:30. In some ways this is analogous to coin tossing. If we toss a
coin we expect that half of the time it will land heads up and half of the time tails up. This probability
is based upon an infinite number of coin tossing where the effects of chance deviations in favour of
heads or tails cancel one another.
All experiments, however, involve finite numbers of observations and therefore some deviation
from the expected numbers (sampling error) is expected due to chance.
To allow us to evaluate these two sources of error we frame a null hypothesis. The word 'null'
means no, so the null hypothesis states that there is no difference between observed and expected
other than that due to sampling error. Putting it another way, deviations between observed and
expected are not due to an error in the predictions of our genetic hypothesis, but solely due to
chance.
In testing this null hypothesis, we need to decide objectively how large a deviation from the
expected should be allowed before the null hypothesis is rejected.
For example, in the case given above, is the difference between the 93:27 observed and the 90: 30
expected (on the basis of the genetic hypothesis) due to chance alone? Or is this difference due to
our genetic hypothesis being in error? Do we accept or reject the null hypothesis? The objective

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P1- 16 Practical 1: DNA, a Gene, an Auxotroph BIOL10005: 2018
statistical test uses the "chi-square (𝝌2) test" described in Section 3.

The null hypothesis is rejected in most genetic experiments when the deviation is so large that it
could be accounted for by chance less than 5% of the time. Such results are said to be significant
and the null hypothesis is rejected. When we reject the null hypothesis at the 5% level we take a
one in twenty chance of discarding a valid hypothesis. It must be remembered that statistics can
NEVER render absolute PROOF of the hypothesis, but merely set limits to our certainty.
If we accept the null hypothesis we also then accept the genetic hypothesis upon which it based.
The genetic hypothesis has not been proven; it simply explains the observed data satisfactorily
and is accepted until it is refuted by contrary data.

3. Statistical Test
a) Degrees of Freedom (df)
Assume a coin is tossed 100 times. We may arbitrarily assign any number of heads from 0 to 100
as appearing in this hypothetical experiment. However, once the number of heads is established,
the remainder is tails and must add up to 100. In other words, we have n-1 degrees of freedom,
where n is the number of classes (in this case 2; heads and tails).

(a) In an experiment involving three phenotypes (n=3), we can fill two of the classes at random,
but the number in the third class must constitute the remainder of the total number of
individuals observed. Therefore, we have 3-1 = 2 degrees of freedom.

(b) A 9:3:3:1 dihybrid ratio has four phenotypes (n=4). There are 4-1 = 3 degrees of freedom.
For most genetic problems concerning inheritance, the degrees of freedom will be one less than
the number of phenotypic classes.
b) Chi-square (c ) test
2

In order to evaluate a genetic hypothesis, we need a test which can convert deviations between
observed and expected values into the probability of such inequalities occurring by chance.
Furthermore, this test must take into account the size of the sample and the number of variables
(i.e. degrees of freedom). The chi-square test (pronounced ki, symbolised c) includes all of these
factors.

c 2
= å (O-E)2
E
df

Where O = observed number in a class

E = expected number in a class

c = sum of the values for all classes

df = degrees of freedom

The value may then be converted into the probability that the deviation is due to chance by
reading Table A1 at the appropriate number of degrees of freedom.

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BIOL10005: 2018 Practical 1: DNA, a Gene, an Auxotroph P1 – 17

Table A1: Chi-square Distribution

PROBABILITY
DEGREES
NON-SIGNIFICANT SIGNIFICANT
OF
FREEDOM 0.95 0.90 0.80 0.70 0.50 0.30 0.20 0.10 0.05 0.01 0.001
CHI SQUARE VALUES

1 0.004 0.02 0.06 0.125 0.46 1.07 1.64 2.71 3.84 6.44 10.83
2 0.10 0.21 0.45 0.71 1.39 2.41 3.22 4.60 5.99 9.21 13.82
3 0.35 0.58 1.012 1.42 2.37 3.66 4.64 6.25 7.82 11.34 16.27
4 0.71 1.06 1.65 2.20 3.36 4.88 5.99 7.78 9.49 13.28 18.47
5 1.14 1.61 2.34 3.00 4.35 6.06 7.29 9.24 11.07 15.09 20.52

Remember you are testing the null hypothesis. The probability values in the table relate to the
null hypothesis; the higher the probability value the greater is our confidence in the null hypothesis.
c) Chi-square limitations
The chi-square test as used for analysing the results of genetic experiments has two important
limitations:
(1) It must only be used on numerical data itself, NEVER on percentages or proportions
derived from the data.
(2) It cannot be used where the expected frequency in any phenotypic class is less than 5.

4. Procedure for handling genetic data: Worked examples

Example 1
A purebreeding strain of black guinea pigs was crossed to a pure breeding white strain. For
reciprocal crosses males and females in the F1 were all black. The F1 progeny are intercrossed
and in the F2 80 black and 20 white guinea pigs were produced.
Procedure
The data suggest an approximate 3:1 ratio in the F2. The simplest genetic hypothesis to explain
this would be:
1. Autosomal inheritance
2. One locus
3. Two alleles (one for black phenotype and one for white)
4. Black colour is dominant to white
5. The cross showing genotypes and phenotypes.
6. Let w be the white allele, w+ be the black allele (w = white, w+ = black).

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P1- 18 Practical 1: DNA, a Gene, an Auxotroph BIOL10005: 2018
Diagram of crosses:

Then,
P w+w+ x ww
(black) (white)
F1 w+w
(black)

F2
Genotypic ratio: 1 w+w+ : 2 w+w : 1 ww

Phenotypic ratio: 3 Black : 1 White

6. Prediction: Expect 3 black : 1 white in phenotypes of F2.

Null hypothesis: There is no difference between the observed results and those expected other
than that due to chance.
Statistical test

c =
2
å (O-E)2
E

df = n (number of classes) - 1

black white

Observed (O) 80 20

Expected (E) 75 25

(O-E)2 25/75 = 0.33 25/25 = 1.00

E

c =
2
å (O-E)2
E
= 0.33 + 1.00 = 1.33 ; p>0.20

df = 1

2 classes therefore, 1 degree of freedom

Conclusion: The probability that the difference between observed and expected numbers is due
to sampling error (chance) alone is greater than 20% but less than 30% which can be expressed as
0.20<p<0.30. Therefore, we accept the null hypothesis and the underlying genetic hypothesis that
gave rise to the prediction (because the observed results are consistent with the hypothesis).
Note: The simplest genetic hypothesis that fits the data should be the one tested first. Only if the
simplest hypothesis is rejected should more complex hypotheses be considered.

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