Lab 4 Seminar

Genetics
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Outline of Seminar
1. Overview of Lab 4

2. Pre-lab: Molecular Genetics - DNA profiling

3. Pre-lab: Molecular Genetics - Gene regulation

4. Pre-lab: Lab 4 setup

 Lab 4: Learning Outcomes
• Determine the mode of inheritance of genetic traits in
humans and model organisms
• Diagrammatically represent and predict the outcome
of genetic crosses
• Explore the mechanism by which bacteria can
transcriptionally control gene expression, by using
reporter genes in the study of biological problems
• Develop experience in practical and computational
skills for the interpretation and analysis of genetic data
Assessment for Lab 4

Weighting (% of final
Lab 4: Genetics Assessment Type
mark)

Pre-lab activity
Pre-lab H5P None
(assessed in pre-lab quiz)
Pre-lab quiz Pre-lab Moodle Quiz 2.5%

Post-lab quiz (conducted during lab time) Post-lab assessment 5%

TOTAL 7.5%
Pre-lab activity – Inheritance VirtualLab
What you need to know about for this virtual lab
1. Mendelian inheritance (and its exceptions, namely codominance)
2. Reciprocal crosses (discussed in workshop)
3. Punnet squares (monohybrid)
4. Determining genotypic and phenotypic proportions

• I will not talk about the ‘Genetic inheritance’ component during the seminar
• The Virtual Lab should be self-explanatory
Questions at this point?
Molecular genetics
1. DNA profiling

2. Gene regulation

Image credit: gopixa/Shutterstock.com

 1. DNA profiling
• While all humans share ~99.9% of their DNA sequence
• The 0.01% is large and can be used to build a ‘unique profile’, like a fingerprint
• DNA profiling is used in:
• DNA forensics to identify suspects and victims
• Paternity testing
• Humans
• Livestock/ Pets
• Genealogy
• Makes use of Short Tandem Repeat (STR) loci
 Inheritance of STRs
• Follow the normal rules of mendelian allelic assortment STR locus 12

Think LAW OF SEGREGATION 5 repeats 11 repeats 15 repeats 15 repeats

5 11 15 15

5 5 11 11 15 15 15 15

Image credit:
Adapted from Elayne Fivenson
(Harvard University)
 Inheritance of STRs
• Follow the normal rules of mendelian allelic assortment STR locus 12

Think LAW OF SEGREGATION 5 repeats 11 repeats 15 repeats 15 repeats

S phase

5 11 15 15

5 5 11 11 15 15 15 15

Image credit:
Adapted from Elayne Fivenson
(Harvard University)
 Inheritance of STRs
• Follow the normal rules of mendelian allelic assortment STR locus 12

Think LAW OF SEGREGATION 5 repeats 11 repeats 15 repeats 15 repeats

S phase

5 11 15 15
Meiosis

5 5 11 11 15 15 15 15

Image credit:
Adapted from Elayne Fivenson
(Harvard University)
 Inheritance of STRs
• Follow the normal rules of mendelian allelic assortment STR locus 12

5 repeats 11 repeats 15 repeats 15 repeats

S phase

5 11 15 15
Meiosis

5 5 11 11 15 15 15 15
Fertilisation
What is the genotype of the
child’s STR12 loci?
Image credit:
Adapted from Elayne Fivenson 11 repeats /15 repeats
(Harvard University)
 DNA profiling case study
• A terrible accident at the hospital maternity ward! ? ? ?
? ?
• You need to determine the true parentage of child 1 and child 2 ?
• Between Mother 1 and Mother 2
M1 M2
• You are part of the Hospital’s pathology laboratory

• You will use three separate STR loci to determine a DNA profile for
each individual C1 C2
The 3 STR loci – Each with 3 variants
The 3 STR loci – Each with 3 variants
The 3 STR loci – Each with 3 variants
 Lab 4 – DNA profiling
During the lab you will be required to:
1. Set up a multiplex PCR reaction within your group for the 3 STR Markers
■ M1, M2, C1, C2
2. Once cycled, run the PCR reaction on a gel to analyse products
3. Deduce the likely inheritance of STR markers among M1, M2, C1, C2
4. At the end of the lab, undertake a short quiz testing your understanding of STR
marker inheritance
 NOT ASSESSED IN
PRE-LAB QUIZ

What is the Polymerase Chain Reaction (PCR)?

• A genetic technique that amplifies a specific section of DNA in a
sample
• Using PCR a single copy of DNA can be amplified to over a billion copies

Image credit:
Wikimedia
Commons
Agarose gel electrophoresis
How do we analyse gel electrophoresis?

DNA Standard Markers

How do we analyse our results?

What is the STR profile of

person Z1?

1A/1B

2B/2B

3A/3C
Questions at this point?
 2. Gene regulation
Background
Not all an organism’s genes will be active at the same time

Cells have the ability to regulate when certain genes are expressed and when they are not

This can be determined by several factors including:

1. Spatial expression patterns
2. Presence / absence of key catabolites
3. Time
 NOT ASSESSED IN
Prokaryotic gene structure PRE-LAB QUIZ
General transcription factors RNA polymerase
Regulatory transcription RNA Transcript
factors
5’ Operator PROM. … Gene 1 Gene 2 Gene 3 … TERM. 3’

• No introns
• Bacteria tend to arrange related genes close to one another – operons
• Makes the transcription process more efficient
 NOT ASSESSED IN
The Arabinose promoter PRE-LAB QUIZ
 NOT ASSESSED IN
The Arabinose promoter PRE-LAB QUIZ
 The practical scenario
Many therapeutics are produced recombinantly
For example: human insulin

Credit:https://ib.bioninja.com.au/standard-level/topic-2-molecular-biology/
27-dna-replication-transcri/universality.html
Expression of insulin must be tightly controlled

Expression not regulated

Expression of insulin must be tightly controlled
Expression regulated

Expression not regulated

Induced

Why do you think

there’s a difference?
Expression of insulin must be tightly controlled

Production of recombinant genes are a stress on the bacterial cell

Typically slows bacterial growth

If you induce production of insulin when cell density is higher:

1. The stress on each cell remains the same BUT

2. There are now more cells available to make the protein
The practical scenario – your task

Imagine you are a biomedical engineer responsible for making a

recombinant therapeutic

You have to test a number of different operator/promoter regions to

work out which one is of the most commercial value

Things to consider:
1. Total output
2. Stringency of regulation
 The practical scenario - your task
You need an easy way to determine the amount of therapeutic
generated.

So you replace the therapeutic gene with another gene that can be
easily quantified – called a reporter gene

Op & Prom Therapeutic gene Terminator

AraPα
Op & Prom gfp Terminator
Reporter
construct
Strength of promoter determined by the amount of GFP produced
AraPβ mutants
Portions of the regulatory machinery can be mutated

Which one has the most activity?

Op & Prom gfp Terminator

Op gfp Terminator

Prom gfp Terminator

??? gfp Terminator

AraPβ 1-4
What are you testing in the lab?
• You will be given four E. coli strains containing
different mutants of the AraPα reporter
construct (called AraPβ 1-4)

• You must test their relative expression of GFP

in comparison to the wildtype (AraPα) and a
negative control (No GFP)

• How might we determine why one mutant

functions differently to the wildtype?
 in silico analysis
Why does this operator work better/ worse than others???

We can use sequencing and computational analysis of the nucleotide sequence to help
figure out why

You will be provided with the operator sequences of each construct

You need to compare the sequence of each construct to the wildtype.

Take note of the differences between them! AND

Where the nucleotide differences occur?

 in silico analysis

Promoter (-10)

Transcription

AraC 5’….CAGAAAAGTCCACATTGAT…3’ AraC 5’….TTGCTATGCCATAGCATT….3’

Binding Site 1 Binding Site 2 3’….AACGATACGGTATCGTAA….5’
3’….GTCTTTTCAGGTGTAACTA…5’
 Where to from here?
You must complete the following tasks:

1.Revise Weeks 7 & 8 Discover content – these will be assessed in post lab quiz
2.Complete the prelab activity (Inheritance VirtualLab)
3.Complete the prelab quiz (refer to Moodle for deadlines)
4.Attend the lab
5.Complete the post-lab quiz (final 30 mins of lab time).

Remember that Week 7 & 8 theory content is assessable

Final Questions?