Masterclass 26 June 2008 - University of Nottingham · 2016. 11. 19. · Her work is mostly lab based and she is responsible ... Nitrile gloves should be worn for all practical manipulations.

Masterclass

26 June 2008

Genetics and Immunology:

Life in a Research Lab

2

General Introduction

Welcome to the Masterclass – Genetics and immunology: Life in a

Research Lab. This information pack contains important information about how to keep safe in the laboratory. It should also give you

some ideas about what you are going to listen to, and what you are

going to participate in, during the course of the afternoon.

Timetable

Table of Contents

PEOPLE INVOLVED 3

GENERAL HEALTH AND SAFETY INFORMATION 3

A REFRESHING LOOK AT DNA 9

ENGINEERING DNA IN BACTERIA 11

PCR AND ITS APPLICATIONS 14

AN ILLUSTRATION OF ELISAS – LIGHTING UP PROTEINS 20

FURTHER INFORMATION 23

Time Activity Location

11-11.15 Arrival/Housekeeping/Agenda/coffee Foyer

11.15-11.35 Mini lecture by Dr. Sara Goodacre B3

11.45-12.35 Lab-based practical 1 Biology A1 lab

12.35-1.15 Lunch Foyer

1.20-1.40 Mini lecture by Dr. Aziz Aboobaker B3

1.45-2.35 Lab-based practical 2 Biology A1 lab

2.40-3pm Info on courses & questions Foyer

3

People Involved

Dr Aziz Aboobaker studied for a BA in Natural Sciences at the

University of Cambridge before going on to complete a PhD. He has also worked as a scientist in Edinburgh, Berkeley (California), and

Nottingham. He is an evolutionary developmental biologist studying

the genetic basis for the diversity of animal body plans, given that so

many of the genes that control what animals look like seem to be the same. He uses flat worms in his research to look at the role of micro

RNAs during the evolution of developmental processes.

Dr Sara Goodacre earned her BA in Natural Sciences from the

University of Cambridge, and completed her PhD at the University of

Nottingham. She worked as a research fellow at the University of

Oxford before returning to Nottingham as an RCUK research fellow.

She is an evolutionary biologist working on spiders. Ongoing projects

include investigating spatial/temporal shifts in population structure in

ballooning money spiders, mating strategies in subsocial desert

spiders and conservation genetics of endangered fen raft spiders.

Dr Jane Grove is a research fellow in the Department of Genetics. Her current research is on DNA repair and recombination in E. coli.

Her previous experience includes projects on anaerobic bacterial

growth, genetic susceptibility to alcoholic liver disease, and human

cancer treatment by gene therapy using virus magic bullet.

Dr Elizabeth Lunt is a research fellow in the School of Biology. She

works in the Cell Biophysics Group developing new techniques for studying proteins, particularly their interactions with one another.

She did a degree in Chemistry and a PhD in Biological Chemistry at

Manchester, worked briefly at Aston University, and then came to

Nottingham. She works in a group that includes biologists, chemists

and engineers. Her work is mostly lab based and she is responsible

for looking after the labs, lab safety and training research students.

Dr Jess Tyson is a senior research fellow in the Institute of

Genetics. She works in the field of Human Genetics, in particular

Cancer Genetics. Her work looks at how variation in our DNA may affect our risk of developing cancer. To prepare for this, she

completed A levels in Biology, Chemistry, Maths and General Studies,

did a degree in Biological and Biochemical Sciences and completed a

PhD on the Genetics of Deafness.

Amanda Hampson and Rachal Allison are postgraduate students (PhD and MRes, respectively). Both are working in the same lab

studying the genetic basis of disease. Ask them for more details

when you see them today!

4

General Health and Safety Information

Before coming to the laboratory

• Read the relevant pages for the experiments you will perform. • Ensure you know the potential hazards.

• If you are unsure of any procedures, make a note of any questions

you want to ask.

Master Class

• Lab coats and safety specs will be provided.

• Wait outside the lab until you are asked to enter.

• Place your bags and coats (including mobile phones and MP3

players) in the cloakroom.

• Ensure lab coats are fastened, long hair is tied back and safety

specs are worn at all times.

End of class • Ensure that you switch off any equipment that needs switching off.

• Make sure that any waste chemicals have been disposed of

correctly.

• Rinse out all glassware and remove any labels before placing on the trolleys provided.

Laboratory Safety

The laboratories are equipped and run so that with appropriate care,

you can work without risking the health and safety of yourself and

others. Accidents are unexpected or undesirable events, but they

are avoidable with due care and attention.

You have a duty of care to work in a way that will not harm

the health and safety of yourself and/or others.

Current legislation requires that chemical, biological and other

hazards etc are assessed and documented. All containers should be

clearly labelled with their contents and any hazards. Also, all

experimental procedures are assessed for risks, any necessary safety

measures put in place, and documentary evidence kept. Therefore,

all containers in the laboratory are labelled with their relevant

hazards and any equipment that may present a hazard or needs

instruction before use (or should only be operated by trained

members of staff) will carry clear notices.

Ensure that you read the schedule before and during the

practical class, listen to instructions from staff and watch

carefully any demonstrations of experimental procedures.

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REMEMBER: Any mishap with a chemical (or apparatus) MUST be

reported to a member of staff IMMEDIATELY so that they can deal with the problem and remove any hazards correctly.

Safety is an integral part of good laboratory practice

Even LOW HAZARD chemicals may be hazardous if misused.

HAZARDOUS chemicals require careful handling at all times because

of one or more of the following characteristics:

(a) flammability

(b) explosive nature

(c) toxic, hazardous, irritant, etc:

with effects on or through the skin.

with effects on or through the respiratory tract.

with effects on or through the eye.

with effects following ingestion.

(d) reactive with water.

(e) reactive with air. (f) detrimental effect on the environment – especially to

aquatic life.

Containers of hazardous chemicals carry a pictogram indicating the type of danger.

Indication of General Nature of Risk:

Toxic Highly Flammable Explosive Biological Hazard

Corrosive Harmful, Irritant Oxidising

The above symbols are black on an orange/yellow

background

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Ensure that you know how to deal with any spillage BEFORE you

embark on an experiment.

Risk is associated with glassware, electrical, mechanical and high/low

pressure apparatus. Be aware of potential risks before using any

equipment.

The wearing of safety spectacles in the laboratory is

mandatory, the wearing of buttoned laboratory coats is

obligatory and eating and drinking in the laboratory is strictly

forbidden.

Nitrile gloves should be worn for all practical manipulations.

Do not ignore the warning signs displayed in the School. They are

there for your protection. The British and European standard safety

signs are used:

Prohibitory Signs: e.g. “No Smoking”, are circular with a red border

and crossbar over a black symbol on a white background.

Warning Signs: e.g. “Caution, risk of ionising radiation”, are

triangular with a black border on a yellow background.

Laser light Risk of Ionising Radiation

Mandatory Signs: e.g. “Eye Protection”, are circular on a blue

background with symbols in white; used when there is an obligation

to wear safety equipment.

7

Emergency Signs: e.g. “Emergency Shower”, square or rectangular,

on a green background with symbols in white.

Fire doors must be kept closed at all times. Know where the fire-

fighting, first-aid equipment, eye-wash and emergency showers are

situated.

Ensure that you know the location of fire exits.

In the event of a FIRE ALARM or an EMERGENCY EVACUATION

of the laboratory, turn off all your equipment as you leave the

laboratory and reassemble in the far end of the School of Biology

car-park away from the main building.

Working Safely in the Masterclass

Before commencing any operation in the laboratory give due care

and attention to how the procedure can be carried out without risk to

yourself or fellow workers. If you are in doubt, consult a member

of staff before you start.

Risks from hazardous chemicals are minimised by handling them correctly.

Check that the container has the correct chemical you require.

Read the hazard information on the label before opening.

• Pour liquids carefully to avoid spillage and splashing.

• Containers should be opened with caution and away from the face.

• Take care not to ingest or breathe in vapours or powders.

• Some containers should only be opened and the chemical used in

a fume hood, especially for those likely to produce fumes or vapours.

• Handle any chemicals in the fume hood that the schedule advises

you to.

• Wear nitrile gloves when handling hazardous chemicals.

• Open wounds, e.g. cuts on hands should be protected by a wound

dressing / band aid.

• Turn back over-long cuffs on clothes and laboratory coats to

reduce the chances of knocking over equipment or contaminating

cuffs / sleeves.

• Do not put pens / fingers etc in mouths and do not eat or drink anything, including sweets or gum.

• Do not rub eyes / face with contaminated hands /nitrile gloves.

8

• Risks are minimised by working tidily, cleanly and avoiding

spillages. • All spillages must be wiped up immediately.

• Dispose of all products, by-products and surplus chemicals

correctly.

• IF IN DOUBT – ASK • Wash hands at the end of your laboratory session.

Spillages / accidents / FIRST AID

• Spillages / splashes on the skin should be rinsed off immediately

with plenty of cold running water, then washed with soap and

warm water.

• Any splashes in the eye(s) should be washed out immediately with

an eye wash bottle and / or with plenty of cold running water.

• All cuts and scrapes on hands should be rinsed with cold running

water.

• Any burns should be held under cold running water for at least 10

minutes.

Seek advice from a member of staff if you have an accident or

if you feel unwell. A trained first aider will give advice,

ensure any injuries are treated and that relevant

documentation is completed.

Safe use of equipment

Glassware

• Do not use any glassware that has chips or cracks – hand it to a

member of staff for disposal.

• Take care when rinsing out glassware for reuse or to send for

washing – if there are any chipped edges or cracks – hand to a

member of staff – do not rinse out.

• In the event of breakages, do not pick up the pieces of glass. Ask

a member of staff to sweep up the pieces with a brush and

dustpan.

Sharps

The glass Pasteur pipettes and microscope slides should be handled

with care and disposed of in the 1.5% Trigene containers provided.

Heat

Bunsen burners – take care not to burn yourself. Do not overheat

microscope slides as they may crack. Ensure that any item that has

just been heated is not put into contact with a potentially flammable

surface

9

A Refreshing Look at DNA

Dr. Jess Tyson

DNA is amazing. We have tonnes of the stuff. If you unravelled all

your chromosomes from all your cells and laid the DNA end to end,

the DNA would stretch from the Earth to the Moon about 6000 times.

As you will know, DNA is made up of nucleotides, and there are 3

billion nucleotides in every cell of your body. Essentially we are 99.9% the same at our DNA level (and interestingly 98% same as

chimpanzees). But we all have sequence differences. Any 2 unrelated

people chosen at random differ at about 1 in every 1200 bases or

“letters”. This natural variation in our DNA can be in regions of the DNA that appears not to affect our health or physical appearance, yet

other changes can play a role in disease.

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Single base changes are known to influence:

Appearance – eye colour, height, hair colour...

Behaviour – thrill seeking, alcoholism…

Disease susceptibility – cancer, diabetes, haemophilia…

Today you will be looking at the genetic code and take part in an

activity that illustrates the effect a DNA change can have, in this case

it’s on your eye colour.

You will each have a pack of refreshers, each colour represents a

different nucleotide. Using the colour code on the handout, open your

packet and write out your sequence. Compare to the person sitting

next to you, see how different at the DNA level you are!

Have a go at translating your DNA sequence into a protein. Do

changes in your DNA sequence always change the protein?

Recently it was discovered that a large amount of variation in

people’s eye colour is due to a combination of single DNA changes in

a gene on chromosome 15. Amazingly just 3 out of the 3 billion

nucleotides cause this difference in eye colour. Part of the sequence

of this gene (OCA2) is shown on your handouts so finally I’d like you

to spell out your eye colour using your refreshers.

These single base changes are not the only differences in our DNA as

you will find out!

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Engineering DNA in bacteria

Dr. Jane Grove

Investigating DNA Repair in living cells

DNA is vital for life.

DNA must be copied and passed to the next generation. DNA damage leads to ageing, disease and cell death.

Current research involves understanding how DNA is replicated in

cells, how proteins repair DNA and how accuracy is ensured.

Normal E.coli cells E. coli cells which can’t repair their

DNA very well after UV treatment which damages their DNA.

The Bacterial Chromosome

E.coli bacteria = a model organism

One circular chromosome

Replication starts at the origin and ends at

the terminus

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RdgC

Genetic Engineering

Genetic manipulation in E.coli is very useful for all

areas of biological research to find out what different

proteins do in cells.

Many organisms have had their genome (all their DNA) sequenced.

Genes can be expressed in E.coli to make lots of the protein which

can be analysed biochemically.

For example, pure protein can form tiny crystals which can be used

to work out their 3D structure by X-ray crystallography.

The amino acid sequences of related proteins can be compared (each

amino acid is represented as a letter). The precise location of

conserved amino acids is shown on 3-D structures which gives an idea about their function in the protein

e.g. B. they can interact holding the protein in a ring

E. they can form a positively charged patch on the surface to bind DNA

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Tools for genetic engineering:

� PCR = polymerase chain reaction: (amplifies a specific sequence)

� Electrophoresis – separates DNA pieces

� Restriction Enzymes: (cut specific DNA sequence eg GAATTC)

� Ligating enzyme: sticks DNA ends together

‘Cloning’

Cloning a gene = make many copies of it so a lot of protein is

produced

� Clone gene into plasmids (mini artificial chromosomes)

which replicate to give many copies

� Cells containing the plasmid also multiply

to produce lots of the protein

� Extract and purify the protein to test its properties

Electrophoresis – separates DNA pieces according to size

� Useful to get the DNA piece you want to clone from other bits of

DNA

Negatively charged DNA is pulled towards the cathode (+)

Load 5ml of sample into each well of the gel

(1ml contains 1000ml)

A ‘Gel’ stained to show the DNA pieces Electrophoresis equipment

-

+

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PCR and its applications

Amanda Hampson and Rachal Allison

PCR = Polymerase Chain Reaction

PCR is a means of amplifying short segments of DNA (usually <2 kb).

It is based on the natural process by which DNA is replicated within

cells during mitosis.

It is used in many laboratory techniques, such as:

• Cloning

• Crime scene analysis

• Diagnostic detection of mutations

PCR is basically the manipulation of conditions so that a DNA

polymerase enzyme repeatedly replicates a specific sequence of DNA.

PCR reagents

There are five reagents essential for PCR:

Target DNA

This is the sequence that is to be amplified, which acts as a template

for the first round of replication. Very little DNA is required. For

example, if the purpose of the reaction is to determine if a patient

carries a specific mutation, sufficient DNA can be extracted from

buccal cells, which can be painlessly scraped from the inside of the

cheek. Alternatively, DNA can be taken from a small blood sample.

Taq polymerase

Taq is a heat stable DNA polymerase enzyme that was originally

isolated from Thermus aquaticus, a thermophilic bacterium that

naturally inhabits hot springs. Like all DNA polymerases, Taq polymerase:

• Requires a primer to initiate synthesis

• Synthesises DNA in a 5’ to 3’ direction

dNTPs

Deoxynucleotide triphosphates (dNTPs) are the substrates for Taq

polymerase, from which the new strands of DNA are synthesised. The

reaction should include equal amounts of dATP, dTTP, dGTP, and

dCTP.

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Primers

Each reaction includes 5’ and 3’ primers that together flank the target sequence and anneal to complementary sequences on

opposing DNA strands. Primers are usually synthetically produced

oligonucleotides that are about 20 bases in length.

Buffer

The buffer maintains the optimum pH and chemical environment for

the polymerase enzyme.

Thermocycling

Tubes containing the various reagents (mixed in water) are placed in

a thermocycling machine. This heats and cools the tubes in a cyclical manner, each cycle consisting of heating and cooling to three distinct

temperatures.

Denaturing temperature The reagents are heated to about 96°C for approximately 15

seconds. At this temperature, double stranded DNA is denatured.

Annealing temperature

The reagents are cooled to about 60°C for around 1 minute. Primers

anneal specifically to complementary sequence in the target DNA at

this temperature.

Extension temperature

The reagents are heated to about 72°C for around 1 minute. This is

the optimum working temperature for Taq polymerase.

Each cycle theoretically doubles the amount of DNA, although the reaction becomes less efficient when residual amounts of reagents

such as dNTPs eventually become limiting.

Typically 30 cycles are performed (roughly 2-3 hours), after which

about 105 copies of the target sequence are present. This is sufficient

DNA to be visualised on an agarose gel.

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Fig. 1 Mechanism of action of the polymerase chain reaction

P1 = primer 1 P2 = primer 2

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PCR activity 1 – Preparing 50µµµµl of PCR reaction mix

Combine the following reagents in a small Eppendorf tube:

Reagent Volume (µµµµl)

Water

Buffer

dNTPs Primer 1

Primer 2

DNA

Taq polymerase

30

5

5 2

2

5

1

The reaction mix is now ready for thermocycling.

When the PCR has finished, you can check the size of the amplified

fragment of DNA by running a small sample of the mix through an

agarose gel and visualising it under UV light (gel electrophoresis).

PCR activity 2 – Analysing PCR products

An example of the way PCR is used in our lab is the method by which

the Myd mouse is genotyped.

The Myd mouse has a mutation in a gene called Large – it has a

section of the gene missing from its DNA, which means the protein

the gene codes for is non-functional.

People who have mutations in the Large gene have muscular

dystrophy – it is therefore important to study the Myd mouse to find

out how the disease is caused in humans. The Myd mouse has a similar phenotype to the people who suffer from this kind of muscular

dystrophy.

The Myd mouse is genotyped via a duplex PCR method, using 2 pairs

of primers instead of the usual 1 pair:

• Primer pair 1 (P1 and P2) only amplify a product from the

normal gene sequence

• Primer pair 2 (P3 and P4) only amplify a product from the

Myd mutant gene sequence containing the deletion

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Fig. 2 Schematic representation of the Large gene. The region

containing the white boxes (4-6) is missing in the Myd mouse – this

region is ~100 kb.

How does this work?

P1 anneals within the missing region of DNA, therefore P1 and P2

only amplify a product when this region is present (a normal gene).

P3 and P4 are too far apart in a normal gene to amplify any product, and so only amplify a DNA fragment when the region is missing (a

Myd mutant gene).

Primer pair 1 product length = 162 bp

Primer pair 2 product length = 421 bp

DNA is taken from mice about 4-8 weeks old and used in a PCR

reaction including both pairs of primers – the size of the DNA

fragment produced tells us the genotype of the mouse. This is much

easier than sequencing the DNA.

P1P3

P2 P4

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The following diagram shows the results of this PCR reaction using

DNA from three different mice (A, B and C). The ladder to the left of the results indicates the size of the bands in bp. What is the

genotype of each of the mice?

Mouse A genotype ………………….

Mouse B genotype …………………..

Mouse C genotype …………………..

500

400

300

200

100

A B C

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An Illustration of ELISAs – lighting up proteins

Dr. Beth Lunt

Enzyme-linked Immunosorbent Assays (ELISAs) combine the

specificity of antibodies with the sensitivity of simple enzyme assays,

by using antibodies or antigens coupled to an easily-assayed enzyme

(the measurement of enzyme activity with a particular substrate).

ELISAs can provide a useful measurement of antigen concentration.

One of the most useful is the two-antibody "sandwich" ELISA. This

assay is used to determine the antigen concentration in unknown

samples. The sandwich ELISA requires two antibodies that bind to

different epitopes on the antigen (a unique shape or marker carried

on an antigen's surface which is recognisable by the antibody). This

can be accomplished using an antibody that recognises a single site

on the antigen and an antibody which will bind to several sites on the

antigen.

One antibody (the "capture" antibody) is purified and bound to a

well-plate. Antigen or sample containing the antigen in unknown amounts is then added and allowed to react with the bound antibody.

Unbound products are then removed by washing, and a labelled

second antibody (the "detection" antibody) is allowed to bind to the

antigen, thus completing the "sandwich". The assay is then quantified by measuring the amount of labelled second antibody attached to the

bound antigen, through the use of a substrate which interacts with

the label via an enzymatic interaction producing a complex which can

be used to detect the quantity of protein bound. Popular enzymes

are those which convert a colourless substrate to a coloured product.

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Plate preparation:

1. 100ul/well of capture antibody is added to each well of a 96-well plate

2. The plate is incubated overnight.

3. Excess liquid is removed

4. Each well is washed 3 times wash buffer. 5. 200ul blocking solution is added to each well for at least 1 hour.

6. Each well is washed 3 times wash buffer.

Assay procedure:

1. Each sample or standard to added to the appropriate wells.

2. Samples are mixed and incubate for 2 hours at room temperature.

3. Each well is washed 3 times wash buffer.

4. Labelled secondary antibody is added to each well

5. Incubate the plate for 2 hours at room temperature

6. Each well is washed 3 times wash buffer.

7. A further labelled substrate is added which interacts with the

labelled antibody.

8. The plate is incubated for 20 min at room temperature. 9. Each well is washed 3 times wash buffer.

10. The enzymatic regent is added.

11. The plate is incubated at room temperature for 20 min.

12. The reaction is stopped by addition of a reagent. 13. The plate is read using a specialised plate reader.

Using the BCA Assay to illustrate the ELISA principle:

The BCA assay is used to detect and measure the amount of protein

present in a sample. Here we are using it to illustrate the final step

of an ELISA – where a colour change is observed on the addition of a

reagent which is proportional to the amount of protein present. This

is equivalent to the detection step in the ELISA where the colour

change or signal produces is proportional to the amount of antigen

bound to the capture antibody.

Equipment (per group):

2 96-well plates.

9 small sample containers.

Pipettes and tips

Gloves and labcoats

Reagents (stock solutions):

Albumin standard solution

Phosphate buffered saline

BCA working reagent

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Preparation of albumin standards:

Dilute the albumin standard solution (stock) following the volumes in the table below. Remember to label the sample containers.

Sample Volume of

PBS (ul)

Volume of

Stock/Sample for

dilution (ul)

Final Albumin

concentration

(ug/ml)

A 0 300 Stock 2000

B 125 375 Stock 1500

C 325 325 Stock 1000

D 175 175 of sample B 750

E 325 325 of sample C 500

F 325 325 of sample E 250

G 325 325 of sample F 125

H 400 100 of sample G 25

I 400 0 0

Assay:

1) Pipette 25ul of each sample A-I into the well plate. Repeat so that

you have 2 rows one above the other where sample A is at the left-hand end.

2) Add 200 ul of BCA working reagent to each well containing

sample.

3) Shake or tap the plate gently to mix.

4) Write down the initial colour changes observed.

5) Cover and leave for 10 min and monitor any further colour

changes.

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Further Information

Thank you for coming to the Masterclass! We hope you enjoyed the

day!

For further information on anything you see today, please email

[email protected], or go to the project

website at www.nottingham.ac.uk/biology/sop.

For additional information about any of the departments involved in

the Science Outreach Project or today’s event, check out the

following webpages:

School of Biology: www.nottingham.ac.uk/biology

School of Pharmacy: www.nottingham.ac.uk/pharmacy

School of Chemistry: www.nottingham.ac.uk/chemistry

Faculty of Engineering: www.engineering.nottingham.ac.uk