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1:​ ​INTRODUCTION

2

2:​ ​ECOLOGY​ ​AND​ ​BIODIVERSITY​ ​OF​ ​CORNWALL

2

3:​ ​METHODS​ ​FOR​ ​IDENTIFYING​ ​SPECIES​ ​IN​ ​THE​ ​FIELD

4

4:​ ​SURVEYING​ ​BIODIVERSITY​ ​IN​ ​THE​ ​FIELD

5

LIZARD​ ​PENINSULA:​ ​HEATHLAND​ ​AND​ ​GRASSLAND

7

GYLLYNGVASE​ ​AND​ ​SWANPOOL:​ ​ROCKPOOLS​ ​AND​ ​BIRDS

7

KENNALL​ ​VALE:​ ​WOODLAND​ ​PLANTS,​ ​FERNS,​ ​AND​ ​FUNGI

8

5:​ ​DNA​ ​DETECTIVE

8

6:​ ​AMPLIFYING​ ​THE​ ​EVIDENCE

11

7:​ ​SEQUENCING

12

8:​ ​REPRODUCTIVE​ ​SUCCESS​ ​IN​ ​COOPERATIVELY​ ​BREEDING​ ​MONGOOSE

14

9:​ ​LABORATORY​ ​TECHNIQUES​ ​FOR​ ​DEALING​ ​WITH​ ​PROTEINS

15

10:​ ​ELISA

16

11:​ ​BLOTTING

18

12:​ ​MICROSCOPY

20

Joanna​ ​Griffith​ ​(2017)

1:​ ​INTRODUCTION
--------------------------------------------------------------------------------------------------------------------------2:​ ​ECOLOGY​ ​AND​ ​BIODIVERSITY​ ​OF​ ​CORNWALL
Physical​ ​environment
● Climate​ ​(in​ ​comparison​ ​to​ ​the​ ​rest​ ​of​ ​the​ ​UK)
○ High​ ​mean​ ​annual​ ​temperature
○ Fewer​ ​days​ ​of​ ​ground​ ​frost
○ More​ ​hours​ ​of​ ​sunshine
○ Higher​ ​annual​ ​rainfall
○ Frost-free​ ​season​ ​is​ ​becoming​ ​longer​ ​(currently​ ​about​ ​260​ ​days)
■ Some​ ​species​ ​favour​ ​longer​ ​frost-free​ ​seasons,​ ​eg
...
​ ​marsh​ ​fritillary,​ ​three-lobed
water-crowfoot
○ Some​ ​species​ ​favour​ ​a​ ​combination​ ​of​ ​warm​ ​(summer)​ ​and​ ​wet​ ​(winter),​ ​eg
...
​ ​four​ ​species​ ​of​ ​heather​ ​in​ ​the​ ​same​ ​area
○ Cross-leaved​ ​heath
■ Whorls​ ​of​ ​4​ ​leaves
■ Leaves​ ​2-5mm​ ​with​ ​a​ ​greyish​ ​tinge
■ Prefers​ ​wet​ ​and​ ​acidic​ ​conditions
○ Bell​ ​heather
■ Whorls​ ​of​ ​3​ ​leaves
■ Leaves​ ​5-7mm,​ ​dark​ ​green
Joanna​ ​Griffith​ ​(2017)

○

○

■ Prefers​ ​dry​ ​and​ ​acidic​ ​conditions
Cornish​ ​heath
■ Whorls​ ​of​ ​4-5​ ​leaves
■ Leaves​ ​7-10mm,​ ​green
■ Prefers​ ​dry​ ​and​ ​basic​ ​conditions
Ling
■ Adpressed​ ​leaves,​ ​1-2mm,​ ​green​ ​or​ ​slightly
purple/brown
■ Prefers​ ​wet​ ​and​ ​acidic​ ​conditions

Habitats​ ​in​ ​Cornwall
● Heathland​ ​on​ ​serpentine​ ​(eg
...
​ ​Goss​ ​Moor)
● Upland​ ​moor​ ​(eg
...
​ ​around​ ​Isles​ ​of​ ​Scilly)
Cornish​ ​wildlife​ ​(some​ ​examples)
● Land​ ​mammals
○ Badger
○ Common​ ​dormouse
○ Lesser​ ​white-toothed​ ​shrew
○
● Marine​ ​mammals
○ Bottlenose​ ​dolphin
● Birds
○ Peregrine
○ Cirl​ ​bunting
○ Chough
○ Balearic​ ​shearwater
● Reptiles
○ Leatherback​ ​turtle
○ Palmate​ ​newt
○ Adder
● Invertebrates
○ Silver-studded​ ​blue​ ​butterfly
○ Large​ ​blue​ ​butterfly
○ Sandhill​ ​rustic
○ Monarch​ ​butterfly
○ Green​ ​darner
○ Prickly​ ​stick​ ​insect

Joanna​ ​Griffith​ ​(2017)

○ Pink​ ​sea​ ​fan
○ Sunset​ ​cup​ ​coral
○ Trembling​ ​sea-mat
● Fish
○ Cornish​ ​sucker
● Plants
○ Cornish​ ​heath
○ Fringed​ ​rupturewort
○ Dwarf​ ​rush
○ Pygmy​ ​rush
○ Twin-headed​ ​clover
○ Long-headed​ ​clover
○ Land​ ​quillwort
○ Strawberry​ ​stonewort
--------------------------------------------------------------------------------------------------------------------------3:​ ​METHODS​ ​FOR​ ​IDENTIFYING​ ​SPECIES​ ​IN​ ​THE​ ​FIELD
Equipment
● Field​ ​guides,​ ​keys
● Field​ ​notebook
● Binoculars
● Hand​ ​lens
● Camera
Dichotomous​ ​keys
● Found​ ​in​ ​ID​ ​books
● Advantages
○ Best​ ​way​ ​of​ ​being​ ​absolutely​ ​sure
○ Only​ ​reliable​ ​way​ ​of​ ​identifying​ ​plants​ ​in​ ​their​ ​vegetative​ ​state​ ​(no​ ​flowers)
○ Correct​ ​scientific​ ​way
● Disadvantages
○ Time​ ​consuming
○ Requires​ ​good​ ​knowledge​ ​of​ ​technical​ ​language
○ Making​ ​one​ ​mistake​ ​can​ ​prevent​ ​you​ ​from​ ​finding​ ​the​ ​correct​ ​organism
Flicking​ ​through​ ​the​ ​book
● Find​ ​picture​ ​of​ ​the​ ​species​ ​in​ ​the​ ​book,​ ​check​ ​range​ ​and​ ​habitat,​ ​read​ ​ID​ ​clues,
measure​ ​if​ ​necessary
● Advantages
○ Quickest​ ​way​ ​once​ ​you​ ​have​ ​some​ ​knowledge​ ​of​ ​families
○ Requires​ ​less​ ​knowledge​ ​of​ ​technical​ ​language
● Disadvantages
○ Can’t​ ​really​ ​be​ ​used​ ​for​ ​plants​ ​in​ ​their​ ​vegetative​ ​state
○ Can​ ​be​ ​very​ ​time-consuming​ ​without​ ​prior​ ​knowledge
○ Easy​ ​to​ ​make​ ​beginner​ ​mistakes​ ​between​ ​similar​ ​species
Online​ ​resources
● Eg
...
​ ​kestrels​ ​hover,​ ​sparrowhawks​ ​flap-flap-glide)
○ Beak​ ​shape
○ Colouration
○ Feeding​ ​style
○ Size
○ Distribution​ ​at​ ​different​ ​times​ ​of​ ​the​ ​year
○ Habitat
○ Behaviour​ ​and​ ​song
--------------------------------------------------------------------------------------------------------------------------4:​ ​SURVEYING​ ​BIODIVERSITY​ ​IN​ ​THE​ ​FIELD
Why​ ​sample?
● To​ ​find​ ​out​ ​if​ ​organisms​ ​are​ ​different​ ​from​ ​or​ ​associated​ ​with​ ​each​ ​other
○ difference/correlation
● Sampling​ ​all​ ​members​ ​of​ ​a​ ​population​ ​is​ ​impractical,​ ​but​ ​sampling​ ​one​ ​or​ ​a​ ​few
members​ ​is​ ​not​ ​representative​ ​of​ ​a​ ​population
How​ ​to​ ​sample
● How​ ​many​ ​samples?
○ Confidence​ ​in​ ​an​ ​estimate​ ​of​ ​the​ ​population​ ​mean​ ​increases​ ​with​ ​sample​ ​size

Joanna​ ​Griffith​ ​(2017)

Therefore,​ ​confidence​ ​in​ ​difference/correlation​ ​increases​ ​with​ ​sample
size
○ “Power​ ​analyses”​ ​can​ ​be​ ​used​ ​to​ ​determine​ ​required​ ​sample​ ​size
● Sampling​ ​design
○ Random
■ Reduces​ ​bias
■ Not​ ​always​ ​practical​ ​(eg
...
​ ​only​ ​random​ ​sampling​ ​in​ ​an​ ​area​ ​where​ ​there​ ​are​ ​lots​ ​of
the​ ​organism​ ​being​ ​studied
■ Pseudoreplication
● Eg
...
​ ​decrease​ ​in​ ​pirate​ ​numbers​ ​is​ ​correlated​ ​to​ ​increase​ ​in​ ​global
temperatures
■ In​ ​reality,​ ​both​ ​are​ ​changing​ ​separately​ ​over​ ​time
● Spatial​ ​autocorrelation
● Correlating​ ​potential​ ​explanatory​ ​variables
--------------------------------------------------------------------------------------------------------------------------■

Joanna​ ​Griffith​ ​(2017)

LIZARD​ ​PENINSULA:​ ​HEATHLAND​ ​AND​ ​GRASSLAND
Lizard​ ​peninsula​ ​is​ ​the​ ​southernmost​ ​part​ ​of​ ​the​ ​UK​ ​mainland
Unique​ ​geology​ ​+​ ​mild​ ​winter​ ​temperatures​ ​=​ ​unique​ ​environment​ ​for​ ​many​ ​plant
species
● Majority​ ​of​ ​the​ ​peninsula​ ​is​ ​designated​ ​under​ ​the​ ​EU​ ​Habitats​ ​Directive​ ​as​ ​a​ ​Special
Area​ ​of​ ​Conservation​ ​(one​ ​of​ ​the​ ​highest​ ​levels​ ​of​ ​protection​ ​possible)
○ Several​ ​types​ ​of​ ​heathland,​ ​including​ ​those​ ​dominated​ ​by​ ​Cornish​ ​heath
(Erica​ ​vagans)
○ Mediterranean​ ​temporary​ ​ponds
○ Numerous​ ​unusual​ ​plant​ ​species,​ ​such​ ​as​ ​land​ ​quillwort​ ​(Isoetes​ ​histrix)
● Data​ ​collection
○ Interested​ ​in​ ​why​ ​species​ ​occur​ ​where​ ​they​ ​do
■ Helps​ ​us​ ​to​ ​understand​ ​the​ ​physical​ ​and​ ​habitat​ ​requirements​ ​of
species
● Conservation
○ How​ ​can​ ​i​ ​collect​ ​data​ ​in​ ​an​ ​unbiased​ ​way?
○ How​ ​do​ ​I​ ​collect​ ​my​ ​data​ ​efficiently​ ​in​ ​such​ ​a​ ​way​ ​that​ ​I​ ​don’t​ ​spend​ ​ages
collecting​ ​data​ ​that​ ​could​ ​easily​ ​be​ ​collected​ ​quickly​ ​with​ ​a​ ​better​ ​survey
design?
○ Micro-habitat​ ​requirements​ ​of​ ​a​ ​plant​ ​species:
■ Select​ ​a​ ​plant​ ​species
● Should​ ​be​ ​relatively​ ​easy​ ​to​ ​identify​ ​but​ ​not​ ​too​ ​common​ ​or
rare
■ Lay​ ​out​ ​a​ ​0
...
25m​ ​quadrat​ ​and​ ​record​ ​whether​ ​the​ ​plant​ ​is​ ​absent
or​ ​present​ ​within​ ​the​ ​quadrat
■ Record​ ​vegetation​ ​height​ ​(mean​ ​of​ ​five​ ​measurements)
■ Approach​ ​1:
● Place​ ​50​ ​quadrats​ ​completely​ ​randomly
○ Good​ ​to​ ​ensure​ ​that​ ​data​ ​collection​ ​method​ ​is​ ​unbiased
■ Approach​ ​2:
● Walk​ ​along​ ​in​ ​a​ ​fairly​ ​random​ ​way​ ​until​ ​you​ ​encounter​ ​your
target​ ​plant​ ​species​ ​(25​ ​times)
● Place​ ​the​ ​quadrat​ ​in​ ​25​ ​random​ ​locations​ ​where​ ​the​ ​plant
species​ ​is​ ​not​ ​present
--------------------------------------------------------------------------------------------------------------------------●
●

GYLLYNGVASE​ ​AND​ ​SWANPOOL:​ ​ROCKPOOLS​ ​AND​ ​BIRDS
●

Swanpool​ ​is​ ​a​ ​small​ ​body​ ​of​ ​open​ ​water​ ​fringed​ ​by​ ​reeds
○ On​ ​the​ ​outskirts​ ​of​ ​Falmouth
○ Designated​ ​as​ ​an​ ​SSSI
○ Only​ ​site​ ​in​ ​the​ ​UK​ ​where​ ​the​ ​trembling​ ​sea​ ​mat​ ​can​ ​be​ ​found
○ Fringing​ ​vegetation​ ​and​ ​swampy​ ​valley​ ​at​ ​the​ ​top​ ​of​ ​Swanpool​ ​(Swanvale
Nature​ ​Reserve)​ ​is​ ​a​ ​haven​ ​for​ ​insectivorous​ ​passerines​ ​in​ ​winter​ ​and​ ​spring,
and​ ​species​ ​such​ ​as​ ​water​ ​rail​ ​and​ ​bittern​ ​can​ ​be​ ​found
Joanna​ ​Griffith​ ​(2017)

Data​ ​collection
○ How​ ​sampling​ ​effort​ ​can​ ​affect​ ​the​ ​data​ ​that​ ​can​ ​be​ ​collected
○ Does​ ​the​ ​size​ ​of​ ​a​ ​rockpool​ ​influence​ ​the​ ​number​ ​of​ ​species​ ​found​ ​in​ ​it?
■ Record​ ​the​ ​number​ ​of​ ​species​ ​in​ ​rock​ ​pools​ ​of​ ​different​ ​sizes
● Select​ ​a​ ​rock​ ​pool
● Measure​ ​the​ ​length​ ​and​ ​breadth​ ​of​ ​the​ ​rockpool​ ​for​ ​a​ ​rough
estimate​ ​of​ ​surface​ ​area
● Record​ ​different​ ​species​ ​seen
● At​ ​5​ ​minute​ ​intervals,​ ​total​ ​the​ ​cumulative​ ​number​ ​of​ ​species​ ​in
each​ ​rockpool
○ 20​ ​minutes​ ​in​ ​each​ ​pool
--------------------------------------------------------------------------------------------------------------------------●

KENNALL​ ​VALE:​ ​WOODLAND​ ​PLANTS,​ ​FERNS,​ ​AND​ ​FUNGI
Woodland​ ​with​ ​rich​ ​undergrowth​ ​and​ ​open​ ​glades
○ Owned​ ​by​ ​the​ ​Cornwall​ ​Wildlife​ ​Trust
○ Contains​ ​a​ ​water-filled​ ​quarry​ ​and​ ​abandoned​ ​gunpowder​ ​mills
○ Ideal​ ​conditions​ ​for​ ​small​ ​and​ ​rare​ ​Tunbridge​ ​filmy​ ​fern​ ​(Hymenophyllum
tunbrigense)
○ Woodland​ ​hosts​ ​other​ ​species​ ​of​ ​fern,​ ​dippers,​ ​grey​ ​wagtails,​ ​and​ ​sometimes
otters
● Data​ ​collection
○ How​ ​much​ ​data​ ​is​ ​needed?
○ Typical​ ​length​ ​of​ ​fern​ ​fronds​ ​of​ ​a​ ​given​ ​species
■ Select​ ​a​ ​fern​ ​of​ ​a​ ​given​ ​species
■ Select​ ​one​ ​frond​ ​at​ ​random​ ​and​ ​measure​ ​its​ ​length
■ Repeat​ ​and​ ​calculate​ ​the​ ​average​ ​length​ ​of​ ​fronds
■ Means​ ​should​ ​be​ ​highly​ ​variable​ ​at​ ​the​ ​beginning​ ​and​ ​then​ ​settle​ ​down
after​ ​5/10​ ​measurements,​ ​depending​ ​on​ ​the​ ​fern
--------------------------------------------------------------------------------------------------------------------------●

5:​ ​DNA​ ​DETECTIVE
●

●

●

Field-based​ ​observations
○ Phenotypic​ ​data
○ Behaviours
○ Numbers
○ Taxonomy
Field-based​ ​observations​ ​AND​ ​laboratory
○ Phenotypic​ ​data​ ​+​ ​genotypic​ ​data
○ Behaviours​ ​+​ ​hormones
○ Numbers​ ​+​ ​relatedness
○ Taxonomy​ ​+​ ​genetic​ ​relatedness
Need​ ​to​ ​be​ ​able​ ​to:
○ Read​ ​research​ ​papers​ ​and​ ​have​ ​some​ ​understanding​ ​of​ ​methods
○ Interpret​ ​research​ ​and​ ​its​ ​limitations
Joanna​ ​Griffith​ ​(2017)

○ Interpret​ ​news​ ​articles
● Lab-based​ ​techniques​ ​can​ ​be​ ​roughly​ ​divided​ ​into​ ​3​ ​main​ ​groups:
○ DNA​ ​techniques
■ DNA​ ​extraction
■ PCR
■ Electrophoresis
■ Real-time​ ​PCR
■ Southern​ ​blotting
■ Chromatography
■ Spectrophotometry
■ Sequencing
■ Viral​ ​transduction
■ Northern​ ​blotting
■ RNAi
■ DNA​ ​microarrays
■ Pyrosequencing
○ Protein​ ​techniques
■ Western​ ​blotting
■ Eastern​ ​blotting
■ ELISA​ ​-​ ​sandwich
■ ELISA​ ​-​ ​indirect
■ ELISA​ ​-​ ​competitive
■ Monoclonal​ ​antibody
■ Polyclonal​ ​antibody
■ Latex​ ​agglutination
■ Protein​ ​microarray
■ Immunohistochemistry
■ Chromatography
■ Spectrophotometry
○ None​ ​or​ ​both​ ​(DNA​ ​+​ ​proteins)
■ Microscopy
DNA​ ​detective​ ​case​ ​study
● Background
○ Illegal​ ​trade​ ​in​ ​wild​ ​animals​ ​and​ ​plants​ ​is​ ​worth​ ​$20​ ​billion​ ​per​ ​year​ ​(Interpol,
2008)
○ In​ ​Brazil,​ ​an​ ​estimated​ ​40%​ ​of​ ​drug​ ​shipments​ ​are​ ​combined​ ​with​ ​wildlife
○ As​ ​a​ ​species​ ​becomes​ ​rarer,​ ​its​ ​value​ ​increases,​ ​increasing​ ​the​ ​incentives
○ Overexploitation​ ​of​ ​wild​ ​populations​ ​can​ ​lead​ ​to​ ​local​ ​extinctions
○ Life-history​ ​characteristics​ ​such​ ​as​ ​longevity,​ ​high​ ​natural​ ​juvenile​ ​mortality,
and​ ​low​ ​reproductive​ ​output,​ ​increase​ ​vulnerability​ ​to​ ​extinction
○ Keystone​ ​species​ ​are​ ​often​ ​affected
■ Eg
...
5M
ethylenediaminetetraacetic​ ​acid​ ​over​ ​a​ ​4-day​ ​period
■ Calcium​ ​dissolves
■ This​ ​step​ ​is​ ​only​ ​necessary​ ​for​ ​bone/ivory/etc
...
​ ​TRIS-HCL)
○ Detergent​ ​(eg
...
​ ​the​ ​Human​ ​Genome​ ​Project​ ​took​ ​13​ ​years​ ​and​ ​cost​ ​about
USD$1​ ​billion
Next-generation​ ​sequencing
●

Joanna​ ​Griffith​ ​(2017)

Developed​ ​in​ ​the​ ​last​ ​decade
Reduced​ ​time​ ​and​ ​cost​ ​of​ ​sequencing​ ​significantly
Parallel​ ​sequencing:
○ DNA​ ​is​ ​fragmented
○ Fragments​ ​are​ ​bound​ ​to​ ​a​ ​bead
○ The​ ​bead​ ​has​ ​a​ ​slight​ ​ionic​ ​charge,​ ​so​ ​pulls​ ​a​ ​film​ ​of​ ​liquid​ ​around​ ​it
○ Multiple​ ​strands​ ​can​ ​be​ ​sequenced​ ​at​ ​the​ ​same​ ​time
○ Free​ ​nucleotides​ ​are​ ​pipetted​ ​in,​ ​and​ ​results​ ​are​ ​presented​ ​as​ ​a​ ​flow-gram
■ If​ ​a​ ​base​ ​binds,​ ​there​ ​is​ ​a​ ​tiny​ ​release​ ​of​ ​energy,​ ​which​ ​is​ ​recorded
Third-generation​ ​sequencing
● $900​ ​per​ ​genome​ ​(as​ ​opposed​ ​to​ ​$1​ ​billion​ ​for​ ​the​ ​Human​ ​Genome​ ​Project​ ​in​ ​2003)
● Takes​ ​6​ ​hours
● Uses​ ​a​ ​machine​ ​the​ ​size​ ​of​ ​a​ ​chewing​ ​gum​ ​packet
● A​ ​single​ ​strand​ ​of​ ​DNA​ ​is​ ​passed​ ​through​ ​a​ ​nanopore,​ ​hydrogen​ ​bonds​ ​result​ ​in
changes​ ​in​ ​current​ ​which​ ​are​ ​converted​ ​to​ ​a​ ​sequence
--------------------------------------------------------------------------------------------------------------------------●
●
●

8:​ ​REPRODUCTIVE​ ​SUCCESS​ ​IN​ ​COOPERATIVELY​ ​BREEDING​ ​MONGOOSE
●

‘Top​ ​males​ ​gain​ ​high​ ​reproductive​ ​success​ ​by​ ​guarding​ ​more​ ​successful​ ​females​ ​in​ ​a
cooperatively​ ​breeding​ ​mongoose​ ​colony’
○ Observed​ ​‘mate​ ​guarding’​ ​behaviour,​ ​then​ ​determined​ ​the​ ​paternity​ ​of
offspring
○ Study​ ​carried​ ​out​ ​on​ ​groups​ ​of​ ​banded​ ​mongoose​ ​(Mungos​ ​mungo)​ ​in​ ​Queen
Elizabeth​ ​National​ ​Park,​ ​Uganda,​ ​between​ ​1998​ ​and​ ​2004
■ Data​ ​collected​ ​from​ ​638​ ​individuals
■ Ages​ ​estimated​ ​from​ ​tooth​ ​wear
■ Animals​ ​given​ ​unique​ ​haircuts​ ​for​ ​identification
■ Genetic​ ​information​ ​taken​ ​from​ ​the​ ​tip​ ​of​ ​the​ ​tail,​ ​using​ ​a​ ​sterile​ ​scalpel
○ Genetic​ ​analysis
■ Tail​ ​tip​ ​samples​ ​incubated​ ​on​ ​a​ ​rotating​ ​wheel​ ​in​ ​300ul​ ​of​ ​lysis​ ​solution
(alkaline​ ​buffer​ ​to​ ​stabilise​ ​DNA​ ​(eg
...
​ ​SDS),​ ​and​ ​proteinase​ ​to​ ​proteolytically
digest​ ​contaminating​ ​protein)
● Salting​ ​out​ ​process
● Extra​ ​phenol/chloroform​ ​step
○ Phenol​ ​is​ ​used​ ​to​ ​denature​ ​and​ ​remove​ ​protein​ ​(by
using​ ​an​ ​equal​ ​mixture​ ​of​ ​phenol​ ​and​ ​chloroform,
efficiency​ ​is​ ​improved,​ ​as​ ​the​ ​phenol​ ​is​ ​then​ ​removed
by​ ​the​ ​chloroform)
○ Advantages:
■ High​ ​purity​ ​of​ ​DNA​ ​achieved
■ Quick​ ​and​ ​easy​ ​to​ ​perform​ ​(added​ ​as​ ​an​ ​extra
step​ ​to​ ​salt​ ​extraction)
○ Disadvantages:

Joanna​ ​Griffith​ ​(2017)

Cannot​ ​be​ ​used​ ​for​ ​extraction​ ​of​ ​DNA​ ​from
complex​ ​mixtures​ ​(eg
...
​ ​E-coli​ ​in​ ​iguanas
● Antigen-antibody​ ​complexes​ ​can​ ​be​ ​used​ ​in​ ​lab​ ​tests​ ​to​ ​detect
specific​ ​antigens​ ​or​ ​antibodies
● Key​ ​words:
○ Antibody:​ ​a​ ​protein​ ​produced​ ​by​ ​a​ ​B-cell​ ​in​ ​response​ ​to​ ​a​ ​specific​ ​antigen,​ ​that
can​ ​bind​ ​to​ ​that​ ​antigen
○ Antigen:​ ​a​ ​substance​ ​that,​ ​when​ ​introduced​ ​into​ ​the​ ​body,​ ​stimulates​ ​the
production​ ​of​ ​a​ ​specific​ ​antibody​ ​by​ ​B-cells
○ Epitope:​ ​the​ ​specific​ ​localised​ ​region​ ​on​ ​the​ ​surface​ ​of​ ​an​ ​antigen​ ​that​ ​is
recognised​ ​by​ ​the​ ​antibody
○ Hapten:​ ​a​ ​small​ ​molecule​ ​that​ ​reacts​ ​with​ ​a​ ​specific​ ​antibody​ ​but​ ​cannot
induce​ ​the​ ​formation​ ​of​ ​antibodies​ ​unless​ ​bound​ ​to​ ​a​ ​carrier​ ​protein​ ​or​ ​other
large​ ​antigenic​ ​molecule
General​ ​protein​ ​techniques
● Immunohistochemistry
● Latex​ ​agglutination
● ELISA
○ Sandwich
○ Indirect
○ Competitive
● Protein​ ​microarray
● SDS​ ​page
● Western​ ​blotting
● Eastern​ ​blotting
--------------------------------------------------------------------------------------------------------------------------■
■

10:​ ​ELISA
●

For​ ​DNA,​ ​we​ ​use​ ​primers​ ​to​ ​target​ ​specific​ ​DNA​ ​sequences
Joanna​ ​Griffith​ ​(2017)

For​ ​proteins,​ ​we​ ​can​ ​use​ ​antibodies​ ​to​ ​target​ ​specific​ ​epitopes​ ​of​ ​proteins
○ Produced​ ​by​ ​B-lymphocytes
○ Other​ ​molecules​ ​can​ ​be​ ​attached​ ​to​ ​antibodies​ ​to​ ​make​ ​them​ ​more​ ​visible
Enzyme-linked​ ​immunosorbent​ ​assay​ ​(ELISA)
● Available​ ​as​ ​fairly​ ​cheap​ ​kits
● Sandwich​ ​ELISA​ ​(eg
...
​ ​immunisation​ ​programme​ ​of​ ​monkeys​ ​at​ ​Looe​ ​Monkey
Sanctuary)
○ Testing​ ​for​ ​the​ ​presence​ ​of​ ​antibodies​ ​in​ ​the​ ​immune​ ​system
○ Process:
■ Specific​ ​antigen​ ​attached​ ​to​ ​wall​ ​of​ ​well
■ Blood​ ​or​ ​serum​ ​sample​ ​added
● If​ ​the​ ​specific​ ​antibody​ ​is​ ​present​ ​in​ ​the​ ​sample,​ ​it​ ​will​ ​bind​ ​to
the​ ​antigens​ ​on​ ​the​ ​well​ ​wall
■ The​ ​sample​ ​is​ ​washed
● Only​ ​attached​ ​molecules​ ​remain,​ ​stuck​ ​to​ ​the​ ​well​ ​wall
■ Secondary​ ​antibody​ ​is​ ​added​ ​(contains​ ​an​ ​enzyme)
● Attaches​ ​to​ ​the​ ​attached​ ​antibody
■ The​ ​sample​ ​is​ ​washed​ ​to​ ​remove​ ​excess​ ​sample​ ​antibody
■ Substrate​ ​for​ ​enzyme​ ​is​ ​added
● Enzyme​ ​reaction​ ​leads​ ​to​ ​a​ ​colour​ ​change​ ​(positive​ ​for
immunisation)
● Competitive​ ​ELISA
○ An​ ​antigen​ ​is​ ​labelled​ ​with​ ​an​ ​enzyme,​ ​and​ ​competitively​ ​binds​ ​to​ ​the​ ​antibody
attached​ ​to​ ​the​ ​wall​ ​of​ ​the​ ​well​ ​in​ ​place​ ​of​ ​the​ ​sample​ ​antigen
■ Clear​ ​sample​ ​=​ ​positive​ ​result
● Advantages​ ​of​ ​ELISA
○ Very​ ​simple​ ​to​ ​use
●

Joanna​ ​Griffith​ ​(2017)

○ No​ ​harmful​ ​reagents
○ Can​ ​detect​ ​very​ ​specific​ ​targets
○ Easy​ ​to​ ​use,​ ​no​ ​specific​ ​training​ ​required
● Disadvantages​ ​of​ ​ELISA
○ Can​ ​be​ ​expensive
○ If​ ​a​ ​commercial​ ​kit​ ​is​ ​not​ ​available,​ ​it​ ​can​ ​be​ ​very​ ​time-consuming​ ​and​ ​difficult
to​ ​set​ ​up
● Microarrays
○ Antibody​ ​microarrays
■ Normal​ ​ELISA​ ​only​ ​tests​ ​for​ ​one​ ​antigen​ ​(the​ ​96​ ​wells​ ​are​ ​replicates)
■ Microarrays​ ​have​ ​a​ ​range​ ​of​ ​different​ ​antibodies,​ ​to​ ​test​ ​for​ ​more​ ​than
one​ ​antigen​ ​simultaneously
○ Protein​ ​microarrays
■ Proteins​ ​are​ ​bound​ ​to​ ​a​ ​solid​ ​support
■ Similar​ ​to​ ​indirect​ ​ELISA
■ Fluorescent-labelled​ ​antibodies​ ​or​ ​other​ ​ligands​ ​can​ ​be​ ​added
○ Cheaper​ ​than​ ​DNA​ ​microarrays,​ ​and​ ​no​ ​need​ ​for​ ​PCR​ ​or​ ​other​ ​techniques
--------------------------------------------------------------------------------------------------------------------------11:​ ​BLOTTING
There​ ​are​ ​over​ ​1,200​ ​species​ ​of​ ​barnacle
Barnacles​ ​have​ ​two​ ​motile​ ​larval​ ​stages,​ ​plus​ ​one​ ​sessile​ ​adult​ ​stage
○ Nauplius​ ​juvenile​ ​stage
■ Feeds​ ​for​ ​around​ ​6​ ​months
○ Cypris​ ​juvenile​ ​stage
■ Non-feeding​ ​stage​ ​lasts​ ​1-3​ ​weeks,​ ​while​ ​the​ ​larvae​ ​find​ ​a​ ​place​ ​to
settle
● Many​ ​barnacles​ ​are​ ​gregarious​ ​(they​ ​prefer​ ​to​ ​grow​ ​with​ ​other​ ​barnacles)
○ How​ ​do​ ​they​ ​manage​ ​to​ ​find​ ​each​ ​other?
○ While​ ​floating​ ​in​ ​the​ ​sea​ ​in​ ​their​ ​motile​ ​cypris​ ​stage,​ ​something​ ​must​ ​attract
them​ ​to​ ​other​ ​barnacles
■ Crude​ ​experiment​ ​found​ ​that​ ​smearing​ ​adult​ ​barnacle​ ​extract​ ​onto
slate​ ​induced​ ​cypris​ ​larvae​ ​to​ ​settle
● How​ ​would​ ​you​ ​find​ ​out​ ​which​ ​proteins​ ​or​ ​chemicals​ ​are
involved​ ​in​ ​this​ ​process?
How​ ​are​ ​barnacles​ ​gregarious?
● Extraction
○ blend/mash​ ​adult​ ​barnacles
○ Add​ ​chemicals​ ​to​ ​disrupt​ ​cell​ ​membrane
○ Add​ ​pH​ ​buffers
○ Add​ ​solvents​ ​for​ ​the​ ​proteins​ ​to​ ​dissolve​ ​into
○ The​ ​equivalent​ ​of​ ​salting​ ​out/phenol​ ​chloroform​ ​extraction​ ​for​ ​DNA
● Electrophoresis​ ​(SDS​ ​PAGE​ ​-​ ​sodium​ ​dodecyl​ ​sulfate​ ​polyacrylamide​ ​gel
electrophoresis)
○ Used​ ​to​ ​separate​ ​the​ ​proteins​ ​out​ ​into​ ​size​ ​bands
●
●

Joanna​ ​Griffith​ ​(2017)

Proteins​ ​are​ ​treated​ ​with​ ​SDS​ ​to​ ​straighten​ ​out​ ​proteins​ ​and​ ​give​ ​them​ ​an
even​ ​charge​ ​(as​ ​proteins​ ​can​ ​be​ ​positive​ ​and​ ​negative​ ​in​ ​different​ ​sections)
○ Polyacrylamide​ ​gel​ ​is​ ​used​ ​instead​ ​of​ ​agarose​ ​gel​ ​as​ ​proteins​ ​are​ ​smaller
than​ ​DNA,​ ​and​ ​polyacrylamide​ ​gel​ ​has​ ​a​ ​more​ ​tightly​ ​cross-bound​ ​structure,
allowing​ ​it​ ​to​ ​better​ ​separate​ ​smaller​ ​molecules
○ Often​ ​conducted​ ​vertically​ ​as​ ​it​ ​allows​ ​the​ ​gel​ ​to​ ​be​ ​moved​ ​into​ ​different
solvent​ ​baths
● transfer/blotting
○ Transferring​ ​molecules​ ​from​ ​electrophoresis​ ​gel​ ​to​ ​a​ ​solid​ ​support​ ​(eg
...
​ ​testing​ ​for​ ​link​ ​between​ ​larval​ ​stage/age​ ​and​ ​levels​ ​of​ ​specific​ ​protein
■ Add​ ​specific​ ​antibody​ ​and​ ​enzyme​ ​to​ ​cypris​ ​larvae​ ​at​ ​different​ ​ages,
add​ ​the​ ​enzyme​ ​substrate,​ ​and​ ​look​ ​for​ ​a​ ​colour​ ​change
■ Immunostaining​ ​(microscopy)
● Barnacles​ ​manage​ ​to​ ​be​ ​gregarious​ ​by​ ​expressing​ ​three​ ​proteins​ ​(98kDA,​ ​88kDA,
and​ ​76kDA)
○ More​ ​attachment​ ​protein​ ​is​ ​expressed​ ​as​ ​they​ ​age
○ Proteins​ ​expressed​ ​on​ ​the​ ​‘head’
Summary​ ​of​ ​blotting
● Process:
○ Sample​ ​mixture
■ Proteins,​ ​DNA,​ ​RNA,​ ​carbohydrate​ ​side​ ​groups
○ Resolved​ ​in​ ​a​ ​gel​ ​using​ ​electrophoresis
■ Agarose​ ​or​ ​SDS​ ​PAGE
○ Transferred​ ​to​ ​a​ ​solid​ ​support​ ​(blotting)
○ Detected​ ​using​ ​a​ ​probe
■ Antibody​ ​or​ ​complementary​ ​DNA)
● Types​ ​of​ ​blotting:
○ Southern​ ​blotting,​ ​used​ ​for​ ​DNA
■ Edwin​ ​Southern,​ ​1975
○ Northern​ ​blotting,​ ​used​ ​for​ ​RNA
○

Joanna​ ​Griffith​ ​(2017)

■ James​ ​Alwine,​ ​1977
○ Western​ ​blotting,​ ​used​ ​for​ ​proteins
■ Harry​ ​Towbin,​ ​1979
○ Eastern​ ​blotting,​ ​Middle-Eastern​ ​blotting,​ ​Eastern-Western​ ​blotting,
Far-Eastern​ ​blotting,​ ​used​ ​to​ ​look​ ​at​ ​post-transcriptional​ ​modifications​ ​to
proteins​ ​(eg
...
19um)​ ​x​ ​wavelength/numerical​ ​aperture
○ There​ ​is​ ​always​ ​a​ ​limit​ ​on​ ​resolution​ ​due​ ​to​ ​the​ ​use​ ​of​ ​light
○ The​ ​shorter​ ​the​ ​wavelength,​ ​the​ ​greater​ ​the​ ​resolution
● So​ ​why​ ​not​ ​create​ ​images​ ​using​ ​something​ ​other​ ​than​ ​visible​ ​light?
● Electron​ ​microscopes
○ Uses​ ​a​ ​beam​ ​of​ ​electrons​ ​instead​ ​of​ ​light
■ Wavelength​ ​100,000​ ​times​ ​smaller​ ​than​ ​light
● Can​ ​achieve​ ​over​ ​200​ ​times​ ​better​ ​resolution,​ ​and​ ​over​ ​1
million​ ​times​ ​more​ ​magnification
○ Transmission​ ​electron​ ​microscopy​ ​(TEM)
■ High​ ​voltage​ ​electron​ ​beam,​ ​passes​ ​through​ ​the​ ​sample
■ Sample​ ​needs​ ​to​ ​be​ ​extremely​ ​thin,​ ​so​ ​it​ ​is​ ​embedded​ ​in​ ​polymer​ ​resin
and​ ​cut​ ​(cells​ ​are​ ​killed)
■ Gives​ ​high​ ​magnification,​ ​but​ ​an​ ​extremely​ ​small​ ​depth​ ​of​ ​field
○ Scanning​ ​electron​ ​microscopy​ ​(SEM)
Joanna​ ​Griffith​ ​(2017)

The​ ​electron​ ​beam​ ​scans​ ​the​ ​surface​ ​of​ ​the​ ​sample​ ​and​ ​records​ ​what
is​ ​reflected
● Electrons​ ​do​ ​not​ ​go​ ​through​ ​the​ ​sample,​ ​so​ ​there​ ​is​ ​less​ ​of​ ​a
limit​ ​on​ ​the​ ​size​ ​of​ ​the​ ​sample
■ Lower​ ​magnification
■ Only​ ​shows​ ​the​ ​surface,​ ​but​ ​gives​ ​a​ ​much​ ​larger​ ​depth​ ​of​ ​field
Summary​ ​of​ ​microscopy
● Types​ ​of​ ​microscopy:
○ Bright​ ​field​ ​microscopy
○ Dark​ ​field​ ​illumination
○ Phase​ ​contrast​ ​microscopy
○ Differential​ ​interference​ ​contrast​ ​(polarised​ ​light​ ​microscopy)
○ Fluorescence​ ​microscopy
○ Confocal​ ​microscopy
○ Electron​ ​microscopy
■ TEM
■ SEM
○ Sub-diffraction​ ​limit​ ​microscopy
■

Joanna​ ​Griffith​ ​(2017)



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