Experimental and Industrial Processes

Process Explanation
Autoradiography  E. coli: grow with thymidine so that they contain radioactive H isotope = marks
DNA
 Enzyme: breaks open cell contents
 Film of photographic emulsion applied to the membrane surface
 Radioactive isotope reacts (dark = DNA)
Test Cross  Determines the genotypes of an organism with dominant phenotype
 Cross with a homozygous recessive
 100% dominant = homozygous dominant
 50% dominant = heterozygous
Quadrat Quadrats: square sample areas
Sampling Quadrat sampling: repeatedly placing a quadrat frame at random positions in a habitat
and recording the number of organisms present each time
1. Base line marked all the way along the edge of habitat using measuring tape
2. Random numbers (random number generator)
 First random no: distance along measuring tape
 Second random no: distance across habitat (at right angle)
3. Place quadrat + repeat
4. Use Chi squared testing to test null hypothesis (two species are distributed
independently)
Gel  Process which separates charged molecules in an electric field according to:
electrophoresis o Size
o Charge
1. Samples placed in wells cast in gel
2. Gel immersed in conductive liquid + electric field applied
3. +/- move in opp directions, small particles move farther and further
All DNA is negative, therefore, when carried out on DNA it just separates it by size
Polymerase Synthetic method of amplifying sequences of DNA (double DNA each time) – occurs in
Chain Reaction a thermal cycler
(PCR) 1. Denaturation: Heat DNA (~90ºC) until H bonds b/w strands = broken
2. Annealing: sample is cooled (~55ºC) to allow DNA primers (designate sequence
to be copied) to anneal to each end of the segment
3. Elongation: Heat sample to optimal temp for heat tolerant Taq polymerase
(enzyme) to function (~75ºC)
Taq polymerase synthesises complementary nucleotides from the primers
Determining 1. Theory proposed
epidemiology 2. Survey data (correlation b/w disease + theoretical cause)
3. Statistical procedures (isolate single factor)
Oscilloscope Graph showing membrane potential, created by placing electrodes on either side of
membrane
Calorimetry Measuring energy content in food
 Complete combustion of food sample
 Beaker w/ known mass of water heated by E released in combustion
 Thermometer in water to measure ∆ T

Process Explanation
Respirometer Sealed contained w/ living tissue (invertebrate or germinating seeds)
Alkali to absorb CO2
 Pipette connected to container
Temperature should be kept constant to avoid volume changes due to
temperature fluctuations
Ethics:
 Removing animal from habitat – can it be safely returned
 Harm/ distress
 Risk of accidents reduced e.g. prevent contact w/ alkali
 Is the use of animals essential?
Chromatography Separating photosynthetic pigments
(1) Extract pigments from leaf (grind in mortar)
(2) Smear dry pigments onto thin layer chromatography strip
(3) Solvent runs up strip
Automated Base (1) Break up: genome is broken into small lengths of DNA to be
sequencing sequenced separately
(2) Single stranded copies: made using DNA polymerase.
 Put small quantities of non-standard nucleotide in reaction
mixture =
 Process stopped before whole base sequence copied
 Produces: 4 samples of DNA copy of varying length w/ one of
the four DNA bases at the end
(3) Coloured fluorescent markers: mark DNA copies (diff colour for diff
end base)
(4) Samples mixed together
(5) Gel electrophoresis: separate copies into lanes according to length/ no
of nucleotides
(6) Laser: scans lane to make markers fluoresce
(7) Optical Detector: detects colours of fluorescence (correspond to no of
nucleotides)  computer uses to deduce base sequence
Karyogram Diagram/ photograph of chromosomes present in nucleus (of eukaryotic)
arranged in homologous pairs in descending length
(1) Phase: MITOSIS = most visible in METAPHASE
(2) Stained: make chromosomes visible = BANDING PATTERN
(3) Micrograph taken
(4) Arranged in pairs from longest to shortest
Process Explanation
IVF (1) Stimulation of multiple egg releases
a. Down regulation: stop all FELP hormones = suspend normal
cycle = control timing + amount of egg production
b. Intramuscular injections: FSH/LH (daily 10 days) = more FSH
conc than normal cycle = more follicle development
c. HCG injection: HCG normally secrete by embryo – stimulates
follicle maturation
(2) Eggs collected: micropipette passed through uterus wall to wash eggs
out of follicles
(3) Fertilised: with sperm in a sterile dish, incubated
(4) Viable (fertilised) blastocysts = selected + developed into embryos
(5) Up to 3 embryos implanted
DNA Profiling 1. DNA samples amplified using PCR
2. Restriction Enzymes: cut DNA into fragments at specific base
sequences
3. Fluorescent marker: bind to triplet in DNA fragments = results can be
seen
4. Gel electrophoresis: produces banding pattern which can be compared
Lactose-free (1) Extract lactase from yeast/ bacteria
milk (2) Bound to surface of alginate beads
(3) Milk passed repeatedly over beads
Industrialised Textiles: processing fibres
uses of Brewing: many processes e.g. clarification of beer
immobilised Medicine/ Biotech: contact lens cleaners, cutting DNA in genetic engineering
enzymes
Old method of (1) Extract insulin from pancreas pig/ cattle
insulin (2) Inject into blood
(diabetes) *allergies
New method of (1) Plasmid extracted from bacteria + cut w/ restriction enzyme
insulin (2) Restriction enzyme cuts human insulin gene from genome
(3) Human insulin gene inserted into plasmid  ligase = recombinant
(4) Recombinant inserted into new bacteria
Somatic cell Adult cloning
nuclear transfer (1) Remove differentiated diploid nucleus from individual to be cloned
(SCNT) (2) Insert into enucleated donor egg cells
(3) Implant into endometrium of surrogate mother + gestate
Treatment for 1. Embryonic stem cells treated to become retinal cells
Stargardt’s 2. Injected into retina
Macular 3. Cells attach + become functional
Dystrophy
Treatment for 1. Hematopoietic Stem Cells (HSCs): harvested from bone marrow,
Leukaemia peripheral blood, umbilical cord blood
2. Chemo/radio therapy = kill diseased WBCs
3. Old WBCs replaced w/ healthy WBCs
4. HSCs transplanted into bone marrow
5. HSCs differentiated into new healthy WBCs
Famous Experiments

Circulatory GALEN: before what we thought was…

systems  Arteries + veins = sep blood networks
 Blood used up in body
 New blood = created by liver

WILLIAM HARVEY: human autopsies + experiments

 Impossible for liver to generate volume of blood needed (therefore, blood
must be circulate + pumped by <3)
 Blood in veins only flows one way (b.c. valves)
 Existence of capillaries
Penicillin: FLOREY & CHAIN
Testing on Growing penicillium (fungus) to secrete penicillin
Mice 8 mice infected w/ streptococcus (causes death from pneumonia)
4 injected w/ penicillin, No Pen = dead, Pen = alive
Bad testing b.c. (1) brief animal tests before human (2) impure samples = could
have been side effects from impurities
Disproving Artificial synthesis of urea
vitalism Ammonia + CO2  ammonium carbonate  urea + water
DNA CRICK & WATSON
structure Stick and ball models to test ideas on DNA structure
1st model: A:T ratio not 1:1 and too much Mg
Realised:
(1) DNA is in double helix
(2) Base pairing
(3) Antiparallel
2nd Model:
(1) Possible mechanisms for replication
(2) Info coded in triplets of bases
Semi- (1) Grow e-coli in diff N isotopes (N-14, N-15)
conservation (2) N-15  N-14 solution
DNA (3) Collect after each rep cycle + centrifuge)
replication 1 gen later: density b/w N-14 and N-15
2 gen later: one as above, second w/ N-14 density
Human Full copy of a human genome
Genome (1) Genomes vary from SNPs but are 99.9%
project (2) Fewer genes than predicted
(3) “junk DNA”  affects gene expression, satellite DNA
Discovery of Improved microscopes = view chromosomes
meiosis Careful observation:
What  often no cells in meiosis = visible
factors?  images not clear enough to see
Examined germ-line cells: horse threadworm
 2 chromosomes in gametes
 4 chromosomes in fertilised egg
 Chromosome number doubles in fertilisation
 Hypothesis: must have a division to halve
Speciation CHARLES DARWIN
Gave example of pop which was recognisably diff but not distinctly diff species (red
grouse + willow ptarmigan) = continuous range of variation
 Modelling HOLST & FROLISCH: Guinea pigs
scurvy Fed w/ whole grains  scurvy-like symptoms
Fed w/ cabbage + lemon juice  cured symptoms
Showed causes + preventative foods
G. PIGS worked as models b.c. one of few mammals for whom Vit. C = essential
Chemical WILLIAM BEAUMONT
Digestions Surgeon of a man w/ gunshot wound to the side = observe digestion (serendipity)
Gastric acid extracted (digest in jar) = overturned that digestion was solely physical
Mendel  First cross  dominant phenotype x recessive phenotype = all dom pheno
 Second cross  self-pollination of offspring ^^ showed 3:1 ratio of
heterozygous cross
Why important to modern scientific methodology?
1. Quantitative Measurements (ratio)
2. Many trials to establish replicability
3. Studied many different parameters
Why did he use pea plants?
1. Clear characteristics
2. Could be manually cross pollinate or if left, would self-pollinate
3. Monogenic traits
Discovery of TIM HUNT: Serendipity
cyclins  Was researching control of protein synthesis in sea urchin eggs
 Discovered a protein which
a. ↑ conc after fertilisation
b. Then ↓ conc (when other proteins continued to increase)
 Further experiments showed that the proteins went through cycles of
increase and decreased concentration which coincided w/ the cell cycle

Experiment Explanation
Theories of ARISTOTLE: SEED + SOIL
reproduction Fertilisation occurs when male seed + menstrual blood = transforms into egg

WILLIAM HARVEY: NATURAL EXPERIMENT:

 Dissected deer uteruses during mating season
 Expected to find eggs immediately after mating
 No signs of devo until 2-3 months later

 Disproved Aristotle but came to incorrect conclusion that fertilisation ≠

linked with coitus
 Why failed? Effective microscopes were unavailable = unable to observe
gamete fusion/ embryo development (esp. deer embryos remain
microscopic LT)
Supercoiling CAIRN
in E. coli See autoradiography for method
Results:
 E. coli cell length: 2 μm
 E. coli chromosome length: 1,100 μm
 Evidence for supercoiling

Nature of Science: Random

N of Science Explanation
Collaboration  Work in multidisciplinary teams
b/w groups  Biologists contribute to research on learning + memory
of scientists