Oivenity ot{ Evalotion

Evolution: Changes in allelic or genotypic

frequencies that occur in a gene pool of apopulation over time

Gene Pool: Sum total of all genes of all breedingindividuals in a population at any onetime

lf only one allde exists at a particular locus = allele is fixed

Population: Localized group of individuals, living in adefined geographic area, and ofthesame sPecies.

Definition of Speciee

Poputation of organimswhich cart interbrcd to produceviable,ffiite offsping, but arc reproductivdy isolatd from othsspec,'es.They have similar behavioral,morphological and physblogical

fmtwes, the same chromosome numbq, and share a common gene

pool.

Lamarck's Theory IWRONGIWhen an organism dweloped a need for a particular structure, theneed induced the development of the structure. Beneficial

characteristics can be inherited by the offspring.

Evidence for Natural Selection

Anatomical Structures

Homologous $ructures- Structural Similarities that suggest that they share a common

ancestor with that feature- Can show how the organism have diversified to colonize different

environments, ie Dvergent Evolution- Example 1: Pentadact!y' Umbs- Ebnes of the forelimbs of vertebrates compose of similar

bones arranged in comparable pattern

- Early land vertebrates (amphibians) possess the pentadactyl

limb structure- All descendard vertebrates, mammals, reptiles, birds, have

limbs that follow the same Pattem

Early Embryonic DeveloPment- Structural Similarities during early stages of embryonic

development- Example 2: Human Embryonic Development- Rudimentary gill ridges even though human embryo receives

oxygen from umbilical cord- lndicates humans evolved from aquatic animal with gill

slits- Long Bony Tail, which becomes coccyx at the end of our

I spineduring adufthoodI Ancestor possessed long tailI

I

lVestigial Structuresl- tlo Clear Function, but resemble structures present in their

I ancestorsl - No Longer subiect to natural selection

I Example 3: Vestigial Structures in HumansI Coccyx (fused tailbones)

I Third Molars (Vvisdom Teeth)

I Ear-Wiggling Muscles

I From mammals such as dogs which can tum theirI ears towards the direction of a sound

I AppendixI Had digestive functions in our arcestorsI Example 4: Vestigial hind'limb bones in Modern Whales,I can be traced back to their tenestrial ancestor

II Molecular / Biochemical Homologies

I - The greater the degree of molecular homologues, the more closely

I rehted the two organisms are

I E<ample 5: DNA/Nucleic AcidI Genetic code is universal

I Descendants will exhibit high overall similarity in DNA withI the oarents

I As descendants evolve, they will accumulate more andI more differences in DNA

I i.e. Chimpanzee has 97.67o similarity, thought to have

! common ancestor 6 million years ago

I - Example 6: ProteinsI - Changes in DNA Sequence reflected in proteins

I i.e. Hemoglobin Beta Chain (146bp)

I i.e. M sequence in P53 Protein

Paleontological Evideflce (Fossil Records)

Example 7: Evolution of Horses- Ehsed on the analysis of fossil records- The Ancestral Species (in wooded habitats)

- genus Hyracotherium- Small, short legs, fleshy pads, 4/3 toes

- Modern Horse (genus Equus)- Change in habitat => grasslands- Teeth grow in lengrth, complex pattern of ridges on

molars and premolars due to more abrasivevegetatbn

- One toe per foot- DevelopmentofbonYhoof- lncrease in size (now can weight half a ton)

of Natrral Selection

o1Great Potential to Reproduce- Produce more than can survive

02

Constancy in Numbers- Umited Resources (food, light, disease etc)- Majority of offspring die before reaching reproductive

maturity

t1Struggle for Existence- Competitionlnevitable

03

Variation within a Population- Each individual will have a unique set of

characteristics- Occur spontaneously before a change in

environment- Arise as a result of spontaneous rnrtations,

controlled by genes

t2

Survival of the Flttest by Natural Selection- Environment selects those well-adapted indMduals

with inherited traits that are best suited to the local

environment- lndMduals with selective advantage able to reach

maturity and reproduce

o4

Differential Reproduction- Those that survive to breed are likely to produce

offspring similar to them- Proportion of indMduats who possess the

favorable alleles inctease- lead to clranges in allelic fnequencies over time

l3

Formation of New Species (see )

- Arise from the accumulation of adaptations- Requires reproductive isolation to occur, in

other wotds no lnterbreeding

tlversity ord Evalution

Biogeographical Records

- Similar environments may exhibit organisms with strikingly similar

appearance, even though the organlsms are only distantly related

- Natural Selection favorcd parallel evolutionary adaptations

- Convergent Evolution- Species from different evolutionary branches may come to

resemble each other if they have similar ecological roles and

natural selection have shaped similar adaptations

- Etample & illarsupials and Placentals- Resemue @ch other- Only difference in that Marsupials' young are bom immature

and held in pouches, while Placentals' young are not bom until

they can safev survive in the external environment

4b

Genetic Drift - Bottleneck Effect- Natural disasters, disease or predators cause drastic

short-term reductions of populatio sze- Survirors represent a small random @rtion of the

original gene pool, leading to under/over representation

of alleles, or even eliminated,- Reduction of genetic variability

- Example 13: Gheetah PoPulation- Two bottlenecks, the end ofthe recent ice age and

mass slaughter by man in the 19th Century

- Limited genetic stock- Sperm abnormality and low sperm count

I Decreased fecunditY

I High cub mortalityI Lower resistance to disease

5

Natural SelectionDirectional Selection- Favors one extreme, shifts the population mean for that

character, usually due to changing environment

- Eliminate the other extreme- Example 7: Evolution of Horses- Example 14: lndustrial Melanism in Peppeled Moth

- Before lndustrialization (UK)

- 100% Pale- Black peppered were conspicuous against light

colored lichen-covered trees- Black variants subject to greater predation

- Duringlndustrialization- Smoke/soot had blackened the lichens and the

bark of tree

I Black well camouflaged, pale was not

I Higher frequency of black-peppered moths

I After lndustrialization

I Light form making a comebackI Less soot on trees

l- Example 15: Antibiotic ResistanceI Mutation produced an individual bacterium while an

I aee urrtrich codes for resistance

I MLrtant at selective advantage

I Large population of penicillin resistance evolved

II Stabilizing Selectionl- Favors the existing mean, extremes selected against

l- Reduction in variance, due to unchanging environment

l- Example 16: Birrh Mass in HumansI lnfants with intermediate birth weight have highest

I survival rate

l- Example t7: Sickle Cell AnaemiaI Hetero4/gotes selected for (Heterozygous

I Advantage)I Not suffering from sickle-cell, and less susceptibleI to mahria

I Sebction Pressure exerted by both sickle cell

I anaemia and malaria

IlDisruptive Selectionl- Two or more extreme phenotypes are favored

l- Example 18: British land SnailsI Dark shells thrive in forest area

I Light shells thrive in grasslands

I Selection Pressure: Predator, thrushes

Processes Leading to Evolution

1

Mutation- Change the frequency of alleles and gene pool

- Source of new alleles, but arise not in any relation toenvironmental necessities

- Mutation rate low in humanVplants, burt very high in

mbroorganisms and viruses- Hence for diseases like HM single drug treatments

do not work Drug cocktails are needed.

2

Gene Flow- Movement of genes from one population to another- Migration of fertile indMduals or transfer of gametes

- Reduce differences between populations

- Constructive / Constraining- Example 9: Copper Tolerance in Bent Grass

- Occuning on abandoned mine sites in UK- Expect soil copper tolerarlce to be 't 00% on mine

sites and 096 on non-mine sites- Constraining:

- Wirds blow pollen with non-tolerant alleles

from up\ rind sectors onto mine site- Constructive

- Winds blow pollen with tolerant alleles frommine site to downwind sectors

3

Non-Random Mating, Artificial Selection- Non-random selective breeding, determined by

deliberate human actions for human benefrt

- Alleles favored by humans will increase in frequency- Man exerts the selec'tim gessure- lnbreeding

- lncrease genetic uniformity- Reduced iartility, disease resistance

- Outbreeding- Develop Hybrids- Decreased genetic uniformitY- New traits, which may be superior to parent

- Example te Wild vs Domestic Bnassica Oleracea- Broccoli (flower cluster), Cabbage (Condensed

shoot), Brussels Sprouts (Lateral Buds)

- Example 11: Gray Wolf and DonEstic Dogs

4a

@netic Nift isthe mdom changeof alldicfrcm generation

to gqteration as a resuft of change alone in a small gene

Pool/ Poqulation.

Genetic Drift - Founder Effect- One or few individuals colonize a habitat isolated from

their place of orQin.- The alleles they carry will be a signfficant faction of the

gene pool- Example 12: Amish Population

- lnbreeding among 20O members of the Amish

Rel(7ion which migrated to PennsyMania

- Allelic frequency for Blis-van Crevald syndrome was0.07, compared to d.0O1 in the general population

Nalural Selection Artificial Selection

Long period of time, manygenerations

Rapid Process, few generations

Traits selected are advantages toorganism, increase chances or

suMval

Traits selected are advantages toman, may not conler advantage for

suMval

Organisms with greater fitnesssdected to reproduce

Organisms with desirablecharacteristics selected to reproduce

Nature o(erts selection Pre$sure Man exerts selection pressure

Maintaining Genetic Variation

Dhbidy

Recessire allele dlowed to persist as they are propagated in

heterozygous individualsDominant alleles mask e{fects of recessive alleles, protecting them

from natural selection

Balanoed Potymorphism

Polymorphism: 2 or morc {orms of an organism that are genetically

distinct but are contained within the same interbreeding population

Balanced Po[morphism: Maintain stable frequencies of two ormore phenotypic formsHeterozygote Advantage- See Example 17Frequency- Dependent Selection- Relative fitness of different genotypes is related to how

frequentty they occur in the population- Fitness of a genotype declines if it becomes too common- Example 19: Scale Eating Cicfilids

- Left-mouthed and Right-mouthed- Prey species guard against attack from whatever

phenotype of scale-eating fish is most common in the lake

- Frequency of both fish oscillates o/er time, achieving

bralancing selection

Neutral lrariations

Most of DNA variation has little or no impact on reprcductive

srrc@ss (non-cording etc.)Not subjected to natural selection as they confer no selective

disadvantage or advantage

Sexual Reproduction

Crossing Over during ProPhase I

lndependent Assortment of homologous chromosome, during

Metaphase I

lndependent assortment of sister chromatids during Metaphase ll

Random Fertilization- Fusion of mde and fumale gametes- New recombination of alleles already present within the gene

pool

Gene FlowlntrodLrction of new alleles due to migration of fertile indMduals from

one Population to another

tliversitl ord Evalutioh

Modes of SpeciationAllopatric Speciation: New species arise as a result of geographical

isolation and subsequent natural selection and genetic drift' The

Physical Barrier blocks interbreeding and gene flow'

Sympatric Speciation: New species that may evolve form individuals

lMng in the same area and usually through formation of a polyploid

organism.

Note: Allopatric Speciation is a long slow proce$s, and Sympatric

Speciation is a relatively much faster process

Example 2G Squirrels of Grand Canyon- South Rim: Hanis'Antelope Squirrel with GreyTail, White Belly

- North Rim: White-Tailed Antelope Squinel with White Tail, Grey

Belly- Process:

1. Geographical lsolation due to the Canyon

2. Founder Effect3. Different Selection Pressure, Natural Selection, Adaptation

4. Change of allelic frequencies

5. Accumulation of Sufficient RlMs and Genetic Diversity

6. Reproductively lncompatible7. Speciation Occuned.

Adaptive Radiation is the anolution of rnany divusely rdatdspec,'es from acomfiPn anc$tor

Example 21 : Darwin's Galapagos Finches- Derived from a small flock of finches ftom Central or South

America- Today, 13 species with similar size and coloration, but with

remarkable variation is the size and shape of their beaks

- Type of food on each island is different, exerts selection pressure

- By diversiEing, the species would stand a better chance of

survival within a diverse niche- Process:

1. lsland colonized by a small population

2. Founder Effect3. Different Selection Pressure, Natural Selection, Adaptation

based on the availability and type of food source

4. Change of allelic trequencies5. Accumulation of Sufflcient RlMs and Genetic Diversity

6. HeproductivelY lncomPatible7. Speciation Occuned.8. A few individuals reach neighboring island, process repeats

Sympatric Speciation: Autopolyploidy- Contains more than 2 chromosome sets, all derived from single

species- Non-disjunction creates diploid gamete

- Fertilized by haploid garnete = Autrotriploids

- Fertilized by diploid gamete = Autotetraploids (More Common)

- Can be induced experimentally by applying cold/heat shock or

' appllng Colchicine- lnterferes with spindle formation

- Autopolyploids often have flowerVfruits of greater size hence it

has greater horticultural or commercial value

Sympatric Speciation: Allopolyploidy- Contains multiple sets of chromosomes from two or more

species- Combination of alleles form tvvo different species with different

chromosome numbers- Reproductively isolated from both parents, as gametes have

different number of chromosomes- Significant in evolution of flowering plant species

- Fxtremely rapid speciation, a single generation required toproduce a new, reproductively isolated species

- Example 2l: Example of Allopolyploid: Bread Wheat- Hybrid of a wild Enkorn wheat and wild goat grass

Speciafon

Baological Species Goncept

Members of a biological species are being reproductively

compatibleEach species has a separate gene pool

Reproductive lsolation- Existence of barriers which impede reproductbn- Reproductive lsolating Mechanisms (BlMs)

- Pre4ygotic Baniers- Habitat lsolation, Temporal lsolation (different mating

seasons)- Behavioral lsolation- Physiological lsolatbn (lncompatible genitalia etc.)

- Postzygotic Baniers- Hybrid lnviability/ SterilitY

Phrlogenetic Sp€cies Concopt

Species is a set of organisms with a unique genetic history

Descended from a common ancestor

Morphobgical Species ConcePt

lf their anatomical traits appear to be very similarNote: difficult to analyze quantitative traits, and some species lookremarkably similar

Ecological Species Conoept

Views a species in terms of its ecological niche

Ecological Niche: Unique set of habitat resources that a specbsrequires, as well as its influence on the environment and other

species

clivenity onl Evalutian

Taxonomic CategorieDomainKngdomPhylumClassOrderFamilyGenus

Species

Beconslructing Pffogeny llsing Molecular HomologyMdqula that arcfunctionally similat in two diffqwlttypes of

organisms are considerd homologous if heir pimary squance issimilar.

1.

Genetic sequences change over the course of many generations

due to the accumulation of mutationsThe more the primary sequen@s of macromolecules in twospecies conespond, the more closely rdated they areThe number of difierences may reflect how much time haspasses since the species divergedExampb 22: Socftrome C- Part of ETC, found in Mitochondria for wery aerobic

eukaryote- Cytochrome C molecules in different species are

homologous- Comparison of the number of substitLltions that have

occurred per amino acid site

AdrantagesMolecular data is genetic. Protein and nucleic acid sequerrceshave a clear genetic basis that is easy to interpret.

Can be used with all organisms, proteins and nucleic acids can

be collected from any organismCan be applied to a huge amount of genetic variation, as there is

a large amount of data that can be assessed by molecular

methods. EG Human genome has over 3 billion base pairs.

All organisms can be compared as all organisms have certain

molecular traits in common, such as rRNA sequences and some

fundamental proteins, ofiering a common basis for comparison

Molecular data is quantifiable and precise, accurate. Facilitates

objective assessment.Provides information about the process of evolutionDatabase of information is large and growing

Helps us to understand phylogenetic relationships that can be

determined by non-molecular methodsAllows reconstruction of phylogeny among groups of present-

day prokaryotes and microorganisms for which we have no

fossil record at all

10. Different genes evolve at different rates, molecular trees can

represent short or long periods of time, depending on what gene

is used.

I{eutral Theory of Molecular EvolutionEvolutionary change at the molwular lard occurs pimarily through

nantral mutations whbh do not affect the phenowe of the organism

- Genetic variation is a result of mutation and genetic drift

- lf most of muiations are neutral, with no effect on fitness, then

the rate of molecular change should be regular like a clock- Molecular Clock

- Measures the number of mutations which accumulate in the

DNA sequrence of different species over time

- lf neutral mutations occur at a relatively constant rate, they

can act as a molecular clock on which to measure

wolutionary time.- More nucleotide differences = More distantly related

- 5 Key Principles- For each protein, the rate of evolution is approximately

constant with regard to neutral substitutions- Proteins that are functionally less important for suMval tend

to g/olve faster than more important proteins

- Amino acid substitutions that do not disrupt the existing

structure and furrction of a protein occur more frequently

- Gene duplication must always precede the emergence of a

gene haMng a new function- Selective elimination of deleterious mutations and random

fixation of neutral alldes occur far more frequently in

evolution than in Natural Selection

Why is the Population the smallest unil that can evolve?Natural selection acts on the indMdual with the environment as

the selection pressure, but allelic frequencies of the individual do

not changeRather: natural selection result in only better adapted individuals

to reach r@roductive maturity and pass on their alleles, and

along with gene flow and genetic drift, this leads to changes in

allelic frequencies of the population3.

4.

5.

6.7.8.

s.

Glassification Ptrylogeny

May or may not identiry Pattems ofevolutionary dationships among

species

ldentification of patterns ofevdutionary rdationships arprE

species

Naming System usedFliromial Nomendature

No Naming Sl6{ern used

Ataxon is aformd grouping oforganisms at any given levd

A clade inchdes an ancestral speciesand all of its descendants

May or may not emphasize on r€centcornmon ancestry

Emphaszes on recent @mmonancesw

No such visual representationRepresented visually by a

pMogenetictree

No n€sted hierarchY Nesred hierarchy

Based on analogous andhomoloqous structures

Based on homologpus structures

Neutrel TheoryVS Darwin's Theory of Natural Selection

t{eutral Theory Darwin

lvtost molecular differences betweenspecies are sdectively neJtral, no

effect on the fitness of the organismand hence unaffected bY natural

Sdecition

Variation within a population affectsfitness, as there are some who are

better adapted and hence are at thesdective advantage

Most evo{utionary change due togenetic drift acting on rreutral alleles.

Neriv alleles are intrcduced byspontarreous mutation, which areeither lost or fixed by genetic drift.

Over tirne, this resutts inaccumulation of these fted ;

mutations and hence evolution

Variation is a pre-condition for naturalsdection, arising spontaneously butDaMin did not give a reason forthis.

(No reference to mutations)

Evolution arises when better adaptedindividuals are sdected for, live toreproductive maturity and Pass on

the alleles to their offspring, such thalover time frequency of the favorable

allele increases.

H2 Biologylsolating, Cloning and Sequencing DNA

gSTEPS INVOTVED IN I'YY'YOGENE CLONING

I lsolation of DNA FragmentwithGOl

2 lnserf on of DNA lnto Ooning Ve<tor

1 . Cleaving Cloning Vector with RE (form linear DNA)

2. Cleave Gene Donor (with GOI) with same RE

3. Sticky-end€d fragments of @l incubated with linear vector,

allowing for annealing4. DNA Ligaseforms permanent phosphodiester bond to

produce recombinant plasmid

Possible ProduckRe.annealed PlasmidRe(ombinant Plasmid (with Gol)Recombinant Plasmid (without @l)

3 lntroduction of recomt{nant ve<toB lnto cultured bacterialhoet cells for ampllfication

H€atShodMethod. Cells made competent by pre'treating with ice-cold

cakium chloride solution. Ligation Mixture Added. Heat Shock (42degc for 2 minutes). Recovery of Cells by incubat'lon in norr-selective growth

medlumEhcroporation. Ligation Mixture Added. Electricityapplied in a pulse

. Disturbs the phospholipid bilayer, fotming temporaryaqueous pores

. Ehddc Potential across the membnne rises

. DNA driven across the membrane through porcs

1 Selection of Cells wlth Clonlng VectorAmpicillimesirtonce gene is outside of the ,iCS

Host Cells are plated onto nutdent agar plates containing theantibiotic Ampicillin

Case l r Ampidllln Sensltlve (Cel! Dhs)Do not contain amf gene, have not taken in plasmid

Case 2: Ampicillln Reriistant (Cell Survives)Possess am$ geneTaken up re annealed plasmid / recombinant plasmH

5 Selectioo of C-ells wiih Recomblnant PlasmidDqendent on phenomenon of insertional it,.dctitl4,tion

Replica Platlng for Antlbiotic SensitvltyTetacycline-resistonce gene is fannd within MG. Cells replica plated onto second agar plate containing

tetracycline. Re.annealed plasmid: o<press tetR gene, survives. Recombinant Plasmid: disrupted tetfigene, d'tes

. Cells with recombinant plasmid retrieved from masterplate

Nutridonal Requir€mentLad. g ene i s f d.r t1d w ith in MG

. Transformed Cells plated onto X-Gal Plates

. Cells with re-annealed plasmids express lacz gene, causingblue colonies

. Cells with recombinant plasmids have disruPted lacz gene,

causing white colonies. Can merge step 4 and 5 by plating directly on a plate

containing X-Gal and Ampidllin

6 Sele<ilon of Cdlswtth GOINucleic Acid Hybridization

. Coloniestransfered to nitrccellulosefilter

. C-ells lysed to release DNA, DNA denatured to gh/e single-stranded DNA

. Single.Stranded DNA lrybridized with a single'strandedradilra<tively labelled DNA/RNA probe. Probe nucleotide sequence complementary to that of

the gene of interest. Fiher subjected to autoradiognptry. Position of hybridization signal on autoradiog6ph

indicates il a colony contains the gene of interest

7 Scallng Up

APPI.ICATIONS OF GEI{E CLONING

Case Study: Human lnsulinlnsutin, synthesized by the Scetls of the islets of Langerhans in the

Panqeos, controlsthe latel of glucose in the blood,

characteristics that facilitate insulin production by recombinant

DNA techniques:. lnsulin not modified after translation by addition of sugar

molecules. Relatively small protein (21aa A Chain, 30aa B Chain)

Synthesis of lnsulin. Two artificial genes construction, one for the A chain(63

nucleotides) and one for the B chain(90 nucleotides). Each gene placed under the control of a strong lac promoter and

next to the lac Z gene (part of the p-galactosidase structural

genes). Both recombinant plasmids containing two artificial genes

transformed separately, amplifi ing the polypeptides produced

. Both genes expressed as fusion proteint consisting the first few

amino acids of B-galactosidase following by the polypeptide

. Each gene was designed such that the insulin and p-

galactosidase segments were separated by a methionine

residue, such that it can be cleaved by cyanogen bromide. Purified A and B chains mixed, reduced and re-oxidized to form

disulfide bonds present in native insulin

Gase Study Human Gtourth Hormone (Somatotrcphin)

Characteristics ofhGH. Produced by Anterior Pituitary Gland. Single polypeptide chain of 191 amino acid

. Gene found on chromosome 17

. Anabolic Effects. Height increase in childhood. lncreased muscle mas' decreased fat mass

. Growth of internal organs

. LackofhGH->dwarfism ;. Extracts from dead humans were limited, and were often

contaminated with prions

Synthesis. Preparation of the somatotrophin cDNA fragment

. mRNA obtained from pituitary gland

. cDNA, which has 1 RE site is digested by Haelll

. Longer segment, from codon 24to 191, is retained for use in

construction of the plasmid. Smaller segment replaced by artificial DN& which has correct

prokaryote translation signals (for translation in E. coli). Exprcssion

. Gene ligated into a plasmid carrying the lac promoter. . lntroduction into cells, selection and identification of cells

. Lactose induces expression, hormone secreted by bacterium

H2 Biologylsolating, Cloning and Sequencing DNA

gDescribe the polyrnerase chain reoction (PCR) and exploin the

odvantages ond limitations ofthis procedura

Reagents:. Thermal Cycler. DNATemplate

. Contains Sequence oflnterest. Thermostable DNA Polymerase -Taq Polymerase

. Stable at high temPerature

. Not denatured by repeated heat treatments in PCR Cycle

. DNAOligonucleotide Primers (Two Sets)

. Flank/ Mark out the region of interest

. One primer complementary to 3'end of target sequence on

one strand, the other primer complementary to 3'end ofother strand

. Free dNTPs in excess and equimolar quantities

. Reaction Buffer. Buffering Salts - Maintain pH

. Detergent - Prevent aggregation of enrymes

. MgCl2 - Cofactos for DNA Polymerase

DenaturationBrief Heat Treatment (30s)

94- 95 degc

Hydrogen bondsbroken, forms singlestranded DNA

AnnealingHybridiation

Cooling of DNA (1min)

5G60 degC

Primers anneal

specificallytocomplementarysequence

ExtensionOptimal Temperature forTaq Polymerase (72 dega)2 min

Synthesis ofdaughterstrand by polymerase

Ri n * and Re Peat 2G3O Ti nesNO. Of DNA MolXUleS - 2 Nuribqof rcRcv.t6

Advant ges1. Extremely Sensitive: Can Ampl'r! minute amounts of DNA

2. Speed (Rapid Cychs) and Easy to Use (Automated)

3. Robustness: Can Amplifi DNA that is badly degnded

Disadvanbges'1. Highly Sensitivity to Contamination by non-template DNA

2. Pr'or Sequence is needed for synthesis of primers

3. Taq Polymerase lack 3'- 5'exonuclease activity, resulting in

error rate of 1 in 1 0000 bases

4, DNA Polymerase only amplifr DNA Products up to a few

thousand base pairs

5. Cannot ampliflr protein

Explain how gel elearophotesis is used to analyze DNA

Gel Electrophottsis is a technique to separate charged molecules

by their differential nrtes of movement in an electric field.

. Current Applied through a semi solid porous gel matrix

. DNA has phosphate backbone, negatively charged, migrates

towards positive electrode. Molecular tieve to retard the movement of DNA for Size

Fractionation, as shorter DNA less impeded by pores

Steps1. Preparation of Gel, solidifcation of molten agarose

2. Use of Electrophoresis Buffer/ Electrophoresis Chamber

3. Loading of DNA Samples intoWells (NearCathode). LoadingDye(BromophenolBlue,XyleneCyanol). Makes DNA visible and easyfor loading, monitors rate of

DNA Migration, Glycerol increases density of DNA so they

sinkto the bottom ofthe well

4. Staining the gel with DNA Binding dye like methylene blue or

ethidium blue forvisualization of DNA Position

Outline the process of nucleic acid hybridimtion and explain how it can

be used to detect ond onolyze rcstriction frogment length

polymorphism

Nucteic Acid"Wbtidizotion is the process @ which two complementory

single-stronded nucleic ocid chains bose-poir and reform a double -

stranded helix

Characteristics of Probe:1. SingleStranded DNAoTRNA

2. Labelled: Radioactive, Fluorescent, Chemical Markers

Characteristics of Hybridization1 . Highly Sensiti\re, small amounts of complementary sequences

are needed

2. Highly Selective probes only bind to nucleic acids contain part

of or all ofthe complementary sequence

3. Stringency can be controlled by varying the incubation

temperature. Low Temp (210 degC) - Perftctly Complementary. Loh,cr Temp - Less Stringent Pairing

UsesofNAH1. Detect and quantiry specific nucleotide sequences

2. Compare nucleotide sequences

3. Study Gene Expression, Localize Genes of lnterest in cells etc

ONE:Southem Blotting4. DNASize Fractionated byGel Electrophoresis

5. DNA denatured by exposing the gel to alkaline conditions, can

occur before or during blotting6. Replica of DNA Bands formed by blotting the DNA on the gel

onto a nitrocellulose membrane7. Membrane incubated with labelled, single stranded probes

8. Probes will hybridize to complementary sequences on the DNA

g. Such DNA Molecules detected by autoradiography / chemkal /fluorescence (labelled probes appear as bands)

1. Autoradiography-> X-nyfilm placed over membrane

2. Fluorescence -> Fluorescent Microscopy

10. Size of DNA can be determihed by comparison with DNA

Markets that were electrophoresed alongside the sample

TIUO: I{ortlrem Blotting - RNA instead of DNA

THREE: ln situ HybridizationUses nucleic acid probes to detect specific nucleotide sequences i

situ, ie without the extraction of the DNA/RNA

For DNA, chromosomes must be denatured temporarily via high pH

For RNA, tissue must be fixed to retain RNA in an exposed form

FOUR Colony Hybridlzation (see ISG 1)

H2 Biologylsolating, Cloning and Sequencing DNA +

Restriction Fragment I'ength PolymorphismVailotions in the length and/or number of restriction frogments

generuted by digestion by rcstiction endotucleae, orising fran:l.SNpsaffead RESies

a. Single Nuclati de Ch on ges

b. oeateldelete RE sites

2.VNTRs baween RE Sites

a. Dtfference in number ofTandem Repeots between RE Sites

b. Altet the rclative locotion of RE Sites

RFIP Analysis - OnewiewDifferent individualVphenotypes have different RFLP Alleles, hence

digestion of DNA by the same RE will produce different banding

patterns after southern blotting, due to different number offragments or different lengths.

RFLPs are detectable as codominant allelet as the wild type allele

produces one type of banding pattern, and the mutant allele

produces a different pattern. Hence, heterozygous individuals will

show the banding of both alleles.

llse of RFI,P hrotysis for D/,rca* Dctecliotr (Sicldc Cefi Arsermia)

Only when mutations changing the allele to the mutarfi allele

creates or desroys a restriction enzyme recognition site

Case Sardy: Skkle{ell AnaemiaPoint mutation changing a nucleotide A for L changing Valine toGlutamate, eliminating a RE site for Ddel in the Bglobin gene

Digestion by Ddel will produce:

2CopiesofNormal Gene 201bp+ 175bp

2 Copies of Muant Gene 376bPHeterozygous 201bP+ 175bP+376bP

Use of RFIP Analysts for @nontk lilopplng (Linkqe filapping)

Aimed at mapping the chromosomal locus of a gene causing a

genetic disorder, as wetl as detecting for the predisposition of thegenetic disoder.

Requircs two gene loci, one disease causing and one genetic

marker, which are tightly linked (aka close on the same

chromosome) that they are inherited together. Suitable RFLP loci

can be determined by studying patterns of co-segregation of the

disease trait and the RFLP allele in an affected family

Therefore, the RFLP is said to @-*$fglotc with the genetic disorder,

thot tlle inheritance of the disease can be correlafud with the

inheitance of the genaic mo*er. lndividuols who inherit the genetic

mo*er hwe a high chonge of having the disorder.

Note: Absolute Llnkage not possible unless genetic marker is within

the gene, and thus a genetic marker only has a statistically

significant correlation with the disease, and is not absolute.

ll* of RFIP Anatyis lor DNA Fingeryi* UNIR Atrolysis)

Number of copies of repeats in a give VNTR locus is highly variable

and hence the locus is highly polymorphic, ie many loci have 5Gr

alleles each.

Analysis of multiple VNTR loci generate a unique DNA fingerprintdue to the large number of possible combinations, which is specific

to the individual. This s done by using REs to cleave at RE sites

flanking the VNTR locut hence size of remaining fragments varies

based on the number oftandem repeats.

Usage in Paternity Testing:. A child will inherit oneVNTR allele from each parent

. Hence, any bands found in the child after southern blotting

must correspond to at least one parent

Note:. Large sample of DNA required. DNA must be intact and non-degraded

Discuss the gools and implications of the human genome Woiect,

inctuding the benefits and difficult ahical concernsfor humans.

Goals:. ldentifo allthe human genes

. Construct a detailed physical map ofthe entire human genome'

with all the locations of the gene loci. Determine the nucleotide sequence of all the 24 human

chromosomes. Storethis information in databases. lmprove tools for data analYsis

Benef,ts:. Pharmecogenomics

. Genetic differences affect how we react to dru95

. Knowledge of genetic code of an individual may allow

doctors to prescribe the right drugs at the right doses for

maximum effectiveness. Concerns:

. Privacy and Confidentiality of Genetic Information

. Fairness in the use and interpretation of genetic

information. RiskAssessment

. Detect genetic predispositiontocertaindiseases

. Earlypreventionorcureofthesediseases

. Concerns:. Psychological impact and stigmatization. Faimess in the use and interpretation of genetic

information. lnsights through Comparative Genomics

. Compare the genome of different organisms to identiry

genetic similarities and diffierences. lnsightsongenediscovery. Development of animal models for human diseases

. EvolutionaryRelationshiPs. Concerns

. Animal Rights

OdrerEdrical Concetrrs. Commercialiation and ownership of products due to patents

. Withdraw accessibility to data and materials

OwnershiP of Genes. Philosophical lmplications: FreeWill vs Genetic Determinism

. Whether a person's genes will determine the future of theperson, or whether he or she has control over his or her

future. Reproductivelssues

. Use of genetic information in reproductive decision making

- Selection offutusesbygenetictesting

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