Biology: Genetics & Inheritance III: Genetic Basis For Variation I

Genetic Basis for Variation I Genetic Basis for Variation II M
olecular Biology Techniques
Biology: Genetics & Inheritance III

Genetic
Basis for Variation I

Common Terms
Homologous A diploid organism has 2 sets of chromo., 1 set from each parent. HC are
Chromosome similar in size, shape, centromere position, and staining pattern; they have the
same genes that determine a certain trait at corresponding loci.
Locus Position of a gene on a c

hromosome; alleles of same gene occupy same locus.
Gene An ordered sequence of DNA nucleotides located at a particular locus, on a

particular chromo., that encodes a specific functional product (protein, RNA).
Allele 1 of a no. of alternative forms of a gene that possesses a unique nucleotide

sequence → Only 1 allele can occur at a given locus. Differences in nucleotide
sequences of each allele, when translated, may produce a different gene
product → Different observed traits.
Homozygous Diploid condition where alleles at a g

iven locus on a pair of HC are i dentical.
Heterozygous Diploid condition where alleles at a g

iven locus on a pair of HC are d
ifferent.
Genotype Combination of alleles located on HC at corresponding loci.
Phenotype Characteristic arising from i nteraction b/w g

enotype and e
nvironment.
Dominant Allele that shows its phenotype regardless of whether it is homozygous

Allele dominant or heterozygous → 1 copy sufficient for its expression in phenotype.
Recessive Allele that shows its phenotype only if homozygous recessive → 2 copies
Allele needed for its expression in phenotype.
Codominance 2 different dominant alleles of the same gene are equally expressed in
heterozygote’s phenotype, so it is d
ifferent from those of either homozygote’s
→> 2 possible phenotypes.
Incomplete Heterozygote’s phenotype is i ntermediate between those of both

Dominance homozygotes’.
F1 Generation Offspring produced by crossing parental generation.
By Jermaine Wong (2020)

F2 Generation Offspring produced by crossing 2 F1 organisms.
Self- Crossing of an organism with itself (plants → can produce pure line over many
Fertilization generations).
Test Cross Crossing b/w an individual of unknown genotype/heterozygote and a

homozygous recessive organism (tester).
Pure Breed Organism is homozygous for traits concerned.
Wild Type Typical genotype found in a natural population of that species.
Linkage ≥2 genes on same chromosome that do not assort independently in meiosis →

Inherited together (further apart = more likely to undergo crossing over)

Labelling Alleles
- Add apostrophe to lowercase letter to distinguish [e.g. C and c’ / W and w’]
- Multiple Alleles: Give gene uppercase letter and each allele uppercase (dominant)
or lowercase (recessive) superscript [e.g. IA IB Io → small ‘o’ as recessive]
- Same for codominant alleles
- Sex-Linked [e.g. hemophilia: XHXH/XHXh/XhXh, XHY/XhY]

Genes & the Environment
- Phenotypic variation may result from interaction b/w genotype and environment
- E.g. phenylketonuria (PKU) is an autosomal recessive genetic disorder
characterized by deficiency in enzyme phenylalanine hydroxylase, needed
to metabolize phenylalanine to tyrosine → Phenylalanine accumulates and
is converted into a toxic substance
- Can be controlled by diet (i.e. diet low in phenylalanine)
- Environmental effect is usually greater on polygenes
- Environment may induce mutations affecting phenotype (e.g. overexposure to UV
light may cause chromosomal damage that leads to skin cancer)
Temperature
- E.g. coat color in Himalayan rabbits (cool → black fur), sex determination in Nile
crocodile hatchlings, type of wings in fruit flies (hot → vestigial wings can grow as
long as long wings)
UV Light
- E.g. human skin contains melanocytes, cells that produce more melanin when
exposed to UV light
Photoperiod
- E.g. wavelength of light and duration of light exposure affect seed germination
Food/Nutrition
- E.g. larvae of honey bees fed with royal jelly become queen bees, while females
fed with worker jelly become worker bees


Monohybrid Crosses
- Inheritance of o ne characteristic controlled by one gene
- Hybridization: Crossing of two varieties
- Pure-breeding (homozygous recessive) parents are the parental generation, and
their hybrid offspring are the F1 (first filial) generation; self-pollination of the F1
generation produces the F2 (second filial) g eneration
- Monohybrid Ratio: 3:1 F2 phenotypic ratio
Mendel’s First Law: Law of Segregation
- Separation of a pair of parental factors (genes) during
meiosis, so that 1 factor (gene) is present in 1 gamete
- States that two alleles for one characteristic separate
during formation of gametes → Parent with two copies of each allele can pass on
either allele → Characteristic not evident in parent can appear in F1 generation
Genetic Diagram
- Only include a key/legend if question does not provide it

- All gametes need to be circled
- Phenotypes need to be linked to genotypes in Punnett square + ratio outcomes
Test Cross
- Reveals genotype (homozygous dominant AA / heterozygous Aa) of an organism
that exhibits the dominant trait
- Organism is crossed with individual expressing homozygous recessive (aa) trait
- F1 phenotypic ratio is a
ll dominant phenotype → Homozygous dominant
- F1 phenotypic ratio is 1 :1 → Heterozygous

Important Ratios in Complete Dominance Monohybrid Cross

- Aa x Aa → 3:1
- Aa x aa → 1:1
- AA x aa → All dominant phenotype

Dihybrid Crosses
- Inheritance of t wo different characteristics controlled by two different genes
- Dihybrid Ratio: 9 :3:3:1 F2 phenotypic ratio
- Alleles of each characteristic found on different chromosomes (unlinked
genes): Whether A/a is inherited is independent of whether B/b is inherited
Mendel’s Second Law: Law of Independent Assortment
- States that the alleles of different genes on different pairs of chromosomes assort
independently of each other because…
- Homologous pairs of chromosomes align randomly on either side of
metaphase plate during metaphase I
- Alignment of each homologous pair of chromosomes is independent of
other homologous pairs of chromosomes
Genetic Diagram



a.
- Stem Color: Allele for purple stem → From cross (ii), purple stem x green stem results in all
offspring with purple stems, so allele for green stem p’ inherited from parent has been
masked by allele for purple stem P
- Leaf Type: Allele for cut leaves → From cross (i), cut leaves x potato leaves results in all
offspring with cut leaves, so allele for potato leaves c’ inherited from parent has been
masked by allele for cut leaves C
*b.
- Analyze phenotypic ratio of each character in a dihybrid cross separately using known
monohybrid cross ratios

Test Cross
- Organism is crossed with individual expressing homozygous recessive traits for
both characters (aabb)
- Offspring phenotypic ratio is 1 :1:1:1 → Heterozygous (AaBb x aabb)
Important Ratios in Complete Dominance Dihybrid Cross
- AaBb x AaBb → 9:3:3:1
- Linked genes with no crossing over: Alleles for two characteristics do not
segregate independently → 3:1 F2 phenotypic ratio (same as monohybrid)
- AaBb x aabb → 1:1:1:1

Multiple Alleles
-
Condition where a single characteristic appears in several different forms as it is
controlled by 3
or more a lleles
- E.g. Rabbit Coat Color: C is dominant to cch > ch > c
Incomplete Dominance
- Heterozygote’s phenotype is i ntermediate between those of both homozygotes’
- E.g. Flower Color in Snapdragons → Red: CRCR, White: CWCW, Pink: CRCW


Codominance
- 2 different dominant alleles of same gene equally expressed → Both phenotypes
are equally expressed in heterozygote → Different phenotype from those of either
homozygote’s (no intermediate) → >2 possible phenotypes
- E.g. MN Blood Group → Type M: LMLM, Type N: LNLN, Type MN: LMLN
- E.g. ABO Blood Group: IA and IB are codominant (equally expressed in phenotype),
and dominant to Io → Type A: IAIA, IAIo; Type B: IBIB, IBIo; Type AB: IAIB; Type O: IoIo
- AB phenotype is not intermediate between A and B phenotype: Individuals
with AB phenotype have both A and B antigens on surface of their RBCs
- Monohybrid Ratio (2 heterozygotes): 1:2:1 ratio (e.g. 1 IAIA : 2 IAIB : 1 IBIB)
Lethal Alleles
- Causes death if organism is homozygous for that allele; organisms carrying lethal
alleles have impaired biochemical or physical functioning
- E.g. In chickens, the dominant allele C is responsible for profound developmental
changes that result in aberrant ‘creeper’ forms with short, crooked legs
- Homozygous dominant genotype CC is lethal

- 2:1 ratio replaces 3:1 ratio as embryos with the homozygous dominant genotype
CC die → 1 part less
- Deviation from standard Mendelian ratios (NOT 4/16 parts) indicates that gene of
interest may be a lethal gene

Pedigree Analysis
- Pedigree: Diagram that describes the interrelationships of parents and children
across generations → Squares represent males and circles represent females
- Analyzes pattern of inheritance of a specific genetic trait → Shaded shapes
- Determines mode of inheritance (dominant, recessive, etc.) of genetic diseases

Linked Genes: Autosomal Linkage

- Linked Genes: Situated on the same chromosome → Alleles __ and __ tend to
segregate together, while alleles __ and __ tend to segregate together
- The closer the gene loci of 2 genes on the same chromosome, the higher
the chance that their alleles will be inherited together as 1 linkage group
(further gene loci, higher chance of crossing over)
- Do not usually undergo independent assortment + random segregation
→N O expected 9:3:3:1 / 1:1:1:1 ratio → Variety of ratios
- E.g. In fruit fly Drosophila, genes for body color and wing length are linked; if the
genes are completely linked (i.e. no crossing over takes place between them as
they are very close to one another), a 3:1 ratio is obtained from GgLl x GgLl
- Rarely achieved as crossing over may occur → Recombinant phenotypes
Crossing Over
- E.g. In D rosophila, GgLl (grey body, long wings) x ggll (black body vestigial wings)
- Completely Linked Genes: 1:1 ratio → 1 grey, long : 1 black, vestigial
- Crossing Over by Chance: 1:1:1:1 ratio → 1 grey, long : 1 black, vestigial : 1
grey, vestigial : 1 black, long
- Greater frequency of parental than recombinant phenotypes as crossing over is
a chance event determined by distance between gene loci of 2 genes [QD] *look at
individual allele for each characteristic → link alleles together
- 2 parental phenotypes have approx. equal numbers, while 2 recombinant
phenotypes have approx. equal numbers
- Number of recombinants depends on proximity of linked genes

How Recombinants are Formed
1. During prophase I of meiosis I, crossing over occurs b/w non-sister chromatids of
HC to form r ecombinants
2. At chiasma, portion of chromatid containing allele __ breaks and rejoins portion of
chromatid containing allele __
3. New linkage group where allele __ and allele __ are linked on same chromosome
4. Gamete with chromosome containing allele __ linked to allele __ fuses with
gamete with chromosome containing allele __ linked to allele __
➢ The further the gene loci of 2 genes on the same chromosome, the higher the
chance of c rossing over occurring → Higher % of recombinants


Genetic Diagram
*Draw ‘stick’ diagrams + Circle gametes
Let: G represent allele for grey body (dominant)
g represent allele for black body (recessive)
L represent allele for long wings (dominant)
l represent allele for vestigial wings (recessive)

[1]

[2]

[2] for correct
genotype linked
to correct
phenotype

Calculate %
Label ‘parental’
and ‘recombinant’
- Larger number of parental phenotypes than recombinant phenotypes indicates

that parental genes are linked → G and L are on the same chromosome and g
and l are on the same chromosome

Linked Genes: Sex Linkage
- Human Cells: 22 pairs of autosomal chromosomes + 1 pair of sex chromosomes
- Females (XX): H omogametic; Males (XY): Heterogametic
- vs. Birds → Females (ZW): Heterogametic; Males (ZZ): Homogametic
- Sex Linkage: Genes carried on sex chromosomes that result in a characteristic
being expressed mainly in 1 sex
- In humans, genes are usually carried on X chromosome
→ X-linkage
- Y chromosome is smaller than X chromosome
- Heterogametic chromosomes: Portion of X chromosome
has no homologous region on Y chromosome
- Characteristics determined by genes carried on non-homologous portion of X
chromosome appears in males even if they are recessive (no dominant allele to
mask the effect of recessive allele)
- Male offspring obtain these characteristics from their m others only
- Female offspring obtain these characteristics from b oth parents
H h B b
- E.g. Hemophilia (X , X ), Red-green color blindness (X , X ) → Sex-linked r ecessive

Genetic Diagram (X-linked dominant/recessive allele for)

- Recessive gene causing hemophilia/color blindness is exchanged from 1 sex to
another at each generation (e.g. father → carrier daughters → hemophilic sons)
- Hemophilic/color blind females only arise from a cross between a hemophilic/color
arrier female and a hemophilic/color blind male (e.g. XHXh x XhY)
blind c
Reciprocal Cross
- Determines if a particular characteristic is sex-linked → 2 separate crosses are
conducted using the s ame c haracteristic, with the s
exes reversed
- Different phenotypic ratios for reciprocal crosses shows that characteristic is
sex-linked (if not sex-linked, results will be the same)


Explain why heterozygotes for PKU appear normal at birth. [ 2]

- Heterozygotes have the normal/dominant allele → Cell can synthesize sufficient levels of
the enzyme phenylalanine hydroxylase (from question stem) → Level of phenylalanine is
not high enough to cause disease
Suggest societal advantages of mass screening for PKU. [2]
- Prevent irreversible brain damage in PKU sufferers (from question stem)
- Reduce cost of care for PKU sufferers if treated earlier / Provide counseling for parents

Suggest how similar breeding experiments with many different pairs of characters can be used to
map the position of genes on the chromosomes of fruit flies. [3]
- Recombinant Freq. = total no. of recombinant offsprings/total no. of offsprings x100%
- The g reater the % of r ecombinants, the g
reater the d
istance between genes
- Distance between linked genes is expressed in map units, where 1 map unit is equivalent
to 1
% recombinant frequency
- If expected phenotypic ratio, such as 1:1:1:1, is obtained when double heterozygotes are
test crossed, there is n o linkage


Explain why heterozygous plants for Tt gene have the same phenotype as homozygous dominant

plants. [3]
- T allele codes for a f unctional enzyme + t allele codes for the non-functional enzyme
- In a h eterozygote, exp. of dominant T allele m asks phenotypic effect of recessive allele t
- Expression of 1 T allele is sufficient to produce functional enzyme for plants to grow tall

Suggest why the IA and IB alleles are dominant over the io allele. [3]
- Expression of dominant IA or IB allele m asks phenotypic effect of r ecessive io allele
- Presence of recessive i allele does n
o
ot lead to any production of antigens
- Presence of dominant IA / IB allele leads to production of A / B antigens respectively
on surface of red blood cells
- Heterozygotes with IAi / IBi genotypes have A / B antigens respectively on surface of their
red blood cells

Suggest an explanation for the tortoiseshell coat (XBXO) in terms of activity of X chromosome.
- X-inactivation is random at early development → Produces a mosaic of black and orange
coat to give tortoiseshell coat in females [not in syllabus]

Genetic Basis for Variation II

Discontinuous Variation
- There are distinct and discrete phenotypes, generally caused by different alleles
of a single/few genes (e.g. ABO blood groups, color and texture of peas)
- Phenotypic expression is usually u naffected by environmental factors
Continuous Variation
- Phenotypic d ifferences vary along a continuum (no distinct groupings)
- There is a range of phenotypes, caused by polygenic inheritance, where multiple
genes are involved (e.g. height, skin color)
- There is an additive effect, where each gene has a small overall effect on single
phenotypic character
- Results in normal distribution curve, where intermediate phenotypes are more
common than extreme ones
- Phenotypes are affected by e nvironmental factors



Epistasis
- Definition: Form of gene interaction where a gene at one locus alters phenotypic
expression of a gene at another locus
- Two genes interact with one another to influence one trait (different from
dihybrid cross, where 2 genes code for 2 separate traits)
- Gene whose phenotype is expressed is epistatic; gene whose phenotype
is masked is h ypostatic (e.g. gene A is epistatic to gene B)
- Epistasis is interaction between alleles at different gene loci; dominance is
interaction between alleles at s ame gene locus
- Recessive Epistasis: Recessive allele of one gene, in homozygous condition,
masks phenotypic effect of alleles of the other gene
- Dominant Epistasis: Dominant allele of one gene, in homozygous or heterozygous
condition, masks phenotypic effect of alleles of the other gene

Example 1: Comb Types in Chicken (9:3:3:1 ratio)
- A single trait, comb type is determined by two genes → Gene interaction
- R (rose comb) is dominant to r, P (pea comb) is dominant to p’, R and P are
codominant, rrp’p’ gives single comb

Replace with
F1 phenotype __ x __
F1 genotype __ x __
Meiosis
Gametes (n) O O O O


Example 2: Flower Color in Sweet Pea (9:7 ratio)

Replace with
F1 genotype __ x __
Meiosis
Gametes (n) O O O O

Biochemical Pathway: Recessive Epistasis
- C_ genotype codes for functional enzyme C, which converts colorless precursor
into colorless intermediate → Absent in homozygous recessive c’c’ genotype
- P_ genotype codes for functional enzyme P, which converts colorless
intermediate into purple pigment → Absent in homozygous p’p’ genotype
- When either functional enzyme C or P is absent, purple pigment is not produced,
so flower remains white
➢ c’c’ genotype is epistatic to P/p’ locus
➢ p’p’ genotype is epistatic to C/c’ locus


Example 3: Coat Color in Labrador Retrievers (9:3:4 ratio)

Replace with
F1 genotype __ x __
Meiosis
Gametes (n) O O O O

Biochemical Pathway: Recessive Epistasis
- B_ genotype codes for functional e nzyme B, which produces b lack pigment
- bb genotype codes for functional e nzyme b, which produces b rown pigment
- C_ genotype allows for color deposition on coat → No color deposition in c’c’
genotype → G old phenotype
➢ c’c’ genotype is epistatic to B/b locus


Example 4: Kernel Color in Wheat (15:1 ratio)

Replace with
F1 genotype __ x __
Meiosis
Gametes (n) O O O O

Biochemical Pathway: Duplicate Dominant Epistasis
- A_ or B_ genotype codes for functional enzyme A or B, which converts colorless
precursor to colored pigment
➢ A_ genotype is epistatic to B/b locus
➢ B_ genotype is epistatic to A/a locus


Example 5: Fruit Color in Squash (12:3:1 ratio)

Replace with
F1 genotype __ x __
Meiosis
Gametes (n) O O O O


Biochemical Pathway: Dominant Epistasis

- W_ genotype codes for inhibitor W, which inhibits conversion of colorless
precursor into y
ellow intermediate → White intermediate
- G_ genotype codes for inhibitor G, which inhibits conversion of white or yellow
intermediate into green product → White or yellow product
➢ W_ genotype is epistatic to G/g locus


Example 6: Feather Color in White Leghorn Chicken (13:3 ratio)

Replace with
F1 genotype __ x __
Meiosis
Gametes (n) O O O O

Biochemical Pathway: Dominant Epistasis
- C_ genotype codes for functional enzyme C, which converts colorless precursor
into colored pigment → Colored feathers
- I_ genotype codes for inhibitor I, which inhibits conversion of colorless precursor
into colored pigment → White feathers
➢ I_ genotype is epistatic to C/c’ locus


How to Solve Epistasis Question

1. Identify if it is epistasis
a. Two genes influence one trait (may have 2 or 3 phenotypes)
b. Modified 9:3:3:1 ratio when two heterozygotes are crossed
2. Match genotypes to corresponding phenotypes in Punnett square
3. Figure out which gene locus (genotype) is epistatic to which gene locus

Chi-Square Test
- Used when sample results need to be compared to expected results to determine
if any difference is due to chance alone or some other factor

1. List expected phenotypic ratio
- The expected phenotypic ratio of F1 generation test-crossed is…
2. State null hypothesis (H0)
- H0: There is no significant difference between observed and expected results →
Any difference is due to chance a lone
3. Calculate 𝝌2 value
- Total number of offspring = __
- Expected f requency per unit = __
F1/Offspring Phenotype Observed Freq. (O) Expected Freq. (E) (O-E)2 / E
calc. to 2 d.p.
df = Σ = 𝝌2 =
4. Determine degrees of freedom (df)

- df = n – 1 = __
5. Determine probability (p) using 𝝌2 and df value to check against 𝝌2 table
- 𝝌2 = __ df = __
p > 0.10 / p < 0.001 / __ < p < __
6. Reject or do not reject H0
- Reject: Since p __, at 5% level of significance, p < 0.05 → We reject the null
hypothesis → There is significant difference between observed and expected
results → Any d ifference is n ot due to chance alone, but some other factor
- Probability is very low that any difference is due to chance alone
- Do not Reject: Since p __, at 5% level of significance, p > 0.05 → We do not
reject the null hypothesis → There is no significant difference between observed
and e xpected results → Any d ifference is due to chance a lone
- Probability is very high that any difference is due to chance alone

T Test
- NOT necessarily related to genetics → Can come out for any question in syllabus
- Used to compare m ean of two unpaired samples
- Suitable for s
mall sample size + assumes samples follow normal distribution

- Standard Deviation (s): Measure of s pread of values within data set

- s1 and s2 are used to calculate t value

1. State null hypothesis (H0)
- H0: There is no significant difference between means of two samples → Any
difference is due to chance alone
2. Calculate x
e.g.
3. Calculate Σ (x − x)2
e.g.
4. Determine no. of observations (n1 and n2)
- n1 = __ ( e.g. n1 for males = 4)
- n2 = __ ( e.g. n2 for females = 4)
5. Calculate standard deviation (s1 and s 2 )
- s1 = __ (e.g. s 1 for males =
√ = 5.80)
101
3
- s2 = __ (e.g. s 2 for females =

√ = 5)
75
3
6. Determine degrees of freedom (v)

- v = n1 + n2 – 2 =
__ (e.g. v = 4 + 4 – 2 = 6)
7. Calculate t value
|173.5 − 165.5|
- t = __ ( e.g. t = = 2.09
5.80 2 5 2
√ 4
+4
8. Determine probability (p) using v and t value to check against t distribution table
- v (df) = __ t = __
- p > 0.10 / p < 0.001 / __ < p < __
9. Reject or do not reject H0
- Reject: Since p __, at 5% level of significance, p < 0.05 → We reject the null
hypothesis → There is significant difference between means of two samples →
Any difference is n ot due to c hance alone, but some other factor
- Do not Reject: Since p __, at 5% level of significance, p > 0.05 → We do not
reject the null hypothesis → There is no significant difference between means of
two samples → Any d ifference is due to c hance alone


𝝌2 vs. T Test

Explain the likely genetic basis of resistance in each of these species. [5]
- A: Resistance is determined by complete dominance involving 2 alleles at 1 gene locus,
where 1 allele confers pesticide resistance while the o ther does not
- Homozygous dominant and heterozygous genotypes show 1 type of resistance,
while homozygous recessive genotype shows the other type of resistance
- B: Resistance is determined by incomplete dominance involving 2 alleles at 1 gene locus
- Heterozygote shows intermediate resistance, which is a third phenotype
- In A and B, there are 2 and 3 distinct phenotypes respectively, which indicate
discontinuous variation
- In C, there is a continuum of pesticide resistance on a bell-shaped curve, which indicates
continuous variation
- C: There is polygenic inheritance with multiple genes involved → Additive effect where
different alleles of each gene have small overall effect on pesticide resistance

**Explain the term epistasis in this context. → Describe biochemical pathway
- Allele __ codes for functional enzyme/inhibitor __, which…
- Allele __ codes for functional enzyme/inhibitor __, which…
- __ genotype is epistatic to __ locus
- Phenotype that shows epistasis (e.g. aa genotype to B/b locus: lack of functional enzyme
A results in white flowers despite presence of functional enzyme B)

Suggest why pea plants are good experimental organisms for carrying out such crosses. [2]
- Pea plants have many observable traits with contrasting forms, e.g. round vs wrinkled
seeds + produce many offspring in one cross

Molecular Biology Techniques

Polymerase Chain Reaction (PCR)
- Allows specific DNA segment to be a mplified i n vitro (test tube)
Structure of DNA
- DNA strands denature and separate at high temperatures as hydrogen bonds
between complementary bases are broken
- DNA strands reanneal through complementary base pairing upon cooling
DNA Replication
- DNA p rimers provide free 3’OH group for DNA polymerase to extend chain
Components of Reaction Mixture for PCR
DNA containing target DNA seq. to be amplified; usually genomic /
Template DNA
fragmented DNA or plasmid
Synthetic s ingle-stranded DNA primers (20-30 nucleotides) a

nneal via
CBP to specific 3’ ends of ss target DNA seq. to initiate DNA synthesis
2 different primers each have seq. complementary to seq. flanking
target region → Determine region to be amplified
DNA primers provide f ree 3’OH group for chain extension by T aq
polymerase
Oligonucleotide DNA
Primers

- Knowledge of seq. flanking DNA segment needed to artificially
synthesize DNA primers
- DNA primers in large excess to increase chance of binding to
target DNA (if not, template strands reanneal to each other)
- DNA primers become part of amplified segments
Thermostable DNA polymerase isolated from thermophilic prokaryotes

in hot springs; stable at 95ºC, functions optimally at 72ºC
Taq Polymerase
- Vs. normal DNA polymerase: Denatured at high temperature + need to
be replaced after each cycle → T aq allows PCR to be fully automated
Deoxyribonucleoside dATP, dTTP, dCTP and dGTP are substrates for DNA replication
Triphosphates (dNTPs)
Buffer cont. Mg2+ Cofactor for proper DNA polymerase function


Procedure
1. Place all components into PCR tube. Place tube into thermocycler. Run a standard
program that heats tube to different temperatures for different periods of time
2. PCR: The following 3 stages constitute 1 cycle, which is then repeated (25-30x)
Brief heat treatment of up to 9 5ºC to d
enature and s
eparate ds DNA into ss
Denaturation DNA by b reaking H bonds b/w complementary bases of each strand →
Exposes bases for complementary base pairing
Cooling of DNA at 6 4ºC for e xcess DNA primers to anneal via complementary
Primer
base pairing to specific 3
’ ends of each s s target DNA seq. (flanking target
Annealing
region to be amplified) → Determine region to be amp
Taq polymerase synthesizes c omplementary DNA strand by catalyzing

Extension formation of phosphodiester bonds between dNTPs at 72ºC; e xtension occurs
via C
BP from 3’ end of DNA p
rimer, which provides f ree 3’OH group
*QD from information given in question as temperature may be d ifferent!
Results from PCR
- Each cycle doubles no. of DNA molecules being replicated, where n cycles yield 2n
DNA molecules → Increases exponentially ( e.g. after 25 cycles, 226 copies of ori DNA)
- 25-30 cycles are usually run → Fewer: Not enough DNA for analysis; More: Too
many mistakes in rep.

No. of copies of ss DNA = 2n+1

Advantages of PCR
- Amplifies limited amount of source DNA so that there is sufficient amount of DNA
for analysis (if limited DNA, bands may not be visible)
- Taq polymerase is thermostable and allows PCR to be automated and fast
Applications of PCR


Limitations of PCR
- Taq polymerase lacks 3’ to 5’ proofreading ability: Errors occurring early in PCR
are compounded with e ach replication cycle
- Synthesis of DNA primers requires info. on sequences flanking target region to
be a mplified [if not, no amplification/wrong DNA fragments amplified]
- Size limit of DNA fragments to be amplified (~3kb2) [Taq polymerase tends to ‘fall
off’ DNA template strand before chain extension is complete if too long]
- Genomic DNA too large to be amplified entirely by PCR
- Exponential amplification of minute amounts of contaminant DNA to significant
amounts affects reliability of results

Gel Electrophoresis
- Separates mixtures of DNA/RNA/proteins according to molecular size
- Agarose gel electrophoresis separates DNA according to size
- Agarose: Polysaccharide derived from seaweed that forms gel when dissolved in heated
aqueous solution → The higher the conc. of agarose, the better the resolution of the gel
Procedure
1. Buffer Solution: Contains i ons that conduct electricity to generate electric field
2. Loading Dye: Dense loading dye mixed with DNA sample contains
- Glycerol to help it sink to bottom of w ells located near negative electrode
- 2 different-colored dyes to color invisible DNA sample and indicate if DNA
has been l oaded correctly into well
- Act as v isual markers for migration of DNA fragments in gel
- Dark blue dye (corresponds to 100bp) runs ahead of DNA sample
to indicate when to stop electrophoresis so samples x run out of gel
- Light blue dye (corresponds to 1100bp) indicates position of larger
DNA fragments on gel
3. DNA Ladder: Mixture of DNA fragments of known sizes that act as a standard to
compare with DNA fragments of unknown sizes in sample and estimate their size
4. Negatively-charged DNA moves from - ve to + ve electrode in electric field
5. Meshwork of polymer fibers impedes movement of longer DNA fragments more
than shorter ones → Migrate s lower and end up nearer to the w ell
6. Ethidium Bromide (carcinogen): DNA-binding dye to visualize D NA under U V light
Purpose
- Estimates size of DNA fragments by comparing position of band relative to bands
of DNA ladder
- Estimates amount of DNA by comparing intensity at which band fluoresces +
thickness of band (not accurate!)
Applications
- Analyzes DNA fragments (e.g. restriction fragments for RFLP analysis)


Southern Blotting
- Usually carried out after gel electrophoresis to detect and confirm DNA fragments
containing specific nucleotide sequences
➢ Alkaline solution d enatures d
s DNA fragments in gel into s s DNA
- Paper towels draw alkaline solution upwards through gel towards themselves via
capillary action
➢ Ss DNA in gel is drawn upwards and binds to nitrocellulose membrane in same
position as they were in gel
➢ Nitrocellulose membrane is incubated with radioactive, ss DNA probe with
specific sequence that is complementary to part of target DNA sequence
➢ DNA fragments containing target DNA sequence will hybridize to probe by
complementary base pairing (where A forms 2 H bonds with T…)
➢ Nitrocellulose membrane is w ashed to remove any u nhybridized probes
➢ X-ray film is placed over nitrocellulose membrane to visualize banding pattern via
autoradiography
- Radioactivity of bound probes exposes film to form image corresponding
to bands that have base-paired to probes

Restriction Enzymes
➢ Recognizes and binds to specific nucleotide seq. called restriction sites as its
active site is c omplementary to the nucleotide sequence
- Most restriction sites are 4-6 nucleotides long + palindromic
➢ Breaks p hosphodiester b onds b/w nucleotides on b oth DNA strands to give…
- Sticky Ends: Restriction enzymes make staggered cut → Ss overhangs
- Blunt Ends: Restriction enzymes make simple cut at single point
Natural Function
➢ Used as defense mechanism by bacteria against bacteriophage → Cleave foreign
DNA into non-infective fragments
- Bacterium’s own DNA is not cleaved as it is methylated at restriction sites that
are recognized by its own restriction enzyme
- Methylation: Enzyme adds methyl groups to A or C residues in DNA seq. at
restriction site → X complementary in conformation and charge to restriction EAS
→ Restriction enzyme x recognize and cleave DNA

Restriction Fragment Length Polymorphism (RFLP)
- DNA Polymorphism: Differences in DNA sequences b/w homologous (non-coding)
regions of different individuals
- RFLPs are caused by single nucleotide polymorphisms (SNPs) with difference in
single base pair, or variable number tandem repeats (VNTRs) in homologous
regions of different individuals


Application: Disease Detection (e.g. SNP in sickle-cell anemia)

- Sickle-cell anaemia is caused by single base substitution in gene that codes for
β-globin chain of haemoglobin → S ickle-shaped RBC
- Point mutation at MstII restriction site in β-globin gene for HbS → No longer
recognised and cleaved by s ame restriction enzyme, MstII
- RFLP analysis can be used to infer genotype of individuals
Procedure
1. Extract g
enomic D
NA from individuals.
2. Amplify via P
CR with p
rimers complementary to seq. flanking target region. (if x enough)
3. Digest with same r estriction enzyme. (VNTR: cuts on either side of tandem repeat locus)
4. Run g
el electrophoresis to separate DNA fragments according to size.
- Negatively-charged DNA moves from - ve to +ve electrode in electric field
- Meshwork of polymer fibers impedes movement of longer DNA fragments more than
shorter ones → Migrate s lower and end up n
earer to the w
ell
5. Conduct S outhern blotting with radioactive 5. Stain with ethidium bromide and
ss probe complementary to part of target DNA visualize b
anding pattern under U
V light.
seq. DNA fragments hybridize to probe by C BP.
Visualize b anding pattern via autoradiography.
- HbS allele yields l arger fragment when visualized via autoradiography
- A single large band indicates 2 HbS alleles in a sufferer, 2 bands (1 intermediate, 1 small)
indicate 2 HbA alleles in a normal individual, and 3 bands (1 large, 1 intermediate, 1
small) indicate 1 HbS and 1 HbA allele in a n ormal individual

Application: DNA Fingerprinting in Forensic Science / Paternity Testing
➢ Different alleles/markers produce different bands in gel, resulting in a unique
banding pattern for different individuals
➢ Polymorphic nature of DNA: Different no. and location of restriction sites +
different no. of t andem repeat seq.
➢ DNA fingerprints of 2 individuals can be compared to see how closely-related
they are → S imilar banding patterns occur in closely-related individuals
[describe procedure above: contextualize (e.g. ...primers complementary to seq. flanking
PKU allele, radioactive ss probe)]



(i) What is the probability that III5 and III6 will have a child with the disease. Explain. [4]
- Both I V5 and IV7 have the d isease and are homozygous for RFLP allele 2
- III5’s mother (II3) has 1 copy of RFLP allele 2, while his father (II4) does not have the marker
→ III5 has a probability of ½ of being heterozygous for RFLP allele 2 + III6 has a probability
of ½ of being heterozygous for RFLP allele 2 (similar reasoning)
- Probability of III5 and III6 having an a ffected child = ½ x ½ = 1/4
- Probability of child having d isease = ½ x ½ x ¼ = 1/16
(ii) IV6 was found to suffer from the disease although genetic screening using RFLP marker only
identified him as being a carrier. Suggest a possible explanation. [ 2]
- During formation of gametes in III4, crossing over occurred at locus between disease
allele and RFLP marker such that disease allele became l inked to RFLP marker 3


Biology: Genetics & Inheritance III: Genetic Basis For Variation I

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Biology: Genetics & Inheritance III: Genetic Basis For Variation I

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Genetic Basis for Variation I​ ​Genetic Basis for Variation II​ M

​ olecular Biology Techniques

Biology: Genetics & Inheritance III

Locus Position of a ​gene​ on a c

Gene An ordered sequence of DNA nucleotides located at a particular locus, on a

Allele 1 of a no. of ​alternative forms​ of a ​gene​ that possesses a unique nucleotide

Homozygous Diploid condition where ​alleles​ at a g

Heterozygous Diploid condition where ​alleles​ at a g

Genotype Combination​ of ​alleles​ located on ​HC​ at ​corresponding loci​.

Phenotype Characteristic​ arising from i​ nteraction​ b/w g

Dominant Allele that shows its phenotype regardless of whether it is homozygous

Incomplete Heterozygote’s phenotype is i​ ntermediate​ between those of both

F​1​ Generation Offspring produced by crossing parental generation.

By Jermaine Wong (2020)

F​2​ Generation Offspring produced by crossing 2 F​1​ organisms.

Test Cross Crossing b/w an individual of unknown genotype/heterozygote and a

Pure Breed Organism is ​homozygous​ for traits concerned.

Wild Type Typical genotype found in a natural population of that species.

Linkage ≥2 genes on same chromosome that do not assort independently in meiosis →

By Jermaine Wong (2020)

By Jermaine Wong (2020)

Important Ratios in Complete Dominance Monohybrid Cross

By Jermaine Wong (2020)

By Jermaine Wong (2020)

By Jermaine Wong (2020)

Linked Genes: Autosomal Linkage

By Jermaine Wong (2020)

- Larger number of parental phenotypes than recombinant phenotypes indicates

By Jermaine Wong (2020)

Genetic Diagram (​X-linked dominant/recessive allele for)

By Jermaine Wong (2020)

Explain why heterozygotes for PKU appear normal at birth. [​ 2]

By Jermaine Wong (2020)

Explain why heterozygous plants for Tt gene have the same phenotype as homozygous dominant

By Jermaine Wong (2020)

By Jermaine Wong (2020)

Example 2: Flower Color in Sweet Pea (​9:7​ ratio)

By Jermaine Wong (2020)

Example 3: Coat Color in Labrador Retrievers (​9:3:4​ ratio)

By Jermaine Wong (2020)

Example 4: Kernel Color in Wheat (​15:1​ ratio)

By Jermaine Wong (2020)

Example 5: Fruit Color in Squash (​12:3:1​ ratio)

By Jermaine Wong (2020)

Biochemical Pathway: Dominant Epistasis

By Jermaine Wong (2020)

Example 6: Feather Color in White Leghorn Chicken (​13:3​ ratio)

By Jermaine Wong (2020)

How to Solve Epistasis Question

df = Σ = 𝝌​2​ =

4. Determine degrees of freedom (df)

By Jermaine Wong (2020)

- Standard Deviation (​s​): Measure of s ​ pread of values​ within data set

- s​2​ ​= __ ​(e.g. s​ 2​​ for females =

6. Determine degrees of freedom (v)

By Jermaine Wong (2020)

𝝌​2​ vs. T Test

By Jermaine Wong (2020)

Molecular Biology Techniques

Genetic Basis for Variation I Genetic Basis for Variation II M

olecular Biology Techniques

Locus Position of a gene on a c

Allele 1 of a no. of alternative forms of a gene that possesses a unique nucleotide

Homozygous Diploid condition where alleles at a g

Heterozygous Diploid condition where alleles at a g

Genotype Combination of alleles located on HC at corresponding loci.

Phenotype Characteristic arising from i nteraction b/w g

Incomplete Heterozygote’s phenotype is i ntermediate between those of both

F1 Generation Offspring produced by crossing parental generation.

F2 Generation Offspring produced by crossing 2 F1 organisms.

Pure Breed Organism is homozygous for traits concerned.

Genetic Diagram (X-linked dominant/recessive allele for)

Explain why heterozygotes for PKU appear normal at birth. [ 2]

Example 2: Flower Color in Sweet Pea (9:7 ratio)

Example 3: Coat Color in Labrador Retrievers (9:3:4 ratio)

Example 4: Kernel Color in Wheat (15:1 ratio)

Example 5: Fruit Color in Squash (12:3:1 ratio)

Example 6: Feather Color in White Leghorn Chicken (13:3 ratio)

df = Σ = 𝝌2 =

- Standard Deviation (s): Measure of s pread of values within data set

- s2 = __ (e.g. s 2 for females =

𝝌2 vs. T Test

Synthetic s ingle-stranded DNA primers (20-30 nucleotides) a

Thermostable DNA polymerase isolated from thermophilic prokaryotes

Buffer cont. Mg2+ Cofactor for proper DNA polymerase function

Taq polymerase synthesizes c omplementary DNA strand by catalyzing

No. of copies of ss DNA = 2n+1

Application: Disease Detection (e.g. SNP in sickle-cell anemia)