Chapter 3
Genetic Polymorphism
T. Abed Al Kareem Noureddine 1

Activity 1: Mutations and Environment
• What are the different types of mutations?
• Chromosomal mutation (Chapter 5)
• Gene mutation
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Gene mutation:
• Definition: is the accidental changes of DNA (nucleotide sequence) that
may lead or not to change in phenotype.
Note: new allele is called mutant allele.
• Causes:
1. Spontaneous mutation: due to error in natural biological process such as DNA
replication
2. Induced mutation: due to environmental factors that causes mutations (called
mutagens)
UV radiation- polycyclic aromatic hydrocarbons (PAH)- free radicals- smoking..
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Transmission and impact of mutation
• Are all mutations could be
transmitted to off springs?
The transmission of a mutation
depends on the type of cell where
mutation had occurred.
Somatic cells: mutation can not be
transmitted to off springs
Germline sex cells: mutation can
be transmitted to off springs.
• Why mutations in somatic cells
may be significant although
they are not transmitted to
offspring?
Mutations in somatic cells may
develop cancers such as cancer in
respiratory system (due to PAH),
skin cancer due to UV
radiations…
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Consequence of mutation
• Consequence of mutation: Are all mutations harmful?
Mutation can be:
1. Harmful: lead to new disease. Example: thalassemia
2. Favorable: mutations lead to new allele that results in new favorable
phenotype. This cause diversity of living things (example dark moth)
3. Silent: change in DNA that has no change in phenotype
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Introduction to Genetic Polymorphism
Introduction:
• Definition: Genetic polymorphism is presence of 2
or more variants in a certain sequence of DNA.
• Cause: Genetic polymorphism is due to mutation.
• Note: Genetic polymorphism could be in coding
regions of DNA (genes) or non-coding regions
(document 1)
• Coding regions of DNA: is portion of DNA that
codes for protein
• Non-coding region: portion of DNA that does not
code for protein (example: telomere, centromere,
introns in the gene, satellites...)
• Note that terms coding and non coding regions of
DNA different than terms coding and coding strand
of DNA
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Document 2: Mutations and multiple alleles
Revision:
• Relation between gene and phenotype:
DNA (gene) → protein → phenotype
normal allele → normal protein → normal phenotype
Mutant allele → altered protein → altered phenotype
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DNA protein synthesis
G A A T T T A T G C C C G
C T T A A A T A C G G G C
Triplet
Double
Stranded
DNA
• Transcribed Strand
• Template
• Non-coding
• Used in protein synthesis
• Non- Transcribed Strand
• Non- Template
• coding
• Not used in protein synthesis
C U U A A A U A C G G G C
mRNA
Codon • Complementary to
transcribed
• Identical to non transcribed
but T replaced with U
Transcription
Translation
Polypeptide Leu__ lys __ tyr __ Gly
Note: when writing an aminoacid sequence
of polypeptide don’t forget to draw the
peptide bond between each 2 consecutive a.a
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Remarks regarding protein synthesis
• Remarks:
• RNA contains uracil (U) instead of thymine (T)
• Stop codons: UAA /UAU/ UAG where translation stops
• Change in DNA leads to change in mRNA that leads to change in
amino acid sequence and thus change in 3d structure of protein
leading to a new phenotype.
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Normal DNA of gene
Normal mRNA
Normal protein (functional)
Normal phenotype
mutant DNA of gene
mutant mRNA
altered protein (functional)
abnormal phenotype
mutation
Diagram showing relation between gene and
phenotype in normal and mutated gene
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Types of genetic mutation
G A A T T C
C T T A A G
G A T T C
C T A A G
G A A T T C
C T T A A G
G A G A T T C
C T C A A A G
G A A T T C
C T T A A G
G A C T T C
C T G A A G
Deletion addition substitution
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Consequences of different types of mutations
• Substitution mutations are point mutations that affect one nucleotide
thus may lead or not to a change in the sequence of 1 amino acid.
• Addition (insertion) or deletion mutations are frameshift mutations
disturb the DNA reading frame, so it changes the whole sequence
after the mutation
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Consequences of different types of mutations
Type of mutation Normal Mutant Consequence
DNA producing aminoacid DNA producing aminoacid
Substitution CCA GAG ACT CCA GTG ACT Missnese mutation
Pro __ Glu__Thr Pro __val__Thr
CCA GAG ACT CCA GAC ACT Silent mutation
Pro __ Glu__Thr Pro __ Glu__Thr
CCA GAG ACT CCA TAG ACT Non-sense mutation
Pro __ Glu__Thr Pro stop
deletion TAC ACC ACG A.. TAC CCA CGA.. Frame shift mutation
Tyr __ Glu __ Thr Tyr__pro_Arg
insertion TAC ACC ACG A.. TAC GAC CAC GA.. Frame shift mutation
Tyr __ Glu __ Thr Tyr__ Asp__ His
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Note:
If you were asked to explain how the change of nucleotides leads to
certain disease presented in text. Your answer should include:
1. site and type of mutation
2. Consequence of mutation (at level of mRNA and at level of
polypeptide)
3. The effect of change in the amino acid sequence in polypeptide
(causes a change in the 3d structure of protein ( or in case of non-
sense mutation: a truncated protein is resulted) that becomes inactive
4. Go back to text: indicate the function of protein and relate the effect
of inactivation of protein to the disease
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2013 2nd- exercise 2

Answer:
We establish the mRNA sequence by replacing T by U
• Normal mRNA: UAU ACC CCC GAA CCU GAC AUC
• Amino acids sequence : Tyr-Thr-Pro-Glu-Pro-Asp-Ile
• Diseased m RNA: UAU ACC CCC AAA CCU GAC AUC
• Amino acids sequence : Tyr-Thr-Pro-Lys-Pro-Asp-Ile
2. The mutation by substitution at the level of the first nucleotide of the 280th codon of the
DNA where G is replaced by A is transcribed at the level of mRNA by a new codon which
is translated into a new amino acid, lysine instead of the glutamic acid. This new amino acid
sequence affects the tridimensional structure of the enzyme PAH which becomes inactive
(nonfunctional). Since this enzyme is responsible for the transformation of phenylalanine
into tyrosine. This transformation doesn’t occur any more leading thus to the accumulation
of phenylalanine which in high amount becomes toxic and causes phenylketonuria.
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Genes and multiple alleles
• An example of multiple alleles in humans is ABO
blood group.
• 3 different molecules (A, B, o).
• Each molecule is made up of: substance H with
presence or absence different type of sugar.
• Antigen A: substance H + N-acetyl galactosamine
• Antigen B: substance H + galactose
• Antigen o: substance H
• The type of sugar motif added depends on the type
of transferase enzyme present.
• Different enzymes are due to presence of different
alleles as result of different types of mutations
occurred from wild type allele
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Draw out the definition of genetic polymorphism and its possible
causes.
• Gene is polymorphic when it has more than 2 alleles. The diversity of
alleles are due to mutations.
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Document 3: Polymorphic genes in
population
• Define polymorphic genes:
Gene is said to be polymorphic when it has more than 2 alleles of
frequency (>1%)
• Examples of polymorphic genes
• Blood group
• Major Histocompatibility Complex
• Beta-globin gene
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Major Histocompatibility complex MHC
Structure and Function:
• MHC is also called HLA (Human Leukocyte Antigen) because these
proteins were initially described on surface of leukocytes
• Structure: large glycoprotein carried by all nucleated cells.
• Function:
• Determines graft acceptance and rejection
• Other function will be discussed in Immune System
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Major Histocompatibility complex MHC
Transmission:
• MHC is coded by 6 genes (haplotype) carried by chromosome 6.
• Each gene has many alleles and alleles of each gene are codominant.
• That’s why these gene are highly polymorphic
• Haplotype: set of genes very close to each other that are inherited as
one block
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• Define a wild type allele.
A wild type allele is the allele that codes for the most common
phenotype.
No- 1 p: 63
Every individual has 6 genes that are involved in coding for MHC (A B
C DP DQ DR) existing in 2 copies (one of paternal origin and one of
maternal origin). Each gene has many different alleles and all are
codominant. This makes it practically impossible for 2 indviduals to
have exact combination of the all 6 HLA loci. On the other hand
identical twins have same genetic information so have the same alleles
at each of the loci in the MHC complex
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Beta globin gene
• Hemoglobin contains 2 kinds of polypeptides
• 2 alpha
• 2 beta
• Beta globin gene is coded by gene that has 150 alleles
• Most of them are normal
• Few of them codes for abnormal beta globin leading to disease.
• Solve number 3 and 4 p: 63
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3) polymorphic gene of B- globin gene is due to the presence of diverse
alleles in the human population. These alleles are the results of different
types of mutation insertion, deletion, or substitution of DNA nucleotide.
4)The severity of B- thalassemia depends on the site, type and extent of
mutation of beta globin gene. In general, substitution mutations are less
severe than deletion or insertion, especially if the substitution leads to
an amino acid characteristically similar to that of the original amino acid
(example lysine and arginine). However deletion of a long stretch of
beta globin gene leads to severe thalassemia.
Moreover, one must note that the severity of disease depends whether
the individual carries one or 2 mutant alleles
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Document 4: Detection of Genetic
Polymorphism
• Introduction:
• As defined in previous document, genetic polymorphism is the presence of 2
or more alleles of the same gene.
• And we’ve seen that the origin of different alleles is mutation and thus having
different sequences of nucleotides in a given locus
• So how can we detect the existence of change in nucleotide sequence?
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Detection of Genetic polymorphism
• Genetic polymorphism could be assessed by:
1. Study phenotypic variations: (for genetic polymorphism at level of
coding regions) (ex: presence of different blood groups shows that
there are mutations at level of gene responsible of blood group)
2. Restriction Fragment Length Polymorphism (RFLP) that could
detect genetic polymorphism at level coding or non-coding region
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Principle of RFLP technique:
• A mutation in certain site leads to creation or deletion restriction site
of specific restriction enzyme leading to variation in number and size
of fragments of obtained by restriction enzyme. The obtained
fragments are separated according to size using gel electrophoresis
In certain cases, southern blot is done using radioactive probe to
observe only specific part of DNA fragments.
• These will be explained in details in the following slides.
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Restriction Enzymes
• Restriction enzymes:
• Bacterial enzymes
• Recognize specific nucleotide
sequence (recognition site)
• Cuts a specific location in it
(cleavage site)
• Different enzymes may have
different recognition and cleavage
sites.
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• Example:
Restriction Enzymes
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1. Determine the number and the length of the
restriction fragments obtained as a result of
cutting allele A by Hae III enzyme. ( Given
Hae III restriction site doc. 2)
2. Determine the consequence of mutation on the
produced restriction fragment upon using Hae II
enzyme on allele 2
Document 1
Document 2

Answer:
1. Allele A has 2 restriction sites of enzyme Hae III where it will cuts after nucleotides 198
and 240. Therefore, the enzyme cuts the allele into 3 fragments. The length of each
fragment is:
• Fragment 1 of 198 base pairs (bp) (before the site 198),
• Fragment 2: 240-198= 42 bp
• Fragment 3: 470-240 = 230 bp
2. Hae III enzyme cuts the DNA when encountering the sequence GGCC. The cutting is
done between GG and CC (document 2). Document 3 shows that the restriction site at the
level of the nucleotide 240 does no longer exist for allele B due to the mutation by
substitution. Instead of the GGCC sequence for allele A there is a GGCT sequence for allele
B. As a result, the enzymatic treatment of allele B will give 2 fragments instead of 3:
• a fragment of 198 base pairs (bp) (before the site 198),
• a fragment of 272 bp instead of the two fragments (42 and 230 bp)
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The restriction process and the obtained fragments are obtained
in a test tube, so how can we detect these fragments?
Gel Electrophoresis
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Gel Electrophoresis
• How can we detect these fragments?
➢By Gel Electrophoresis
• Principle:
• DNA is –ve charged due to phosphate group
• During electrophoresis bands migrate from –ve pole
to +ve pole
• Migration depends on size: smaller fragments
migrates further
• Role of Ethidium Bromide (Et-Br)
➢Dye that binds to DNA and fluoresces under UV
light.
• Restriction map: is the generated pattern of
bands
• Note: The enzyme that gives 2 different
restriction maps of the 2 alleles is able to
detect the genetic polymorphism
-
+
migration
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• Application 1: Given the following sequences of 2 different alleles of
same gene
Allele N:……GGCCTGAATTCGT…..TGACGGCCT..A
Allele d:……GGCCTGAATTCGT…..TGACGGGCT..A
1 147 513
1 147 513
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600
600
Document 2
1) By referring to the table of restriction enzymes (in
previous slides) , compare the action of Hae III and
Eco RI on the 2 different alleles (doc. 1).
2) Deduce the enzyme that is able to detect the genetic
polymorphism in this gene.
3) Given the following results of electrophoresis of 3
individuals A, B and C under the effect of Hae III enzyme
(document 2). Specify the genotype of each individual.
Document 1
For Teacher: Read the notes
in slide notes!

• Answer:
1. Hae III has 2 restriction sites (at nucleotides 148 and 518) on allele 1 and
gives 3 restriction fragments more than that of allele 2 that has 1 restriction
site (148) giving only 2 fragments. One of fragments obtained from allele 1 is
equal to that obtained from allele 2. The sum of other two fragments obtained
from allele 1 is equal to that obtained from the other fragment of allele 2.
On the other hand, EcoRI has same number of restriction sites (1 at
nucleotide 153) and restriction fragments obtained (2) on both alleles.
2. Since obtained results in the 2 different alleles by Hae III are different (3
fragments in allele 1 and 2 fragments in allele 2). Then separating them by gel
electrophoresis will show 3 different bands for allele 1 and 2 different bands
of allele 2 thus obtaining different restriction maps. Therefore we can detect
genetic polymorphism for this gene by Hae III.
On the contrary, EcoRI shows same number of fragments for the 2 alleles of
same size, thus separating them through gel electrophoresis will give 2 bands
for each in same position, thus same restriction map is obtained. For that,
EcoRI is not suitable for detecting genetic polymorphism for this gene.
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Answer (continue)
• Individual A: Nd since it has band 452 that is present only in the d allele
and bands 370 and 82 that are specific for the N allele.
• Individual B: dd since he only has 452 thick band (that corresponds for the
d allele) which indicates presence of 2 copies of this allele and lacks the
band that correspond for N allele (370 and 82)
• Individual C: NN since he has thick bands of 370 and 82 that corresponds
for the normal allele (N) which indicates existing 2 copies of this allele and
lacks band corresponding for d allele (452 bp)
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Restriction Fragment Length Polymorphism
(RFLP)
• Objective of technique: Detect
genetic polymorphism in coding or
non-coding regions
• The mutation at level of non-coding
region of DNA does not alter the
phenotype but changes the restriction
map.
• Therefore, the restriction map is
independent of gene function.
• The variation at level of non-coding
regions is used to determine genetic
identity of individual!
• DNA fingerprinting (document 5) is
an application of RFLP technique
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• What two ways could be used to assess genetic polymorphism in
population?
• Study the phenotypic variation (for coding regions)
• RFLP (for coding and non coding regions)
• Note: RFLP technique allows to determine the exact genotype of individual in case
he had dominant phenotype (he could be homozygous or heterozygous)
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Document 5: Genetic Identity of individual
• Introduction:
Because of the large number of polymorphisms observed in humans, it
is virtually certain that each of us is genetically unique (except identical
twins). How can we determine genetic identity of individual?
Determination of Genetic identity is made by RFLP followed by
southern blot.
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Southern blot
• General Terms:
• Probe: a known sequence of radioactive or fluorescent DNA used to
hybridize the denatured DNA molecule by complementarity
• Denaturing: separation of DNA strands by high temperature or using
NaOH
• Hybridization: binding of probe to studied bands/ gene by
complementarity.
• Blotting: transferring
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Southern Blot
1. DNA is cut with restriction
enzymes
2. DNA fragments separated by gel
electrophoresis
3. Fragments then transferred
(blotted) and fixed on filter paper.
4. On this membrane the DNA are
denatured then a radioactive 32P
probe is added to hybridize
specific sequence by
complementarity
5. Hybridization of DNA with probe
is visualized by autoradiography.
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Notes
• There are two types of probes that could be used:
• Mono-locus Probe: A probe that binds to a sequence that exist only at one
locus. It is used in case of detecting a certain gene.
• Multi-locus Probe: A probe that binds to a sequence that occurs frequently
throughout genome. This type of probe is used in DNA fingerprinting.
• Among all fragments obtained from the action of restriction
enzyme, only the one where probe can bind partially or totally
would appear in results
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Example:
• In the following example the restriction
enzyme Hae III cuts in 3 position leading
to 4 fragments of sizes 198, 52, 230 and
30 bp. In gel electrophoresis the 4 bands
would appear (unless given in text that
there is minimum size for band to appear
in the gel electrophoresis). However upon
blotting, only the zone where the probe
binds partially or totally, the bands would
appear. For that in the final results only
bands appear (42 and 230 bp that
corresponds for allele A as there is
restriction site cute at level of 240) and
fragment of size 272 bp that corresponds
for allele B.
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DNA Fingerprinting
• DNA fingerprinting is an
application of RFLP (using
restriction enzymes, gel
electrophoresis, southern blot) and
it is used for:
• Paternity Test
• Forensic science (crimes, victims
after plane crush..)
• Probe used is multi-locus
(complementary to sequence
occurs frequently throughout the
genome in non coding regions)
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DNA fingerprinting in paternity test
• Each band of child’s DNA
fingerprint should be shared with
either the father’s or the mother’s
DNA fingerprint.
• Solve exercise IV + V p: 73
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• P:73 official book: Exercise IV
• The DNA isolated from blood traces left at the scene of the crime and
the DNA of the suspect are cut with the same restriction enzyme,
electrophoresed and blotted onto a membrane. The membrane is then
hybridized to a 32P labelled probe which is complementary to a
repetitive sequence. The two DNA fingerprints are compared. If the
two patterns are identical, then one can safely conclude that the traces
of the blood at the scene of the crime are those of the suspect.
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• Exercise V
No since every band of the child’s DNA finger print is present either in
the mother or the father’s DNA fingerprint so the child belong to this
couple.
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Fluorescent In Situ Hybridization (FISH)
• Aim:
• Locate locus of gene on metaphasic
chromosome using fluorescent probe
• Detect chromosomal mutations (deletion,
translocation..)
• Detect certain types of viral infection
• Steps:
• Denaturation of DNA
• Hybridization: add monolocus probe
• Observation: The chromosome shows a
fluorescent dot where the studied gene (or
sequence of interest) is located.
• FISH technique and other mapping
techqniques allowed to construct
genetic maps
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Genetic Map
• Genetic map representation of
arrangement of genes on
chromosome
• It can be constructed by using
many different mono-locus
probes
Chromosome X genetic map
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