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Gene Mutation and DNA Polymorphism

1) A gene mutation is defined as a change in the nucleotide sequence of a gene. The major types of mutations include point mutations, insertions/deletions, and dynamic mutations involving triplet repeats. 2) DNA polymorphisms are genetic variations that occur in at least 1% of the population. The major types are RFLPs, STRs, and SNPs. 3) DNA polymorphisms can be used for individual identification applications like paternity testing and analyzing the origin of cells or chromosomes. They are also useful for gene mapping and indirect gene diagnosis.

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65 views53 pages

Gene Mutation and DNA Polymorphism

1) A gene mutation is defined as a change in the nucleotide sequence of a gene. The major types of mutations include point mutations, insertions/deletions, and dynamic mutations involving triplet repeats. 2) DNA polymorphisms are genetic variations that occur in at least 1% of the population. The major types are RFLPs, STRs, and SNPs. 3) DNA polymorphisms can be used for individual identification applications like paternity testing and analyzing the origin of cells or chromosomes. They are also useful for gene mapping and indirect gene diagnosis.

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AgricultureAlex
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Gene mut at i on and

DNA pol ymor phi sm


Gene Mutation
DNA Polymorphism
Outline of this chapter
Gene Mutation
Gene Mutation
Definition
Major Types
Definition
Definition
A gene mutation is a change in the
nucleotide sequence that composes
a gene.
Somatic mutations
Germline mutations
Somatic mutations
Somatic mutations
are mutations that occur in cells of the
body excluding the germline.
Affects subsequent somatic cell
descendants
Limited to impact on the individual and
not transmitted to offspring
Germline mutations
are mutations that occur in the germline cells
Possibility of transmission to offspring
Major Types
Point mutations
Insertion or deletion
Fusion gene
Dynamic mutation
Point mutation
substitution of one base with another
Point mutation
Transition
Transversion
purine replaces purine
pyrimidine replaces pyrimidine
or
A
C
purine replaces pyrimidine
pyrimidine replaces purine
or
G
T
A
C
G
T
What are the consequences?
Silent mutation (same sense mutation)
Missense mutation
Nonsense mutation
Stop codon mutation
Within coding region:
Splicing mutation
Within noncoding region:
Regulatory sequence mutation
Silent Mutation
Silent Mutation
changes one codon for an amino acid to
another codon for that amino acid
no change in amino acid
Missense
Missense
mutation
mutation
A point mutation that exchanges one codon
for another causing substitution of an
amino acid
Missense mutations may affect protein
function severely, mildly or not at all.
CGT
AGT
Hemoglobin
Four globular proteins
surrounding heme group
with iron atom: two beta
chains and two alpha
chains
Function is to carry
oxygen in red blood cells
from lungs to body and
carbon dioxide from cells
to lungs
Single base change in hemoglobin gene
Single base change in hemoglobin gene
causes sickle cell anemia
causes sickle cell anemia
Wild-type
allele
mutant
allele
Wild-type
phenotype
mutant
phenotype
Nonsense mutation
Nonsense mutation
A point mutation changing a codon for an
amino acid into a stop codon (UAA, UAG
or UGA).
5 ATG GGA GCT CTA TTA ACC TAA 3
met gly ala leu leu thr stop
5 ATG GGA GCT CTA TGA ACC TAA 3
met gly ala leu stop
Nonsense mutation
Nonsense mutation
Premature stop codons create truncated
proteins.
Truncated proteins are often nonfunctional.
Some truncations have dominant effects
due to interference with normal functions.
Stop codon mutation
A point mutation changing a stop codon
into a codon for an amino acid
5 ATG GGA GCT CTA TTA ACC TAA 3
met gly ala leu leu thr stop
5 ATG GGA GCT CTA TTA ACC TTA 3
met gly ala leu leu thr LEU
Splicing Mutations
Disruption of existing splice sites
intron is not removed from mRNA
Creation of novel splice sites in exons
Hemoglobin E
Hemoglobin E
Regulatory sequence mutation
Occurred in promoter
Occurred in enhancer
Occurred in UTR
Insertion or deletion mutations
Insertion or deletion mutations
The genetic code is read in triplet nucleotides
during translation.
Addition or subtraction of nucleotides not in
multiples of three lead to a change in the
reading frame used for translation. Amino
acids after that point are different, a
phenomenon called a frameshift.
Addition or subtraction of nucleotides in
multiples of three leads to addition or
subtraction of entire amino acids but not a
change in the reading frame.
Frameshift
Frameshift
Mutation
Mutation
5 ATG GGA GCT CTA TTA ACC TAA 3
met gly ala leu leu thr stop
5 ATG GGG AGC TCT ATT AAC CTA A 3
met gly ser ser ile asn leu .
Insertion or deletion of
Insertion or deletion of
codons
codons
5 ATG GGA GCT CTA TTA ACC TAA 3
met gly ala leu leu thr stop
5 ATG GGA TTA TTA GCT CTA TTA ACC TAA 3
met gly leu leu ala leu leu thr stop
Triplet repeat expansion (Dynamic Mutation)
Stretches of triplet repeats (i.e. CAGCAGCAG) have
variable lengths across individuals.
These lengths can vary to a small extent without
consequence.
However, once a repeat length reaches the critical
length, huge expansions can occur which will disrupt
gene expression or function.
Triplet repeat diseases display genetic anticipation
(the progressively earlier appearance and increased
severity of a disease in successive generations) due
to the continual expansion of the repeat as its passed
from one generation to the next.
Trinucleotide Expansion
Fragile X Syndrome

CGGCGGCGGCGGCGGCGG Amplification
CpG
island
FMR-1
(CGG)
n
FMR1 gene
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Normal exon1
AUG
Premutation
exon1
Fullmutation
exon1
AUG

AUG
A Fragile X syndrome pedigree
The number below each individual indicates the number
of CGG repeats in each copy of FMR1.
Myotonic
Myotonic
dystrophy: a triplet repeat disease
dystrophy: a triplet repeat disease
5 -37 copies of CTG repeat normal phenotype
50-1000 repeats myotonic dystrophy
Genes with 40+ copies are unstable and can gain
(or less commonly lose) repeat copies in
successive generations.
DNA Polymorphisms
Definition
Major types
Application
What makes us different from
each other?
The answer is
POLYMORPHISMS
POLYMORPHISMS
Genetic polymorphism
The occurrence in a population of two or more
genetically determined forms in such frequencies
that the rarest of them could not be maintained by
mutation alone
Definition
A polymorphic locus is one at which there are at
least two alleles, each with a frequency greater than
1%. Alleles with frequencies less than 1% are
referred to as mutants.
Mutations and Normal Variants
Gene A
Allele 1 (Normal) 99.5%
Allele 2 (Mutant) 0.5%
Gene B (Polymorphic)
Allele 1 (Nl Variant) 50%
Allele 2 (Nl Variant) 30%
Allele 3 (Nl Variant) 20%
Gene C (Polymorphic)
Allele 1 (Nl Variant) 45.5%
Allele 2 (Nl Variant) 30%
Allele 3 (Nl Variant) 15%
Allele 4 (Mutant) 0.2%
Allele 5 (Mutant) 0.3%
Polymorphism
Polymorphism presents at different level:
Phenotype polymorphism
Protein polymorphism
e.g. ABO blood type or immunoglobulin
Chromosomal polymorphism
e.g. minor variant in chromosomal structure
DNA sequence polymorphism
Chromosomal Polymorphism
Definition : more than one normal allele at
a genomic locus in the population, with
the rarest allele exceeding a frequency of
1%.
Characteristics:
The frequency of the rarest allele is more than
1%
Inherited in Mendelian pattern in the families
No functional consequences
DNA Polymorphism
Major types
Restriction fragment length
polymorphism (RFLP)
Short tandem repeat polymorphism
(STR)
Mini-satellite repeat polymorphism
Micro-satellite repeat polymorphism
Single nucleotide polymorphism(SNP)
RFLPs
polymorphism that alter the length of restriction
fragments
Result from
changes (e.g. SNPs) that introduce or delete a
restriction enzyme site
Two alleles
Genotyping by Southern or PCR-RFLP
Restriction Fragment Length Polymorphisms
Restriction Fragment Length
Polymorphism (RFLP)
Genotyping
Genotyping
-
-
by Southern
by Southern
Isolate DNA
Digest DNA w/ restriction enzyme
Size fractionate DNA
Denature DNA
Blot SS DNA to membrane
Prepare a probe
Label
Denature
Hybridize probe with membrane
Autoradiography
Sout her n Bl ot -RFLP
---- AAGCTT-----------------AAGCTT-----------AAGCTT -----
300bp 200bp
---- AAGCTT-----------------AGGCTT-----------AAGCTT -----
500bp
A1 B1 C1
200bp
300bp
500bp
2/2 1/2 1/1
A2 B2 C2
2/2 1/2 1/1
C3 B3 A3
1/1 1/2 2/2
Genotyping-by PCR-RFLP
Design primers
PCR
Digest PCR product with RE
Gel electrophoresis
PCR-RFLP
-------------------------AAGCTT -------------
300bp 200bp
Allele 1
-------------------------AGGCTT -------------
500bp
Allele 2
A B C
2/2 1/2 1/1
200bp
300bp
500bp
Short Tandem Repeats (STR)
Short Tandem Repeats (STR)
Variable number of tandem repeats
Multiple alleles
Genotyping
Short Tandem Repeats
Short Tandem Repeats
Polymorphism (STR)
Polymorphism (STR)
....CACACACACACACA....
....CACACACACACACACACACA....
....CACACACACACACACACACACACA....
....CACACACACACACACACACACACACACACACA....
(CA)
7
(CA)
10
(CA)
12
(CA)
14
Short Tandem Repeats
Short Tandem Repeats
Polymorphism (STR)
Polymorphism (STR)
Allele 1 (CA)
5
210bp
.....CACACACACA......................
100bp
100bp
Allele 2 (CA)
6
212bp
Allele 3 (CA)
7
214bp
Allele 4 (CA)
8
216bp
Allele 5 (CA)
9
218bp
Allele 6 (CA)
10
220bp
Resul t
2/2 1/3 5/5 2/5 2/3 4/6 1/4
220bp
218bp
216bp
214bp
212bp
210bp
5/5 4/4 3/5 2/5 4/5 2/4 1/5 3/4 3/5 2/5 2/4
11/12 1/4 4/11 4/8 1/11 4/6 12/13 4/7 M 3/4 7/14 7/7 3/10 3/6 3/4 3/11 3/5 1/4
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Definition:
the variation of only one base pair on one polymorphic locus
the most simple form
the most common source of genetic polymorphism in the human genome (90%)
SNPs occur in humans
In coding regions every 1000-3000 bp
In non-coding regions every 500 - 1000 bp
Less polymorphic than microsatellites, but the sheer number of them compensates
Even more highly automatable than microsatellites
Genotyping: ASO or DNA microarray / DNA chip
SNP
(si ngl e nuc l eot i de pol ymor phi sm)
Application of DNA
Application of DNA
Polymorphism
Polymorphism
Individual identification (Paternity test)
Analyzing the origin of extra chromosome
Identifying the origin of cells
Gene mapping
Indirect gene diagnosis
Paternity testing
M1 M2 M3 M4 M5
Y
Y N N N

Crime scene
Sample:
Victim + perhaps
Victim
A B C
Suspects
15 14 45 35 23 45 34
Downs Syndrome

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