MUTATION AND DETECTION TECHNIQUES

Question 1

• A single gene locus causing mutation has a
  major physiologic impact and considered to
  be deterministic of a disease.
• Seen less commonly in general population
• Linked to Mendelian disease.
• Correlation on between mutation and disease
  can be established.
• Single base pair change in nucleotide
  sequence of genes are called point mutation

Answer 1

Mutation

Question 2

• Genetic alteration that contributes to complex
  disease has smaller effect
• Present in at least 1% of the population.
• No correlation
• Single base pair change in nucleotide
  sequence of genes are called single
  nucleotide polymorphism (SNP)

Answer 2

Polymorphism

Question 3

TYPES OF MUTATION

Answer 3

1. SPONTANEOUS MUTATION
2. INDUCED MUTATION

Question 4

a. Hydrolytic reaction
b. Replication error

Answer 4

SPONTANEOUS MUTATION

Question 5

a. UVL
◉ thymine dimer formation by UV light
b. Ionizing radiation
◉ X rays, � rays, and β particles
c. Chemical mutagen
◉ deamination of nitrous acid

Answer 5

INDUCED MUTATION

Question 6

thymine dimer formation by UV light

Answer 6

UVL

Question 7

X rays, � rays, and β particles

Answer 7

Ionizing radiation

Question 8

deamination of nitrous acid

Answer 8

Chemical mutagen

Question 9

◉ read as “snip”.
◉ a change in a single nucleotide in the genome that causes variations in
DNA sequences between members of the same species.
◉ occurs when two individuals in the population differ by a single base in the
DNA sequence.

Answer 9

SINGLE NUCLEOTIDE POLYMORPHISM

Question 10

◉ has to be in >1% of the population.
◉ Otherwise variation is a rare mutation.

Answer 10

FACTORS TO CONSIDER IN POLYMORPHISM

Question 11

Is SNP’s important?

Answer 11

◉ important in studying human
health.

Question 12

Is SNP’s normal?

Answer 12

◉ it occurs normally throughout a person’s DNA (3 X 10! bp).
◉ can be caused by spontaneous
mutation over time

Question 13

SNP commonly found in the DNA

Answer 13

between genes

Question 14

may predict individuals response to drugs, susceptibility to environmental
factor (e.g. toxins), risk to a particular
disease, association with complex
disease (e.g. heart disease, diabetes,
cancer)
◉ track inheritance of disease
(pedigree)

Answer 14

SNP importance

Question 15

almost once in every 1,000
nucleotides, meaning there are
roughly_____ in a persons
genome.

Answer 15

4 - 5 M SNPs

Question 16

◉ can act as biological markers.

◉ occurs within a gene then that
particular gene is described as having
more t

◉ May lead to VARIATIONS IN AMINO ACID sequence which plays a role in disease by
affecting gene function. (e.g. sickle cell
anemia)
han one allele.

Answer 16

SINGLE NUCLEOTIDE POLYMORPHISM

Question 17

◉ caused by a single gene, several different mutations
can result in the same disease but with varying degrees
of severity and phenotype.

Answer 17

SINGLE GENE DISORDER

Question 18

◉ main factor is genes but the cause includes other
factors that aren’t genes.

Answer 18

MULTIFACTORIAL DISORDERS

Question 19

◉ abnormal chromosome number or constitution.

Answer 19

CHROMOSOMAL DISORDER

Question 20

a. Conventional karyotyping
b. Fluorescent in situ hybridization
(FISH)
c. Spectral karyotyping (SKY)

Answer 20

CHROMOSOMAL STUDIES

Question 21

◉ chromosome abnormality
◉ indicative in: sexual disorders, congenital
anomalies, developmental retardation, learning
disabilities, infertility, multiple miscarriage,
stillbirth, malignancy

Answer 21

Conventional karyotyping

Question 22

◉ provide specific localization of genes
on chromosomes.
◉ indicative in: trisomy and
microdeletion using specific probes.

Answer 22

Fluorescent in situ hybridization
(FISH)

Question 23

◉ homologous pairs of chromosomes are
manipulated in such a way that they have
distinctive colors.

Answer 23

Spectral karyotyping (SKY)

Question 24

TYPES OF DETECTION METHOD
2.1 HYBRIDIZATION BASED

Answer 24

a. Fluorescent in situ hybridization
b. Allele-specific oligonucleotide (ASO)
hybridization

Question 25

◉ dot blot method

◉ relies on binding effects of nucleotide
mismatches.

◉ stringent condition are applied so that a
single-base mismatch is sufficient to prevent
hybridization of the non-matching probe.

Answer 25

b. Allele-specific oligonucleotide (ASO)
hybridization

Question 26

◉ requires 2 probes, one specific for each allele
i. + probe : complimentary to normal
sequence
ii. m probe : complimentary to mutant
sequence

Answer 26

Allele-specific oligonucleotide (ASO)
hybridization

Question 27

complimentary to normal
sequence

Answer 27

+ probe

Question 28

complimentary to mutant
sequence

Answer 28

m probe

Question 29

1 : normal (+/+)
2 : heterozygous (m/+)
m : mutant control
+ : normal control
N : negative control

Answer 29

Chromogenic probe detection

Question 30

◉ a special FISH technique (dual
probes).
◉ detecting all genomic imbalances.
◉ comparison of total genomic DNA of
given sample (e.g. tumor DNA) with total
genomic DNA of normal cells.

Answer 30

Array based Comparative Genomic
Hybridization (aCGH)

Question 31

◉ identical amount of both tumor and normal DNA is
labeled with two different fluorescent dyes then the
mixture is added and hybridized to a normal lymphocyte
metaphase slide.

◉ a fluorescent microscope equipped with CCD camera
and an image analysis system are used for evaluation.

◉ Used to determine copy number alterations of
genome in cancer and those cells whose karyotype is
hard or impossible to prepare or analyze.

Answer 31

Array based Comparative Genomic Hybridization
(aCGH)

Question 32

◉ reverse dot blot method.
◉ investigate multiple genomic sites.
◉ unlabeled probes are bound to substrate
◉ specimen DNA is labeled and hybridized to immobilized probes.

Answer 32

Array Technology

Question 33

◉ allows rapid detection of SNP or
mutation.
◉ less laborious
◉ uses a chromatogram

Answer 33

DNA sequencing

Question 34

◉ application of PCR that allows the direct
detection of any point mutation, single base
changes or small deletions in human DNA.

◉ involves amplification of DNA with single
nucleotide polymorphism (SNP)

◉ uses SNP-specific primers (instead of
DNA probes)

Answer 34

Allele specific PCR

Question 35

◉ under stringent conditions (specific temperature, pH, or salt
concentration), PCR is less efficient in the presence of mismatch between the
template and primer.
◉ Successful amplification with an SNP-specific primer signals the presence of
the specific SNP in a sequence
◉ Sequences of the normal allele, mutant allele, and their corresponding
primers.
◉ The primers are specific to the alleles (normal or mutant)

Answer 35

Allele specific PCR

Question 36

CLEAVAGE BASE
◉ a technique that is used to study genetic
variation or polymorphisms among individuals
using restriction enzymes.

◉ PRINCIPLE: any genomic DNA can be
differentiated according to the presence or
absence of restriction enzyme sites. Restriction
enzymes recognize and cut at the particular site

Answer 36

Restriction Fragment Length
Polymorphism (RFLP)

Question 37

CLEAVAGE BASED
STEPS:
1. Restriction Digest
2. Electrophoresis: Agarose or Polyacrylamide
3. Denaturation: NaOH (dsDNAàssDNA)
4. Blotting: Nitrocellulose paper (Southern
blot)

Answer 37

Restriction Fragment Length
Polymorphism (RFLP)

Question 38

STEPS:
Restriction Fragment Length
Polymorphism (RFLP)

Answer 38

1. Restriction Digest
2. Electrophoresis: Agarose or Polyacrylamide
3. Denaturation: NaOH (dsDNAàssDNA)
4. Blotting: Nitrocellulose paper (Southern
   blot)
5. Baking and blocking
6. Probe hybridization and Visualization:

Question 39

autoclaving (fixes
DNA), bovine serum albumin or casein
(blocking: prevent binding of labelled probe
non specifically to charged membrane)

Answer 39

Baking and blocking

Question 40

labelled RFLP probe, autoradiography
(radioactive isotopes)

Answer 40

Probe hybridization and Visualization: