MOLEBIO REVIEW
CHROMOSOMAL MUTATIONS
CHROMOSOME MORPHOLOGY
Chromosomal Structure and Chromosomal
Mutations
o Human genome : all of the genes founf
in a single individual
o Diploid : offspring with 46
chromosomes
o Haploid : 23 chromosomes
 Phenotype : a trait or group of traits
resulting from transcription and translation
of these genes
 Genotype: DNA nucleotide sequence
responsible for phenotype
 Mutation : DNA sequence change that is
present in a relatively small proportion of a
population
 Variant : inherited sequence alterations
 Polymorphism: change in DNA sequence that is
present in at least 1-2% of a population.
 Balanced polymorphism: single-base
substitution in the gene that codes for
hemoglobin
 Genome mutations: changes in the number of
chromosomes
 Euploid: cell or cell population with a normal
complement of chromosomes
 Aneuploid: genome mutations result in cells
CHROMOSOMAL STRUCTURE AND ANALYSIS
 Centromere: where the chromosome attaches to
the spindle apparatus for proper segregation during
cell division
 Position effect: well-known phenomenon that a
gene inserted or moved into a different
chromosomal location may be expressed
(transcribed and translated) differently than it was
in its original position
 Winding of DNA onto histones is the first step
 Histones: proteins in cells
 Nucleosome: formed when DNA is wrapped around
a set of 8 histone proteins
 10-micron fiber : comprises bead-on-a-string
arrangement of nucleosomes
 Linker region : he part of the double helix that is
exposed between the histones
 Nonhistone proteins: remainder of the proteins
involved in DNA compaction
 Heterochromatin: closed chromatin
 Euchromatin: open chromatin
 Centromere: site of attachment of the
chromosome to the spindle apparatus
 Kinetochore: protein complex that assembles at
the centromere sequences
 Alpha satellite: repetitive sequences
 CHROMOSOMES are:
o Metacentric : arms of the
chromosomes are of equal size
o Submetacentric:
o Acrocentric: divided the chromosomes
into long arms and short arms
o Telocentric: centromeres are at the
ends of the chromosome
 Q banding : resulting fluorescence pattern
visualized after staining
o Caspersson, Zech, and Johansson
(1970)
-first demonstrate this method
 G bands: chemical dye Giemsa stains in
patterns
 High-resolution banding: number of visualized
bands can be increased from about 300 to
about 500 per chromosome by staining
chromosomes before they reach maximal
metaphase condensation
 R banding: Harsher treatment of chromosomes
(87°C for 10 minutes, then cooling to 70C)
before Giemsa staining will produce a pattern
opposite to the G banding pattern
 C banding: alkali treatment of chromosomes
results in centromere staining
o C bands: associated w/
heterochromatin, the “quiet” or poorly,
transcribed sequences along the
chromosomes
  Centromere staining: absent in G band patterns
 NUCLEOLAR ORGANIZING REGION STAINING
(NOR staining) -- Chromosomes treated with
silver nitrate will stain specifically at the
constricted regions, or stalks, on the acrocentric
chromosomes.
 DAPI (h 4′,6-diamidino-2- phenylindole)
o First described in 1976 as way to detect
mycoplasmal contamination in cell
cultures.
o binds to the surface grooves of double-
stranded DNA and fluoresces blue
under ultraviolet light (353-nm
wavelength).
o DAPI can be used to visualize
chromosomes as well as whole nuclei
DETECTION OF GENOME AND CHROMOSOMAL
MUTATIONS
KARYOTYPING
 Karyotyping is the direct observation of
metaphase chromosome structure by
arranging metaphase chromosomes
according to size.
 Karyotyping requires collecting living cells
and growing them in culture in the
laboratory for 48–72 hours
 Used to detect translocations
 Karyotype: complete set of chromosomes in a
cell
 Mitogen : phytohemagglutinin added to
stimulate cell division
 Chromosome spread: cell nuclei are disrupted
w/ hypotonic saline
 Translocations can be of several types:
o Reciprocal translocation – parts of two
chromosome exchange
o Balanced translocations – when
translocation does not result in gain or
loss of chromosomal material
o Unbalanced translocations – results
when germ cells are not assorting
properly during meiosis
o Robertsonian translocation – breakpoint of both translocation partners.
movement of most of one entire
chromosome to the centromere of
another chromosome
 Deletion : loss of chromosomal material
o Large deletion can be detected using
karyotyping
o Microdeletions : not always easily seen
 Insertion : gain of chromosomal material
-Inserted sequence can arise from
duplication of particular regions w/in
the affected chromosome or from
fragments of other chromosomes
 Inversions – result from excision, flipping,
and reconnecting chromosomal material
w/in the same chromosome.
o Pericentric inversions – include
centromere in the inverted region
o Paracentric inversions – involves
sequences w/in one arm of the
chromosome
 Isochromosome : metacentric chromosome
that results from transverse splitting of the
centromere during cell division
 Ring chromosome – deletion of genetic regions
from both ends of the chromosome and a
joining of the end to form a ring
 Derivative chromosome – abnormal
chromosome consisting of translocated or
otherwise rearranged parts from two or more
unidentified chromosomes joined to a normal
chromosome.
FLUORESCENCE IN SITU HYBRIDIZATION
Interphase FISH
-widely used method to detect protein, RNA and DNA
structures
- bound probe can be visualized under a fluorescent
microscope in the nucleus of the cell.
 Probes: e designed to be specific to a particular
chromosome or chromosomal regions so that
the image under the microscope will correlate
with the state of that chromosome or region.
 Advantage: growth of cells in culture is not
required
 Commonly used to study prenatal samples,
tumors, and hematological malignancies
 Dual fusion probes - 0.8–1.5 Mb in size, are
designed to bind to regions spanning the
 Dual color break-apart probes: 0.6–1.5 Mb, are
another approach to lower background as well
as to identify translocation events where one
chromosome can recombine with multiple
potential partners.
 - the probes are designed to
bind to the intact
chromosome flanking the
translocation breakpoint.
 Centromeric probes (CEPS) - designed
to hybridize to highly repetitive alpha
satellite sequences surrounding
centromeres
o detect aneuploidy of any
chromosome
 tricolor probe – combination of CEP
and dual color probes
 telomere - unique set of repeat
sequences located just before the end
of the chromosome
 telomeric probes - are useful for the
detection of chromosome structural
abnormalities such as cryptic
translocations or small deletions that
are not easily visualized by standard
karyotyping

Metaphase FISH
 whole chromosome paints - probes that
cover the entire chromosome
o valuable for detecting small
rearrangements that are not
apparent by regular chromosome
banding
 spectral karyotyping - can distinguish all 23
chromosomes by chromosome-specific
colors
 COMPARATIVE GENOME HYBRIDIZATION
(CGH)
o Detects intrachromosomal
amplifications or deletions
 DNA from test and
reference samples is
labeled and used as a probe
on a normal metaphase
chromosome spread
 Cyanine dyes are used as
fluorescent labels for test
and reference DNA for CGH
 Two distinct dyes
 Cy3 – green
fluorescent at
550nm
 Cy5 – red
fluorescent (650-
667 nm)