Cy To Genetics
Cy To Genetics
Cy To Genetics
The human genome consists of 2.9 billion nucleotide base pairs of DNA organized into 23 chromosomes.
Alterations of the DNA sequence may affect not only the phenotype of an individual but the progeny of that
individual as well. A transmissible (inheritable) change in the DNA sequence is a mutation or polymorphism.
DNA mutations can affect a single nucleotide or millions of nucleotides, even whole chromosomes, and thus
can be classified into three categories: gene, chromosome, and genome mutations.
Euploid
A cell or cell population with a normal complement of chromosomes
Aneuploid
Aneuploidy is mostly observed as increased numbers of chromosomes because
the loss of whole chromosomes are generally not compatible with survival.
CHROMOSOME MORPHOLOGY
The centromere consists of tandem repeats of 171 base pair
sequences flanking sets of single repeat units, or monomers repeated in
groups in a higher-order repeat array. The kinetochore is a protein
structure that connects centromere chromatin to spindle apparatus.
VISUALIZING CHROMOSOMES
Fluorescent dyes
Quinacrine and quinacrine mustard.
Chemical dye
Giemsa
Human chromosome could be identified by its characteristic banding pattern. Q-banding, Required fluorescent
microscope, not widely used. G bands, similar to those seen in Q banding. R bands can also be visualized
after staining with acridine orange. Alkali treatment of chromosomes results in centromere staining, or C
banding.
Staining – dyes bind to the DNA or protein of a chromosome and allow visualization by light microscopy.
Giemsa – routine stain
Special staining
● Q – banding / quinacrine flourescence staining – routine karyotyping but replaced with G-banding which
allow permanent stain preparation.
● Rapid identification of Y chromosome
● C- banding – to evaluate constitutive heterochromatin or to determine if the chromosomes has two
centromeres.
● Centromeres appear as single dark spot
● R- banding – the telomeres should stain as dark and their absence as the result of a deletion is more
● obvious.
KARYOTYPING
is the direct observation of metaphase chromosome structure by arranging metaphase chromosomes
according to size. It can also be used to detect chromosomal mutations such as translocations, which are the
exchange of genetic material between chromosomes.
Karyotype is the complete set of chromosomes in a cell.
SPECIMEN
● Routine studies – heparinized blood.
● Hematologic disorders – bone marrow samples
● Fibroblast cultures from skin biopsies
● Not routinely done due its invasive in nature
○ liver, kidney, lung, and muscles
● Amniotic fluid – prenatal analysis
○ done between 16 – 18 weeks of gestation (20-30ml)
●
● Chorionic villus sampling – tissues from the developing placenta. 10 - 14 weeks of gestation
● Cordocentesis or percutaneous umbilical cord sampling > 20 weeks of gestation
All samples
1. must be collected in a sterile manner.
2. Must be transported to the laboratory as soon as possible.
Blood, bone marrow, amniotic fluid and chorionic villi: room temperature
Solid tissues: wet ice
Cell Culture Technique
Bone marrow and blood
aliquoted directly in culture medium
Amniotic fluid cells, CV and solid tissues
grown as a monolayer in situ.
Amniotic fluid – centrifuge
5-10 days – typical culture timE, harvested
Deletion
is a loss of chromosomal material. Large deletions
covering millions of base pairs can be detected using
karyotyping
Microdeletion is not usually detected.
Insertion
is a gain of chromosomal material.
Inversions
result from excision, flipping, and reconnecting chromo-
somal material within the same chromosome.
Pericen- tric inversions
include the centromere in the inverted region, whereas
paracentric inversions involve sequences within one arm of the chromosome. An iso- chromosome is a
metacentric chromosome that results from transverse splitting of the centromere during cell division.
INTERPHASE FISH
● are used commonly to study prenatal samples, tumors, and hematological malignancies
● fixed cells are permeabilized and exposed to a probe.
● The probe is a 60- to 200-kb fragment of DNA attached covalently to a fluorescent molecule.
● Probes are designed to be complementary to a particular chromosome or chromosomal locus so that
the image under the microscope will correlate with the state of that chromosome or locus.
● dual-color probes, or dual-fusion probes.
Centromeric probes (CEN probes) are designed to hybridize to highly repetitive alpha satellite sequences
surrounding centromeres. These probes detect aneusomy of any chromosome.
Telomeric probes are useful for the detection of chromosome structural abnormalities, such as cryptic
translocations or sub- telomeric deletions that are not easily visualized by standard karyotyping.
● Paraffin-embedded fixed tissues are dewaxed in xylene before protease and formaldehyde treatment.
● Probes are available commercially
● Both probe and target must be denatured prior to hybrid-ization.
● FISH slides should not be subjected to prolonged light exposure.
METAPHASE FISH
● allows analysis of small regions not visible by regular chromosome banding.
● Probes that cover the entire chromosome, or whole chromosome paints, are
valuable for detecting these small or complex rear- rangements
● spectral karyotyping can distinguish all 23 chromosomes by
chromosome-specific colors.
1. Separate aliquots of test and reference DNA are labeled with different Cy3 and Cy5 dyes,
2. Application to a normal meta- phase spread
3. Test DNA is partially digested with DNase
4. to produce fragments that will bind efficiently to the de- natured DNA in a metaphase chromosome
spread.
5. Although CGH requires advanced technical expertise, it has shown value in identifying recurrent
genomic im- balances not detected by karyotyping.