(M) Cytogenic techniques (manual based) Flashcards
study of the physical size and structure of chromosomes
cytogenetics
A specialized laboratory discipline that examines the structure and behavior of chromosomes at the cellular level
cytogenetics
Which phase is the best for studying the physical characteristics of chromosomes
cell cultures that are halted near metaphase
why are cell cultures halted near metaphase the best for studying the physical characteristics of chromosomes
chromosomes are most compact
what are the chromosomes stained in the form of?
(cell cultures haltd near metaphase)
dyads or bivalent
study of ______ _______ ______ and number can be vital for understanding a variety of genetic abnormalities that are produced by mutations that alter chromosomes
normal chromosome structure
What are the two techniques used to visualize chromosome changes
- classical / standard cytogenic method
- Fluorescent in situ hybridization (FISH)
this method allows visualization of loss or gain of genetic material that contains at least 4 megabases of DNA
classical / standard cytogenic method
DNA needs to contain at least how many bases for standard cytogenetic method?
4 megabases
This is the gold standard in providing a whole genome scan
standard cytogenic method
What does classical cytogenetics require
viable, nucleated cells in cell cycle
how long are the nucleated cells cultured for classical cytogenetics?
one day to two or more weeks
T or F
All abnormalities can be detected using the classical cytogenetic technique as it has the best microscopic resolutions
F ( certain abnormalities may not be detected due to limitations in microscopic resolutions)
This visualization method allows much smaller changes in chromosomes to be seen
Fluorescent in situ hybridization (FISH)
What is the principal molecular technique currently used in the clinical lab?
Fluorescent in situ hybridization
What is the other name for FISH?
molecular cytogenetics
What was FISH originally developed for?
to enhance the resolution of chromosome observation
This is the complete set of chromosomes in the cells of an organism
Karyotype
how many somatic pairs of chromosomes do we have
22 pairs
how many pairs of chromosome do humans have in total?
23
how many pairs of sex chromosomes do we have?
one pair
T or F
Changes in karyotype is indifferent with the changes of the phenotype of the individual
F (it is correlated with the changes)
how are the different types of chromosomes distinguished?
using dyes
What do dark bands represent
heterochromatins
T or F
heterochromatins are rich in protein-encoding genes
F (have few protein-encoding genes)
What do you call the light bands / staining
euchromatins
what do euchromatins contain
active genes
how are chromosomes identified
(3)
- based on their length
- banding patterns
- location of centromere
how are chromosomes arranged in a karyotype
from largest to smallest, followed by sex chromosomes
A routine specimen for most cytogenetic studies of adults and children
heparinized peripheral blood
how is heparinized peripheral blood sample obtained?
standard phlebotomy technique
what is the handling and transport condition for heparinized peripheral blood smear?
room temp
used for studying hematologic disorders as the sample is the origin of the disease
bone marrow aspirates
what is the handling and transport condition for bone marrow aspirates
room temp
used for fibroblast cultures
skin biopsies
what is the handling and transport condition for skin biopsies
ice
These specimens are not routine, and are obtained after death or fetal loss
tissues like
* kidney
* liver
* muscle
* lung
what is the handling and transport condition for kidney, liver, muscle, and lung
ice
two samples used for prenatal analysis
amniotic fluid
chorionic villus
what is the handling and transport condition for amniotic fluid
room temp
what the handling and transport condition for chorionic villus
room temp
where is chorionic villus extracted from?
developing placenta
This sample is used for rapid karyotyping
fetal blood from umbilical cord
what is the handling and transport condition for fetal blood
room temp
what type of technique should be done to collect the specimens mentioned in the table?
aseptic techniques
What should be the temperature for the samples that needs to be iced?
4 degrees celsius
use this to familiarize yourself with the steps of cell culture and harvesting
- cells are cultured using mitotic agents
- cell division is arrested at metaphase using a mitotic inhibitor like colcemid
- cells are swelled by applying a hypotonic buffer
- Swollen cells are fixed using a modified Carnoy’s fixative
- Cells cultured in suspension are spread on a glass slide by dropping at a certain height
- slides are air dried and are ready for staining
- cells are stained using Giemsa or other stains
- A photograph of the stained metaphase spread is taken through a microscope
- case report is made, may include a copy of the karyotype and sent to the referring physician
substances that stimulates cells like lymphocytes to proliferate
mitotic agents (PHA)
This agent prevents spindle fiber formation and damages those that are already present to prevent anaphase
colcemid
example of hypotonic buffer
0.075 M KCl
at what temp are cells swelled at with a hypotonic buffer and for how long?
37 degrees Celsius for 10-20 minutes
This removes lipids and water molecules and denatures proteins
modified Carnoy’s fixative
what is modified Carnoy’s fixative composed of?
3:1 absolute methanol: glacial acetic acid
what are the slides subjected to, to improve the quality of staining
artificial aging
at what temp is artificial aging and for how long
65 degrees Celsius for 30-60 minutes
T or F
Single cell analysis showing abnormalities is conclusive
F (not conclusive)
meaningful cytogenetic studies require analysis of how many cells?
15-20 cells
how many cells showing loss of or extra same chromosome are needed to represent a true abnormality
two or more
A special descriptive language for reporting classical cytogenetic and FISH results
International System of Cytogenetic Nomenclature (ISCN)
What stain is used for routine technique in staining chromosome spreads
Giemsa
a positively charged dye that binds to the negatively charged DNA molecules
Giemsa
What banding pattern does Giemsa produce
G-banding pattern
What are chromosomes treated with before being stained with Giemsa?
trypsin
What base pairs are heterochromatins rich in?
A-T base pairs
what base pairs are euchromatins rich in?
G-C base pairs
_______ segments of the genome replicate very late in the S phase of the cell cycle
heterochromatin
banding technique that was the first to be developed
Q banding technique
why was q banding technique replaced with g banding ?
due to short-lived fluoresence
What are the chromosomes stained with in q banding technique?
fluorescent dye
examples of fluorescent dye for q banding
Quinacrine
DAPI (4’,6-diamino-2-phenylindole) / Hoeschst 33258
technique mainly applied for the rapid identification of the Y chromosome
Q banding
what part of the y chromosome would fluoresce brightly when Q banding technique is used?
distal Q arm
The reverse of the G banding pattern
R banding
why do heterochromatins produce light colored bands in R banding?
AT-rich regions are denatured with heat
What does R banding detect
deletion in the telomeres
are telomeres light stained or dark stained in R banding?
dark
are telomeres light stained or dark stained in G banding?
light
this technique stains the telomeres by treating it with an alkali solution and staining with giemsa
T-banding
Used to identify specific chromosomes
C-bands
how are specific chromosomes identified in c banding?
using the location of their centromeres
used to identify the presence of dicentric chromosomes
c-banding
identifies telomeres
T-banding
uses severe heat prior to staining
T-banding
well-characterized fragment of nucleic acid
probe
T or F
probes should be small to hybridize with its target
F (should be large enough)
How are in situ hybridization assays labeled
with radioisotpes
how are the radioisotopes visualized
exposure to x ray
or
autoradiography using a light microscope
T or F
Probes can be applied to your chromosome spread
T
used to detect and localize the presence or absence of specific DNA sequences on chromosomes
FISH
these bind only to parts of the chromosomes where they show a high degree of similarity
fluorescent probes
What can fluorescence microscopy identify
aneuploidies, deletions, or translocations
familiarize general FISH protocol
- slide preparation
- place probe on slide, cover with coverslip,
- denaturing step (75’C)
- hybridization (37’C)
- Detection using a Fluorescence microscope
uses of FISH (3)
diagnosis
prognosis
Evaluate remission of a disease (cancer)
T or F
classical cytogenetic techniques are more sensitive in detecting subtle changes in chromosome features
F (FISH)
T or F
FISH distinguishes one disease from another
T
T or F
classical cytogenetic techniques are more labor intensive compared to FISH
T
T or F
FISH results can be quantified automatically
T
T or F
classical cytogenetic technique detects diseased cells more easily
F (FISH)
enumerate the 5 advanced application of FISH
- Whole chromosome painting
- Multicolor FISH
- SKY (spectral karyotyping)
- All-telomere FISH
- Comparative genomic hybridization
this application of FISH uses many probes from a single chromosome and is good for seeing rearrangements
Whole chromosome painting
application of FISH that uses a computer assisted system
Multicolor FISH
Application of FISH that has multiple chromosomal aberrations
SKY
Application of FISH that recognizes the six base repeat present at the ends of all chromosome
All-telomere FISH
All telomer FISH confirms the presence or absence of _____
telomeric regions
Application of FISH that involves co-hybridization of 2 DNA samples
comparative genomic hybridization
this directly compares the DNA content of differentially labeled normal and tumor cell population by their co-hybridization to normal metaphase chromosome spreads
comparative genomic hybridization
what does comparative genomic hybridization detect in a DNA sequence
CNV
Copy number variation
The comparison of labeled normal and tumor cells by their hybridization to series of genomic DNA oligonucleotides aligned on a glass slides and serve as prbes
microarray hybridization
DNA chip technology
how many features can be places on one solid surface
hundreds to thousands
last card if ur a concrete sequential learner <3
magpakaryotype kayo bago kayo mang buntis / mag pa buntis
