LESSON 2: CYTOGENETIC TECHNIQUES

Question 1

Standard display of stained and photographed chromosomes in metaphase spread, arranged in pairs, in order of decreasing length.

Answer 1

Karyotyping

Question 2

Human somatic cells

Answer 2

22 pairs of autosomes

Question 3

Karyotyping Steps

Answer 3

1. Sample
2. Culture
3. Arrest of C.D
4. Cell harvested
5. Cell Fixation
6. Staining
7. Microscopic analysis and photography
8. Karyotype production (manual/automated)
9. Interpretation

Question 4

Preservative-free sodium heparin, mitotic stimulant (phytohemagglutinin), and antibiotics (penicillin, streptomycin).

Answer 4

Culture medium

Question 5

Short-term culture:

Answer 5

2-3 days (blood, bone marrow, chronic villi)

Question 6

Long-term culture:

Answer 6

-3 weeks (other tissue types)

Question 7

Arrest of cell division:

Answer 7

at metaphase by Colcemid for 20 mins

Question 8

Cell Harvested:

Answer 8

centrifuge → incubated for 10 mins. in hypotonic solution (dilute solution of KCl 0.075 mol).

Question 9

Cell Fixation

Answer 9

3:1 methanol/glacial acetic acid mixture (carnoy’s solution) for 30 mins.

Question 10

Staining:

Answer 10

trypsinization of the chromosomes prior to staining weakens the DNA-Protein interactions, add buffer (Na2HPO4 and NaH2PO4) → banding techniques done with the dyes.

Question 11

A molecular cytogenetic technique that uses fluorescent probes that bind only those parts of the chromosome with a high degree of sequence complementarily

Answer 11

FLUORRESCENT IN SITU HYBRIDIZATION (FISH)

Question 12

Used to detect and localize the presence or absence of specific DNA sequences (microdeletion) on chromosomes, and chromosome rearrangements

Answer 12

FLUORRESCENT IN SITU HYBRIDIZATION (FISH)

Question 13

refers to the binding or annealing of complementary DNA or RNA sequences

Answer 13

Hybridization

Question 14

*These methods are most commonly used for in situ hybridization

Answer 14

Frozen sections
Paraffin-embedded sections
Cells in suspension

Question 15

are complimentary sequences of nucleotide bases to the specific mRNA sequences of interest.
Short sequence of nucleic acid (20-40 base pairs) or be up to 1000 bp.

Answer 15

FISH PROBE

Question 16

these types of probes can be produced in two ways:
by bacteria, and
PCR.
*This type of probe is rarely used.

Answer 16

Double-Stranded DNA Probes

Question 17

These types of probes can be produced in two ways:
by reverse transcription of RNA or
PCR

Answer 17

Single Stranded DNA Probes

Question 18

also known as cRNA probes. RNA probes are still most probably the most widely used probes for in situ hybridization.

Answer 18

RNA Probes

Question 19

oligonucleotides are short sequences of nucleotides (RNA or DNA), typically with 20- 30 bases in size that are synthesized in vitro.This is ideal for in situ hybridization because their small size allows for easy penetration into the cells or tissue of interest.

Answer 19

Oligonucleotide Probes

Question 20

binds to highly repetitive sequence alpha satellite sequences of centromere and produces strong signals. Similar sequences in the pericentric region result in cross-hybridization artifacts.

Answer 20

Centromere enumerating probe (CEP)

Question 21

target distinct chromosomal region of interest and utilize single copy rather than repetitive DNA

Answer 21

Locus Specific Identifier (LSI) Probe

Question 22

also known as chromosome painting probes or chromosomes libraries, consisting of thousands of overlapping probes that recognize unique and moderately repetitive sequences along the entire length of individual chromosomes

Answer 22

Whole Chromosome Probes

Question 23

STEP INVOLVED IN FISH:

Answer 23

1. Probe Selection
2. Probe generations
3. Probe labeling
4. Fixation of tissues
   5.Hybridization and Washing
5. Detection
6. Observation

Question 24

the first step of in situ hybridization is a selection of probe types.

Answer 24

Probe Selection

Question 25

two methods of generation of probes.

Answer 25

Nick translation PCR using tagged nucleotides

Question 26

are used for labeling include 3H, 32P, or 35S, but 14C and 125I have also been used.

Answer 26

Radioisotopes

Question 27

labeling Biotin and Digoxigenin are commonly used

Answer 27

Non-radioactive

Question 28

best probe penetration, but may permit the loss of RNA from tissue.

Answer 28

Acetic acid-alcohol mixture:

Question 29

provides the best RNA retention and tissue morphology, but because of extensive protein cross-linking the probe penetration is low.

Answer 29

Glutaraldehyde

Question 30

leads to decreased sensitivity possibly resulting from increased cross-linking or loss of mRNA during embedding.

Answer 30

Paraffin & Formalin:

Question 31

the most widely successful fixative solution. This provides a satisfactory compromise between the variable and good sensitivity.

Answer 31

4% paraformaldehyde solution:

Question 32

is obtained by heating the DNA, which separates the two strands and allows access of the single-strand.

Answer 32

Denaturation

Question 33

the final step of fluorescent in situ hybridization It consists in recognizing the probes with fluorescent antibodies corresponding antigens incorporated in the probes.

Answer 33

Detection

Question 34

is a cytogenetic (chromosome) laboratory technique in which FISH (fluorescence in situ hybridization) is done on chromosomes that have been mechanically stretched.

Answer 34

Fiber FISH

Question 35

a technique used to produce an array of uniformly stretched DNA that is highly suitable for nucleic acid hybridization studies such as FISH; also known as molecular combing or DNA combing.

Answer 35

Chromosome Combing

Question 36

Also known as ‘Multicolor fluorescence in-situ hybridization’. It is a 24-colour, multi-chromosomal painting assay that allows visualization of all human chromosomes in one experiment.

Answer 36

SPECTRAL KARYOTYPING