G-Banding and karyotype analysis

Question 1

What is G-banding?

Answer 1

cytogenetic technique that has been used over the past few decades to report on gross changes to the structure of the human karyotype.

Question 2

Karyotyping
What is a karyotype?

Answer 2

A karyotype essentially refers to the genome of a cell or individual but at the visible-microscopic resolution, which offers a maximum resolution of approximately 3 megabases.

Ordered array of chromosomes, all of which come from the same nucleus

Question 3

What is aneuploidy

Answer 3

type of genetic abnormality which is defined as the gain or loss of one or a few chromosomes.

Question 4

What is approx 50% of misscarriages due to?

Answer 4

approximately 50% of all spontaneous abortions are due to the inheritance of an abnormal number of chromosomes.

Question 5

Describe Mitosis

Answer 5

Prophase – The chromosomes shorten and thicken.
Metaphase – Chromosomes line up in the middle of the cell.
Anaphase – Chromatids break apart at the centromere and move to opposite poles.
Telophase – Two nuclei formed after nuclear envelopes reform around each group of chromosomes.

Question 6

What state must the cell be in for g-banding?

Answer 6

In mitosis
Cell can be pushed with growth factors

Question 7

Interphase

Answer 7

In interphase, the genetic content of a cell is contained within the nucleus, surrounded by the nuclear lamina and nuclear envelope. As cells enter prometaphase of the cell cycle these membrane structures begin to breakdown. The nuclear lamina dissociates and the nuclear envelope breaks down into vesicles. By metaphase the genetic content of the cell exists as condensed chromosomes that are free in the cytoplasm

Question 8

What is required for karyotype anaylsis

Answer 8

Metaphase

Question 9

Prenatal investigations

Answer 9

DNA from foetus is needed

amniotic fluid - as epithelial cells (actively growing) from the foetus tend fall off during development, and are therefore suspended in this liquid.

placental tissue – although there are disadvantages such as a slightly higher risk of spontaneous miscarriage.

foetal blood - associated with a rather high rate of miscarriage and so is only used as a last resort

Question 10

Why is placental tissue not recommended?

Answer 10

Placental tissue is also known to have diverged from foetal tissues quite early on during the pregnancy – which means that it is possible for the genotype of the placenta to differ from that of the foetus

Question 11

When diagnosing an oncology patient what is the assumption?

Answer 11

that most cells in the body are normal, and we must therefore sample and analyse abnormal tissue.

Question 12

Solid tumour analysis

Answer 12

biopsied or completely removed from the patient, and then analysed.

Question 13

Haemotological malignancy (originate from bone marrow) analysis

Answer 13

samples of the bone marrow are required, as this is usually where the disease has originated – though it can be appropriate to sample the blood or lymph nodes in some cases –

Question 14

How can we distinguish if cancer has metasitised or two cancers

Answer 14

Are there the same abnormailities in the leukemiea as present in tumour – we know if cancer has metasised or different cancers.

Question 15

What happens when the tissue has been extracted from patient?

Answer 15

Processed in the lab and cells are encouraged to grow in liquid media.

Cells are needed to be in metaphase for the chromosomes to be analysable. All cancer cells are proliferative so will growth in culture media without growth factors. Might add GF to speed up time.

Question 16

What is used to stop cells from entering anaphase?

Answer 16

A microtubule inhibitor is usually added to prevent cells from entering anaphase and to therefore increase the proportion of cells in metaphase.

Question 17

What happens once cells are in metaphase?

Answer 17

We then swell the nuclei by osmosis to increase the physical space between chromosomes. This reduces the chance of chromosomes overlapping one another once they have been immobilized on a microscope slide. This alone is sufficient for most oncology samples (particularly leukaemias) because the abnormal cells are already actively growing and dividing – unlike the majority of cells in a healthy adult.

Question 18

How does anaylsis on constitutional samples differ?

Answer 18

the cells require hormonal stimuli to kick start division, and are usually synchronized before the mitotic block to encourage a greater enrichment of metaphases. This is done by hijacking the S-Phase checkpoint. dTTP is added to excess which signals to cells to halt the cell cycle (the cell thinks DNA replications is over). dCTP can then be added to over-ride this and release cells back into the cycle.

Question 19

Describe the last steps

Answer 19

cells are then fixed using a mixture of acetic acid and methanol.
, chromosomes are adhered to a glass microscope slide

Chromosomes then require a period of ageing, where they are exposed to sunlight at room temperature for a period of approximately 48 hours – though there are ways to speed this up for urgent samples. To denature proteins, remove residual fixative, enhance adherence to the glass slide and remove water from the chromosomes which significantly improves banding quality.

Question 20

How are the characteristic and reproducible light and dark banding structure generated?

Answer 20

chromosomes need to be digested with a protease (trypsin) and stained with a methylene blue based chemical stain called Leishman’s dye.

Question 21

Theory behind using trypsin

Answer 21

Trypsin degrades histone proteins and causes the local chromatin structure to collapse.
Chromatin architecture is not uniform across the genome. Highly transcribed regions (aka euchromatin) = open conformation in comparison to regions with little or no transcriptional activity (aka heterochromatin).
Histones within highly condensed heterochromatic regions are less accessible to the protease and are therefore relatively shielded from digestion in comparison to histones located in euchromatin.

Exposure to trypsin has therefore helped to collapse chromatin right across the chromosome, but this collapse has been more extreme in regions of euchromatin.

Question 22

Explain different between dark and pale bands

Answer 22

The dark bands you see above have bound more dye than other regions and therefore correspond to the more open regions of chromatin.

The pale regions by comparison have not taken up the dye quite so well – this is because the chromatin is far more collapsed –

Question 23

Explain the staining process in regards to GC and AT regions

Answer 23

In GC regions (stain pale) they are rich in genes and are actively transcribed and chromatin is open. Trypsin has better access to open chromatin. Therefore, level of collapse and histone digestion is greater.

AT regions (stain dark) are less transcribed as more compact. Therfore trypsin doesnt digested as much compared to GC regions. So chromosome is more open and dye can get in leading to darker staining.

Question 24

Describe and draw chromosome morphology and structure

Answer 24

Centromere (marks boundary between each arm)

P arm - top arm, smaller
Q arm - bottom arm, longer

Question 25

Metacentric

Answer 25

as the position of the centromere is roughly in the middle of the chromosome, making p and q arms roughly the same size as one another.

Chromosome 3

Question 26

Submetacentric

Answer 26

The p arm of chromosome 9 is clearly much smaller than the q

Chromosome 9

Question 27

Acrocentric

Answer 27

The p arm is actually satellited, and rich with repetitive sequences, such as the rDNA genes.

Chromosome 21

Question 28

What must be analysed in G-banding?

Answer 28

the size of the chromosome, position of the centromere and banding structure must all be carefully analysed, and compared with the homolog, and what is considered to be “normal” for each chromosome

essential to analyse multiple cells to check for evidence of mosaicism, and compare homologs from each of these cells for subtle abnormalities – which may appear more clearly in some cells than others.