Karyotyping practical

Question 1

What are the two arms of chromsomes?

Answer 1

p arm - short arm
q arm - long arm

Question 2

What pair of chromosomes in humans are not homologous pairs?

Answer 2

23 - the sex chromosomes

Question 3

How are chromosomes arranged in a karyogram?

Answer 3

In homologous pairs
In order from 1-22 then x/y, number one is the longest 22 is the shortest
Often taken from cells in cell division as chromosomes are condensed and visible

Question 4

How is each chromosome subdivided?

Answer 4

Chromosome number (1-22, x/y)
P arm (short) or q arm (long arm)
Then into subregions
Then into bands
These subregions and bands are different densities so stain different colours on the karyotype
Examples 1q2.4 chromosome 1 long arm, subregion 2 band 4.

Question 5

How are chromosomes classified based on their relative position of the centromere?

Answer 5

Metacentric - middle
Submetacentric - between middle and end
Acrocentric - close to end
Telocentric - at end

Question 6

What are the different groups of chromosomes and how are they classified?

Answer 6

Group A - large and metacentric chromo 1-3
Group B - large and submetacentric 4-5
Group c - mediaum and submetacentric 6-12 and X
Group D - medium and acrocentric 13-15
Group E - relaticly short, submetacentric 16-18
Group F - short meta or submetacentric 19-20
Group G - short acrocentric, 21-22 Y

Question 7

What do cytogenetic services offer?

Answer 7

Testing for foetal abnormality or cancer genotypes (translocation chromosomes)
Interpret karyograms for abnormalities including translocations or chromosomal abnormalitites (numerical and structural)

Question 8

What tissues can be used for a karyogram analysis?

Answer 8

Blood
Amniotic Fluid
Bone Marrow
Placenta

Question 9

What is done to a sample before it can be used to make a karyotype?

Answer 9

Cultured - cell replication
Synchronise and stop in metaphase - greater proportion of cells in metaphase by manipulation of culturing.
Colchicine - disrupts spindle fibres to stop at metaphase

Question 10

Why do we want chromosomes in metaphase for karyotyping?

Answer 10

Chromsomes at the most condensed so visible and arranged in homologous pairs.

Question 11

What type of analysis and staining is used to produce a karyogram?

Answer 11

Use G-band analysis
Fixation - 3:1 methanol to acetic acid extractions some histones
Tyrosine - removes proteins from GC rich regions, without these proteins these regions stain pale
Pretreatment with proteolytic enzymes such as trypsin
Rinsed in saline than stained in Giemmsa - gives a dark stain in hydrophobic AT rich regions of DNA.
Giemmsa stain should be added to the area over the metaphase spread, then incubate the slide for 15mins
Rinse with deionized water, and incubate for five minutes until dry.
Using a new smaller pippette adds drops of mounting medium to cover the stained area.
Carefully at the coverslip at a 45 degree angle
View under a light miscroscope

Question 12

What is a karyotype?

Answer 12

A complete chromosome set from a cell.

Question 13

How does chromosome compaction correlate to G-banding stain?

Answer 13

Dark bands - more condensed
Light bands - less condensed, easier for tyrosine to enter and remove protein

Question 14

What is the q-banding process of creating a karyotype?

Answer 14

Chromosomes are treated with quinacrine mustard, patterns are observed by placing a sample under an ultraviolet light microscope.
Chromosomes show will bright flourescent bands

Question 15

What is the process of R-banding to produce a karyogram?

Answer 15

Chromosomes are treated with acridine orange and observed with a light microscope.
results in a darkly stained centromere region.
Also uses a giemsa stain - results in opposite patterning to G-banding.

Question 16

Why is Fluorescence in Situ Hybridisation becoming more common over a traditional karyogram?

Answer 16

Overcome the limitation of the light microscope
Enable visualisation of small mutations that are not normally seen with traditional cytogenetics
Allows for faster analysis, less specialisation is required.

Question 17

What is cytogenetics?

Answer 17

The study of inheritance in relation to the structure and function of chromosomes.

Question 18

How does colchicine work in cells and why is it useful for metaphase spreads?

Answer 18

Is an anti-mitotic drug
Inhibits tubulin formation, this prevents the spindle apparatus from forming, this stops the cell division at metaphase by preventing the elongation of microtubules, spindle fibres are unable to connect to the centromere and unable to pull sister chromatids apart to opposite poles of the cell.

Question 19

Why are certain chromosomal abnormalities severely detrimental or lethal, while other abnormalities
are relatively mild?

Answer 19

The lethality of a mutation depends on the genes it affects.
Some abnormalities are tolarated during development, such as changes in the sex chromosomes, which only have a great affect towards puberty and later gestation.
larger disruptions to the genome are more likley to cause developmental problems that are not compatible with life

Question 20

Why does balanced reciprocal translocation in an individuals genome often result in a healthy phenotype?

Answer 20

Still have their entire genome

Question 21

Balanced translocations between two chromosomes still often result in healthy individuals.
How can such a translocation lead to an abnormal phenotype in the offspring even if the parents are
both healthy?

Answer 21

During the formation of gametes some genetic material may be lost from some gametes, others will have addition genetical material
In the resulting zygote, the genome will not be balanced, which may result in abnormalities that are not compatible with life.
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