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What phase are cells when they are G banded?



How many chromosomes in humans?

22 autosomal pairs
1 sex pair


How are chromosomes visualised?

Cultured human cells (need dividing cells)
Extract nuclei and fix to a microscope slide
Partial digestion of chromatin (Trypsin)
Followed by staining (Leishmans)
Microscopic analysis


What are metacentric chromosomes?

centromere in the middle


What are Acrocentric chromosomes?

centromere at the end


What are Submetacentric chromosomes?

centromere neither at end or middle


What are Group A chromosomes?

Large metacentric


What are Group B chromosomes?

Large Submetacentric


What are Group C chromosomes?

Medium Submetacentric


What are Group D chromosomes?

Large Acrocentric


What are Group E chromosomes?

Small Submetacentric


What are Group F chromosomes?

Small Metacentric


What are Group G chromosomes?

Small Acrocentric


Where are chromosomes analysed from?

o Blood samples from mother and father - looking at inherited defects
o Samples are foetal epithelial cells from amniotic fluid
o Sometimes placental material
o Most samples are not actively growing and dividing (cancer cells are the exception


What is the G-Banding protocol?

1. Cells are cultured to generate mitotic cells – get the cells growing
2. Arrest cell cycle in metaphase (high mitotic index)
3. Swell nuclei with hypotonic solution (osmosis)
4. Kill cells using fixative (3:1 Methanol:Acetic Acid) – spreads samples out and also stops samples form condensing and reduce risk of staff becoming infected
5. Drop fixed sample on to a glass slide
6. Trypsin digest – creates pale bands – wash
7. Leishmans stain – wash
8. Image analysis


How does staining stain the chromosomes?

Dark bands: AT rich
Light bands: GC rich
Open chromatin is stained more – dark, better access to binding pockets
Staining is highly reproducible between samples of the same patient and between patients


What is ISCN?

international system for cytogenetic nomenclature
Centromere is p10 or q10
Numbering increases away from the centromere
Q is the longer arm
P is the shorter arm


What can differences in band resolution be due to?

1. Cell cycle stage – more condensed = more bands
2. Tissue sample
3. Experimental


How does g banding identify differences?

• Looking at differences between the banding on two homologs and for that to be liked to a clinical phenotype
• Differences can be genetic
• Subjective and analysis is dependent on competency


How is the cell cycle an issue to banding analysis?

• Asynchronous cultures:
o Contains dividing cells at all stages of mitosis, and G0 (quiescence)
o Want longer chromosomes so later through metaphase
o These will however be more overlapping


How does tissue type alter morphology?

• Blood samples give much longer chromosomes
• Foetal material is shorter
• Stem cells are even shorter – not fertility related
• Resolution may correlate to state of differentiation – more differentiated = more resolution


How does experimental technique alter analysis?

• Trypsin is a protease – cleaves peptide bonds
• Increased trypsin causes paler bands
• Overexposure causes a collapse of the chromatin – dye can’t get in


Other experimental factors which influence resolution?

1. Slide aging – let the slides dry, better banding for longer
2. Staining time – too long gives a lower resolution
3. Chromosome spread – overlapping is hard to analyse


FISH indirect labelling

• Potential for greater sensitivity than direct, but slower
• More labour intensive
• Add probe then fluorophore
• Not used clinically – is commonly used in research labs
• (nt – hepatin – fluorophore )


FISH direct labelling

• Allows for rapid diagnostic tests
• Less sensitive than indirect methods, so long probes are required
• Used in the NHS
• (nt – flourophore)


Process of metaphase and interphase FISH

• Make DNA single stranded
o heat sample to 75-78 degrees
o denature DNA a bit but not destroy structures
• Anneal probe
o 37 to 40 degrees
• Many wash steps to remove probes bound to non-complementary regions
• DAPI – used as a counter stain for both methods


What FISH techniques are used?

Chromosome Enumeration
Micro-deletion Probes
Whole Chromosome Paint


What is Chromosome Enumeration?

For common aneuploidies (X turners, Y aggression, 21 downs, 18 Edwards, 13 Patau)
• Probes tend to be very large, sometimes 100s of kb.
• Bright signal allowing rapid hybridisation times
• Bright signals allow for a rapid and less ambiguous analysis
• Probes are specific to the alpha satellite DNA sequences at the loci indicated above


What is Micro-deletion Probes?

Usually unbalanced foetal karyotypes due to “abnormal” inheritance of chromosomes from a parent with a balanced rearrangement
• Diagnostic test for Cri du Chat and SOTOS
• Often multiple diagnostic probes are combined, to save money.
• One probe is used as the +ve control for the other


What is whole chromosome paint?

Abnormal chromosome interrogation when part of the derivative chromosome is of unknown origin
• Useful to investigate structural abnormalities involving unidentifiable chromosome regions