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GiM > Techniques of Cytogenetic Analysis > Flashcards

Techniques of Cytogenetic Analysis Flashcards

1
Q

Whole genome tests

A

G-banding
Next generation sequencing
Microarrays

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2
Q

Targeted testing tests

A

FISH
MLPA
QF-PCR or qPCR

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3
Q

G-banding process

A
  1. Cell culture
  2. Mitotic arrest
  3. Hypotonic
  4. Fixation
  5. Trypsin and Leishman’s stain
  6. Banding AT&GC rich regions
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4
Q

FISH stands for

A

Fluorescent in situ hybridisation

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5
Q

MLPA stands for

A

Multiplex ligation dependent probe amplification

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6
Q

Array CGH stands for

A

Microarray comparative genomic hybridisation

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7
Q

QF-PCR stands for

A

Quantative fluorescent polymerase chain reaction

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8
Q

Process of FISH

A
  1. Label chromosomes
  2. Denature chromosomes
  3. Hybridise
  4. Washing
  5. Visualisation
  6. UV light
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9
Q

Types of probes

A

Unique sequence
Centromeric
Paints

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10
Q

Applications of FISH

A 
copy number imbalance
Aneuploidy
Confirmation/clarification of G-banding
Confirmation of array CGH
Identifying specific abnormalities in cancer
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11
Q

MLPA summary

A

Multiplex ligation-dependent probe amplification
DNA-based
Multiplex PCR
Copy number changes up to 50 different genomic locations simultaneously
Alternative to FISH

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12
Q

MLPA schematic

A
  1. Probe added to test DNA and allowed to hybridise
  2. Ligation reaction
  3. PCR amplification using a single primer pair
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13
Q

Microarray CGH summary

A

Genome-wide screen
Hybridise sample and DNA to microarray chip with 1000s of oligonucleotides
Genomic imbalances (copy number variants) at high resolution
Higher detection rates
Replaces karyotyping as 1st line test

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14
Q

Microarray CGH process

A
  1. 3ml of blood added to control DNA of same sex
  2. Co-hybridise
  3. Add to microaway
  4. Put through scanner
  5. Assess colour ratios
  6. Analyse results
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15
Q

Advantages of array CGH

A

Early diagnosis - 1st line test
High resolution = higher diagnostic hit rate
Greater accuracy of location/size of imbalances
Information on relevant genes

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16
Q

Disadvantages of array CGH

A

low level mosaics not detected
non-pathogenic and uncertain pathogenic changes detected
needs good quality DNA

17
Q

QF-PCR summary

A

PCR amplification of short tandem repeats using fluorescent primers.
Products visualised and quantified as peaks

18
Q

Prenatal aneuploidy detection process

A
  1. DNA extraction from amniotic fluid/chorionic villi
  2. PCR aplification to chromosome 13, 18, 21, X&Y
  3. DNA dosage is up to 4-5markers/chromosome

Size of product is directly related to number of ATTT repeats + repeat number is highly variable

19
Q

Microsatellite tetranucleotide repeat marker (STR)

A

Chromosome 21 maternal and paternal. Each box has a tetranucleotide repeat. Paternal has 4 and maternal has 6. Look at relation between 4 and 6 peaks to see whether chromosome repeated.

20
Q

PCR of tetranucleotide repeat is analysed using what?

A

Fluorescent sequencer - a polyacrylamide gel to separates products by size

21
Q

What is done after the tetranucleotide test is done?

A

Report made results of the test are found within 24hours.

Karyotype follow-up

22
Q

What is qPCR?

A 
Real time PCR
Quantitative comparison vs reference gene & normal control patient
Confirms small CNVs
Used when FISH unsuitable
Primer design
23
Q

What is a RQ?

A

Relative Quantitation
A method of qPCR quantification which compares patient sample vs normal control which is assessed by 2 different primer sets.

24
Q

What do you do to clarify imbalances identified via this testing?

A

Find specific size of imbalance
Trawl genomic databases
OMIM morbid genes or not - check against phenotype
FISH follow up for confirmation, parental studies
Supportive literature (leaflets)

25
Q

What sort of fertility problems can people get?

A 
Recurrent miscarriage (x3) - 2-3% carry balanced chromosome change
Infertility - 10% of infertile males have chromosome change
26
Q

Methods of prenatal diagnosis

A

Amniocentesis (16w)
Chorionic villus biopsy (CVS, 12w)
NIPT (12w)

27
Q

What does CVS stand for?

A

Chorionic villus sampling

28
Q

What does NIPT stand for? and what is it?

A

Non-invasive prenatal testing (NIPT)
Maternal blood sample to extract circulating free foetal DNA
Assesses aneuploidy of 13,18,21
Risk for aneuploidy = invasive test to confirm

29
Q

Indicators for prenatal diagnosis

A

Higher maternal age
Serum screen risk
Abnormal ultrasound scan (USS)
FH/previous chromosome abnormality

30
Q

Cytogenetics and amniotic fluid testing process

A
  1. Portion for DNA extraction (QF-PCR)
  2. Separate cells from remaining fluid
  3. Culture cells (7-14 days) if QF-PCR result abnormal
  4. G-banded analysis
31
Q

Array CGH and prenatal diagnosis

A

Replacement of cell culture if abnormal scan

Pro: increased resolution and detection rate
Con: ethical e.g. small duplication & associated incense + needs parental follow up

32
Q

What genetic change is there in leukaemia?

A

Novel fusion gene

abl (9th chromosome); bcr (22nd chromosome) bcr/abl fusion gene = abnormal protein.

33
Q

FISH detection of translocations

A

Produces fusion signal e.g. abl/bcr probes

So, normal FISH looks like 4 blobs. 2 green 2 red = normal. Abnormal = 2 r/g; one red and one green.

34
Q

What is the MYCN gene

A

A gene responsible for neuroblastoma

GiM (7 decks)

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