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Flashcards in techniques of cytogentic analysis Deck (65):
1

what 3 methods are used to analyse the whole genome

G banding
Next generation sequencing
Microarrays

2

what 3 techniques are used for targeted testing

Qf- PCR
MLPA
FISH

3

define convention genetics

look at full chromosome under microscope- must be in metaphase.

4

Molecular genetics

looks at specific parts of the human genome.

5

How is the procedure of G banding carried out

cell culture
Mitotic arrest- metaphase
Hypotonic salt solution
Fixation
Trypsin and Leishman's stain
Banding AT and GC regions
Lighter bands are AT rich and darker bands are GC rich.

6

what stain is commonly used in G banding

Trypsin or Leishmans stain

7

what is FISH used to analyse a gene or whole chromosome

both.

8

How is the procedure for FISH carried out

DNA is placed on slide along side a DNA probe with a fluorescence marker
denature probe and DNA in samples that they are single stranded.
Hybiridastion can occur and then wash of the probe

9

what are the 3 types of chromosome probes

Unique sequence- light up a small region of the genome

Centromere probe- looks at the number of copies of chromosome, as the repeat sequences around the chromosome are unique.

Paints- cover full chromosome and help locate translocations.

10

what is FISH used to detect

copy number variation- centromere probe
Aneuploidy
Confirmation/ clarification of G banding
Confirmation of array CGH
Identify speciifc abnormalities in cancer.

11

define copy number variation

A DNA segment with a variable copy no. compared with a reference genome

12

what 2 conditions can arise due to copy number variation.

Autism and cancer.

13

Is it both high and low copy number variations that predispose you to certain conditions

Yes
High copy no. of CCL3L1 = susceptibility to HIV
Low copy no. of FCGR3B = susceptibility to inflammatory autoimmune disorders

14

define MLPA

Multiplex Ligation-dependent Probe Amplification

15

what are 4 steps involved in carried out MLPA.

1- denature
2- hybridisation
3- ligate
4- Amplify.

16

How does PCR detect different sequences in MLPA apart

different size
each probe is produced with a different stuffer sequence length.

17

what is microarray CGH used for

genome wide screen.

18

what is MLPA used for

alternative to FISH
Show large numbers of copy variations.

19

what sample is microarray CGH carried out on

3 ml blood EDTA (also 1-2 ml lithium heparin blood for cell culture)

20

How is the procedure for microarray CGH work.

DNA and control DNA are labelled with a different probe
each competes for the same site on the microarray
If one colour shines brighter than another then there is a duplication or deletion.

21

what is the number of changes in a row are classed as significant enough to be classed as a duplication or deletion

3 in a row.

22

what are the advantages of microarray CGH

Early diagnosis
Looks at 8 individuals one microarray chip.
High resolution = increased diagnostic hit rate
Help to find new genomic imbalances.
Greater accuracy of location/size of imbalances
Information on relevant genes

23

what are the disadvantages of microarray CGH

Dosage changes only – not balanced rearrangements or mutations
Low level mosaics not detected
Non-pathogenic & uncertain pathogenic changes detected
Needs good quality DNA
Future technologies – analyse mutations and dosage changes, eg exomes, whole genomes

24

how is the procedure for next generation sequencing carried out.

Extract DNA
Fragment DNA
add molecular barcode
All genome of 1 individual has 1 barcode.
genome is split into different windows, look for dosage differences against controls
look at the control ratio- will show whether it is the same there is a gain or a mutation.

25

what does microarrays look for

dosage problems
deletions and duplications.

26

what does next generation sequencing look for

dosage differences.

27

what is quantitative fluorescent PCR is used look for what

aneuplodies.

28

How is the procedure for quantitative fluorescent PCR carried out.

PCR amplification of short tandem repeats (chromosome repeated DNA sequences) using fluorescent markers.
Amplifies DNA between the beginning and the end of repeats
Assess relative amounts of the patient in comparison to the control.

29

How is prenatal aneuploidy detection carried out

DNA extraction from amniotic fluid or chorionc villi
PCR amplification – primers from chromosomes 13, 18, 21, X and Y
DNA dosage in up to 4-5 markers/chromosome
aneuploidy =>2 markers with abnormal dosage

30

define microsatellite tetranucleotide repeat marker (STR)

satellite repeat sequences which can be analysed in maternal and paternal DNA to determine prenatal aneuplodiy.

31

when is qPCR carried out

When FISH unsuitable

32

How long does G banding take

30 mins - 4 hours/case

33

How long does fish take

10 mins -1 hour/case

34

how long does microarry take

10 mins -2 hours/case

35

what long does NGS/qPCR take

30 mins – 2 hours per case

36

how long does MLPA/QF PCR take

10 mins/30 mins/case

37

what is the use of cytogenetics

Diagnosis
Family studies
Assessing reproductive risks
Cancer management

38

what material can be sued to sample cytogenetics

• Blood- best type of sample
• Amniotic fluid
• Placenta
• Other foetal tissue- for spontaneous miscarriages.
• Bone marrow
• Tumour

39

With what abnormalities are people often reffered for cytogenetic anaylsis.

dysmorphic new born
gender assignment
developmental problems- intellectual, physical and sexual
heart defect
reproductive problems
family studies..

40

what sample is required for G banding

2-5 ml unclothed blood.
stimulate T lymphocytes
Culture 2-3 days.

41

what 2 chromosomes are involved in the down syndrome robertsonian trans location

Trivalent formation
meiotic paring.

42

what is the name of the complex formed when carrier is 2 chromosomes join in robersonian translocations during meiosis pairing.

Trivalent formation at pachytene

43

In what stage of meiosis 1 does the trivalent form a chain

anaphase.

44

do circular structures form in metaphase of meiosis one during robersonian translocation

no- because unlike normal structures the telomeres don’t join.

45

what are 2 types of chromosome problems commonly causes fertility problems in the populations

balanced chromosome change.
chromosome change- (47XXY or Y deletion)

46

what are the 3 forms of prenatal diagnosis

• Amniocentesis (16w)
• Chorionic villus biopsy (CVS, 12w)
• NIPT (12w)- non invasive prenatal technique.

47

which prenatal sampling can be done at the ear lies date (12 weeks) amniocentesis or chronic villas biopsy

chronic villus biposy

48

how can a NIPT (12w)- non invasive prenatal technique.
be carried out on the mother of the child

• Maternal blood sample
• Extract circulating free fetal DNA

49

How many miscarriges result due to invasive sampling methods

1 in 200

50

what causes infertility problems more commonly balanced translocations or chromosome changes (45X)

chromosome changes

51

what are non invasive prenatal testing used to confirm

aneuplodies.

52

when should you condor prenatal testing

High maternal age
serum screen risk- hormones in others blood can be different if there is a trisomy.
abnormal ultrasound scan (USS)-physical abnormalities visible
FH/previous chromosome abnormality

53

How is the procedure for cytogenetics in amniotic fluid carried out.

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

54

How is the procedure for cytogentic and chronic villus carried out

1. Separate maternal from foetal tissue
2. QF-PCR
3. Culture cells (7-14 days) if QFPCR result abnormal
4. G-banded analysis

55

how is amniocentesis diagnosis confirmed.

1. QF-PCR – allele ratios from 2 markers indicative of trisomy 21
cell culture for confirmation

56

In a spontaneous abortion 50% of the times is due to

chromosome abnormality

( Triploidy, 45,X, trisomy)

57

Preimplnatation genetic analyse

Testing abnormalities in the blastomere.
Shows dosage changes in cells
Used for individuals who don’t want to undergo invasive techniques.

58

why is cytogenetics used in cancer.

Disease specific acquired chromosome changes
– Mostly translocations
– Several different acquired abnormal clones
– Diagnosis
– Prognosis
– Treatment (stratified medicine).

59

what material can be used to test cancer

tumour or bone marrow.

60

how is the procedure for testing for leukaemia undertaken

1. 1ml unclotted bone marrow
2. Suspension culture overnight
3. G-banded analysis/FISH
Chromosome quality poor

61

most common translocation in leukaemia

• Translocation between 9 and 22- break on abl (9) and bcr (22)
• Forms bcrabl fusion gene on chromosome 22.

62

how can you test for the Philadelphia chromosome

Probe lights up different for 22 and 9 so if next to each other it shows that the translocation has occurred.

63

how is genetic testing carried out in MYCN gene amplification in neuroblastoma.

multiple repeats and copies of the gene.Increased repeats of the gene means you need chemo to be started immediately.

64

what methods of testing are used on a fresh tumour

Fresh tumour – FISH or G-banding (culture – 1-20 days)

65

what methods of testing are used on a archived tissue (paraffin embedded)

FISH or genotyping