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1

what techniques are used for testing whole genomes?

Next generation sequencing, microarray, G banding

2

what techniques are used for target testing

fish, MLPA, QF-PCR or qPCR

3

what sample is used for prenatal diagnosis?

amniotic fluid and chorionic villus

4

what sample is used for postnatal diagnosis?

blood, products of conception

5

what samples are used for oncology?

solid tumours, leukaemia

6

what occurs in G banding

Cell culture --> Mitotic arrest --> Hypotonic --> Fixation --> Trypsin & Leishman’s stain --> Banding - AT and GC rich regions
AT --> dark bands
CG --> light bands

7

what is fish?

Detection of DNA material on slides using fluorescent
Dyes & UV light

8

what is the process of Fish?

start with the specific region of interest --> chromosome, part of a chromosome and so on
labelling, denaturation, hybridsation, visualisation and then with UV light can see
takes 24 hours

9

what are the three types of probes?

Unique sequence probe --> just light up a small region --> couple of regions not a lot --> specific part of the choromosome
Centromeric --> tell you the total number of copies
Paint -->labels complete chromosomes --> see whole chromosomes

10

what is the application of FISH?

Copy number imbalance

Aneuploidy

Confirmation/ clarification of G-banding

Confirmation of array CGH

Identifying specific abnormalities in cancer

11

what is the consequence of having a High copy no. of CCL3L1 ?

reduces your suscepitbility of HIV

12

what gene copy increase the suceptibility of inflammatory autoimmune disorders

Low copy no. of FCGR3B

13

what are the Molecular cytogenetic methods to assess copy number

FISH

MLPA

Microarray CGH

Next generation sequencing

QF-PCR

qPCR

14

what is MLPA?

Multiplex Ligation-dependent Probe Amplification

DNA-based  extract DNA from sample in question

Multiplex PCR

Copy no. changes in up to 50 different genomic locations simultaneously

Alternative to FISH

15

what is microarray CGF?

Genome-wide screen

Hybridise sample & control DNA to a microarray “chip” 1000s of DNA spots (oligonucleotides)

Genomic imbalances (copy number variants) at high resolution (10-10000x conventional cytogenetics)

detection rates

Replacing karyotyping as 1st line test

16

what are the requirments of microaaray CGF?

3ml blood in EDTA (also 1-2ml lithium heparin blood for cell culture if follow up studies needed)

Control DNA from same sex

17

what is the present structure of Microarray Chips? What software is used?

8x60K oligonucleotide chips

Allows data from microarray scanner to be read & interpreted
Analyst – checks variants flagged for pathogenicity

18

what does array CGH show?

any loss or gain of chromosome. --> 1 is the normal and deviation show either loss or gain at a specific point

19

what is needed for Determining regions of potential copy number change

At least 3 oligonucleotides required for any call

Call imbalances >150000 bases, ie 150 Kb

Database searches to ascertain pathogenicity of imbalances

20

what is the advantage of array CGH?

Early diagnosis -1st line test, reduces need for other tests and avoids the “diagnostic odyssey”

High resolution = increased diagnostic hit rate

Greater accuracy of location/size of imbalances

Information on relevant genes

21

what is the disadvantage of array CGH?

Dosage changes only – not balanced rearrangements or mutations

Low level mosaics not detected

Non-pathogenic & uncertain pathogenic changes detected

Needs good quality DNA

22

what happens in next generation sequencing?

Sequencing libraries are generated by fragmenting genomic DNA (up to1µg per library) & adaptor ligation

23

what is the implications when anaylsising data from next generation sequencing?

increase in test:control ratio = gain
decrease in test:control ratio = deletion

24

what occurs in Quantitative Fluorescence PCR (QF-PCR) ?

is an alternative method in which DNA polymorphic markers on chromosomes, is used to determine the presence of different alleles.

25

what occurs in QF-PCR

PCR amplification of short tandem repeats (STRs) [chromosome-specific, repeated DNA sequences] using fluorescent primers

Products visualised & quantified as peak areas using an automated DNA sequencer

26

what occurs in prenatal aneuploidy detection?

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

27

what is qPCR

Quantitative comparison vs reference gene & normal control patient (amplify & quantify)

Confirming small Copy number variation

When FISH unsuitable

Primer design

28

what does qPCR look for and what is its reference?

Relative Quantitation (RQ) - compares difference in concentration between patient sample & normal control assessed by 2 different primer sets

RQ value is expressed as a ratio relative to 1 - a deletion has an expected value of 0.5 & a duplication an expected value of 1.5

29

how long does each analysis take?

G-banding = 30 mins - 4 hours/case

FISH = 10 mins -1 hour/case

Microarrays = 10 mins -2 hours/case

NGS/qPCR = 30 mins – 2 hours per case

MLPA/QF-PCR = 10 mins/30 mins/case

30

what samples are used in cytogenetics?

Blood

Amniotic fluid

Placenta

Other foetal tissue

Bone marrow

Tumour