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ems -gid > week 6 > Flashcards

week 6 Flashcards

1
Q

what techniques are used for testing whole genomes?

A

Next generation sequencing, microarray, G banding

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2
Q

what techniques are used for target testing

A

fish, MLPA, QF-PCR or qPCR

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3
Q

what sample is used for prenatal diagnosis?

A

amniotic fluid and chorionic villus

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4
Q

what sample is used for postnatal diagnosis?

A

blood, products of conception

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5
Q

what samples are used for oncology?

A

solid tumours, leukaemia

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6
Q

what occurs in G banding

A

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

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7
Q

what is fish?

A

Detection of DNA material on slides using fluorescent

Dyes & UV light

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8
Q

what is the process of Fish?

A

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

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9
Q

what are the three types of probes?

A

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

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10
Q

what is the application 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

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

A

reduces your suscepitbility of HIV

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12
Q

what gene copy increase the suceptibility of inflammatory autoimmune disorders

A

Low copy no. of FCGR3B

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13
Q

what are the Molecular cytogenetic methods to assess copy number

A

FISH

MLPA

Microarray CGH

Next generation sequencing

QF-PCR

qPCR

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14
Q

what is MLPA?

A

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

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15
Q

what is microarray CGF?

A

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

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16
Q

what are the requirments of microaaray CGF?

A

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

Control DNA from same sex

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17
Q

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

A

8x60K oligonucleotide chips

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

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18
Q

what does array CGH show?

A

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

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19
Q

what is needed for Determining regions of potential copy number change

A

At least 3 oligonucleotides required for any call

Call imbalances >150000 bases, ie 150 Kb

Database searches to ascertain pathogenicity of imbalances

20
Q

what is the advantage of array CGH?

A

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
Q

what is the disadvantage of array CGH?

A

Dosage changes only – not balanced rearrangements or mutations

Low level mosaics not detected

Non-pathogenic & uncertain pathogenic changes detected

Needs good quality DNA

22
Q

what happens in next generation sequencing?

A

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

23
Q

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

A

increase in test:control ratio = gain

decrease in test:control ratio = deletion

24
Q

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

A

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

25
Q

what occurs in QF-PCR

A

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
Q

what occurs in prenatal aneuploidy detection?

A

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
Q

what is qPCR

A

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

Confirming small Copy number variation

When FISH unsuitable

Primer design

28
Q

what does qPCR look for and what is its reference?

A

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
Q

how long does each analysis take?

A

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
Q

what samples are used in cytogenetics?

A

Blood

Amniotic fluid

Placenta

Other foetal tissue

Bone marrow

Tumour

31
Q

when will there be referal to a blood cytogenetic study?

A

Dysmorphic newborns

Gender assignment

Developmental problems – intellectual, physical, sexual

Heart defect

Reproductive problems

Family studies

32
Q

what type of blood is needed for G banding?

A

2-5ml unclotted

Stimulate T-lymphocytes

Culture 2-3 days

33
Q

if there is a robertsonian translocation of 21:21 then what is the recurrent rate?

A

100%

34
Q

if there is robertsonian translocation of 14:21 for down syndrome what is the recurrent rate for males and females

A

paternal –> 2% because the less viable sperm don’t survive

maternal –>12 %

35
Q

what are the times you can have prenatal testing?

A

Amniocentesis (16w)  invasive test

  1. Chorionic villus biopsy (CVS, 12w)  invasive test
  2. NIPT (12w)  non invasive
36
Q

what are the types of non invasive prenatal testing?

A

Maternal blood sample

Extract circulating free fetal DNA

Assess aneuploidy of 13, 18, 21 (NGS)

Risk for aneuploidy – invasive test to confirm

Reduces no. of invasive tests

37
Q

indicators for prenatal diagnosis?

A

increase in maternal age

serum screen risk

abnormal ultrasound scan (USS)

FH/previous chromosome abnormality
38
Q

what is the procedure for cytogenics and amniotic fluid

A

Portion for DNA extraction (QF-PCR)

Separate cells from remaining fluid

Culture cells (7-14 days) if QFPCR result abnormal

G-banded analysis

39
Q

what is the produced for cytogenics and chorionic villi?

A

. Separate maternal from foetal tissue

. QF-PCR

Culture cells (7-14 days) if QFPCR result abnormal

G-banded analysis

40
Q

what is advantage of array CGH and prenatal diagnosis

A

Increased resolution

Higher detection rate

41
Q

what is the disadvantage of array CGH and prenatal diganosis?

A

Ethical, eg small duplication & associated autism

Need parental follow up

42
Q

what percentage of spontaneous abortion is due to chromosome abnormality?

A

50%

43
Q

how is cytogenic done for looking at leukemia?

A

1ml unclotted bone marrow

  1. Suspension culture overnight
  2. G-banded analysis/FISH
44
Q

what is the translocation of chronic myleoid leukemia?

A

t(9;22)

abl gene of chromsome 9 and bcl gene of chromosome 22 translocate

45
Q

what test are used for fresh tumor?

A

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

46
Q

what testing is done for archive tissue?

A

(paraffin embedded) – FISH or genotyping

47
Q

what gene is invovledin neuroblastoma?

A

MYCN gene amplification

ems -gid (7 decks)

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