week 6 Flashcards
what techniques are used for testing whole genomes?
Next generation sequencing, microarray, G banding
what techniques are used for target testing
fish, MLPA, QF-PCR or qPCR
what sample is used for prenatal diagnosis?
amniotic fluid and chorionic villus
what sample is used for postnatal diagnosis?
blood, products of conception
what samples are used for oncology?
solid tumours, leukaemia
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
what is fish?
Detection of DNA material on slides using fluorescent
Dyes & UV light
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
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
what is the application of FISH?
Copy number imbalance
Aneuploidy
Confirmation/ clarification of G-banding
Confirmation of array CGH
Identifying specific abnormalities in cancer
what is the consequence of having a High copy no. of CCL3L1 ?
reduces your suscepitbility of HIV
what gene copy increase the suceptibility of inflammatory autoimmune disorders
Low copy no. of FCGR3B
what are the Molecular cytogenetic methods to assess copy number
FISH
MLPA
Microarray CGH
Next generation sequencing
QF-PCR
qPCR
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
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
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
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
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
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
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
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
what happens in next generation sequencing?
Sequencing libraries are generated by fragmenting genomic DNA (up to1µg per library) & adaptor ligation
what is the implications when anaylsising data from next generation sequencing?
increase in test:control ratio = gain
decrease in test:control ratio = deletion
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.
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
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
what is qPCR
Quantitative comparison vs reference gene & normal control patient (amplify & quantify)
Confirming small Copy number variation
When FISH unsuitable
Primer design
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
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
what samples are used in cytogenetics?
Blood
Amniotic fluid
Placenta
Other foetal tissue
Bone marrow
Tumour
when will there be referal to a blood cytogenetic study?
Dysmorphic newborns
Gender assignment
Developmental problems – intellectual, physical, sexual
Heart defect
Reproductive problems
Family studies
what type of blood is needed for G banding?
2-5ml unclotted
Stimulate T-lymphocytes
Culture 2-3 days
if there is a robertsonian translocation of 21:21 then what is the recurrent rate?
100%
if there is robertsonian translocation of 14:21 for down syndrome what is the recurrent rate for males and females
paternal –> 2% because the less viable sperm don’t survive
maternal –>12 %
what are the times you can have prenatal testing?
Amniocentesis (16w) invasive test
- Chorionic villus biopsy (CVS, 12w) invasive test
- NIPT (12w) non invasive
what are the types of non invasive prenatal testing?
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
indicators for prenatal diagnosis?
increase in maternal age
serum screen risk abnormal ultrasound scan (USS) FH/previous chromosome abnormality
what is the procedure for cytogenics and amniotic fluid
Portion for DNA extraction (QF-PCR)
Separate cells from remaining fluid
Culture cells (7-14 days) if QFPCR result abnormal
G-banded analysis
what is the produced for cytogenics and chorionic villi?
. Separate maternal from foetal tissue
. QF-PCR
Culture cells (7-14 days) if QFPCR result abnormal
G-banded analysis
what is advantage of array CGH and prenatal diagnosis
Increased resolution
Higher detection rate
what is the disadvantage of array CGH and prenatal diganosis?
Ethical, eg small duplication & associated autism
Need parental follow up
what percentage of spontaneous abortion is due to chromosome abnormality?
50%
how is cytogenic done for looking at leukemia?
1ml unclotted bone marrow
- Suspension culture overnight
- G-banded analysis/FISH
what is the translocation of chronic myleoid leukemia?
t(9;22)
abl gene of chromsome 9 and bcl gene of chromosome 22 translocate
what test are used for fresh tumor?
FISH or G-banding (culture – 1-20 days)
what testing is done for archive tissue?
(paraffin embedded) – FISH or genotyping
what gene is invovledin neuroblastoma?
MYCN gene amplification
