Flashcards in techniques of cytogentic analysis Deck (65):
what 3 methods are used to analyse the whole genome
Next generation sequencing
what 3 techniques are used for targeted testing
define convention genetics
look at full chromosome under microscope- must be in metaphase.
looks at specific parts of the human genome.
How is the procedure of G banding carried out
Mitotic arrest- metaphase
Hypotonic salt solution
Trypsin and Leishman's stain
Banding AT and GC regions
Lighter bands are AT rich and darker bands are GC rich.
what stain is commonly used in G banding
Trypsin or Leishmans stain
what is FISH used to analyse a gene or whole chromosome
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
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.
what is FISH used to detect
copy number variation- centromere probe
Confirmation/ clarification of G banding
Confirmation of array CGH
Identify speciifc abnormalities in cancer.
define copy number variation
A DNA segment with a variable copy no. compared with a reference genome
what 2 conditions can arise due to copy number variation.
Autism and cancer.
Is it both high and low copy number variations that predispose you to certain conditions
High copy no. of CCL3L1 = susceptibility to HIV
Low copy no. of FCGR3B = susceptibility to inflammatory autoimmune disorders
Multiplex Ligation-dependent Probe Amplification
what are 4 steps involved in carried out MLPA.
How does PCR detect different sequences in MLPA apart
each probe is produced with a different stuffer sequence length.
what is microarray CGH used for
genome wide screen.
what is MLPA used for
alternative to FISH
Show large numbers of copy variations.
what sample is microarray CGH carried out on
3 ml blood EDTA (also 1-2 ml lithium heparin blood for cell culture)
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.
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.
what are the advantages of microarray CGH
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
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
how is the procedure for next generation sequencing carried out.
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.
what does microarrays look for
deletions and duplications.
what does next generation sequencing look for
what is quantitative fluorescent PCR is used look for what
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.
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
define microsatellite tetranucleotide repeat marker (STR)
satellite repeat sequences which can be analysed in maternal and paternal DNA to determine prenatal aneuplodiy.
when is qPCR carried out
When FISH unsuitable
How long does G banding take
30 mins - 4 hours/case
How long does fish take
10 mins -1 hour/case
how long does microarry take
10 mins -2 hours/case
what long does NGS/qPCR take
30 mins – 2 hours per case
how long does MLPA/QF PCR take
10 mins/30 mins/case
what is the use of cytogenetics
Assessing reproductive risks
what material can be sued to sample cytogenetics
• Blood- best type of sample
• Amniotic fluid
• Other foetal tissue- for spontaneous miscarriages.
• Bone marrow
With what abnormalities are people often reffered for cytogenetic anaylsis.
dysmorphic new born
developmental problems- intellectual, physical and sexual
what sample is required for G banding
2-5 ml unclothed blood.
stimulate T lymphocytes
Culture 2-3 days.
what 2 chromosomes are involved in the down syndrome robertsonian trans location
what is the name of the complex formed when carrier is 2 chromosomes join in robersonian translocations during meiosis pairing.
Trivalent formation at pachytene
In what stage of meiosis 1 does the trivalent form a chain
do circular structures form in metaphase of meiosis one during robersonian translocation
no- because unlike normal structures the telomeres don’t join.
what are 2 types of chromosome problems commonly causes fertility problems in the populations
balanced chromosome change.
chromosome change- (47XXY or Y deletion)
what are the 3 forms of prenatal diagnosis
• Amniocentesis (16w)
• Chorionic villus biopsy (CVS, 12w)
• NIPT (12w)- non invasive prenatal technique.
which prenatal sampling can be done at the ear lies date (12 weeks) amniocentesis or chronic villas biopsy
chronic villus biposy
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
How many miscarriges result due to invasive sampling methods
1 in 200
what causes infertility problems more commonly balanced translocations or chromosome changes (45X)
what are non invasive prenatal testing used to confirm
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
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.
How is the procedure for cytogentic and chronic villus carried out
1. Separate maternal from foetal tissue
3. Culture cells (7-14 days) if QFPCR result abnormal
4. G-banded analysis
how is amniocentesis diagnosis confirmed.
1. QF-PCR – allele ratios from 2 markers indicative of trisomy 21
cell culture for confirmation
In a spontaneous abortion 50% of the times is due to
( Triploidy, 45,X, trisomy)
Preimplnatation genetic analyse
Testing abnormalities in the blastomere.
Shows dosage changes in cells
Used for individuals who don’t want to undergo invasive techniques.
why is cytogenetics used in cancer.
Disease specific acquired chromosome changes
– Mostly translocations
– Several different acquired abnormal clones
– Treatment (stratified medicine).
what material can be used to test cancer
tumour or bone marrow.
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
most common translocation in leukaemia
• Translocation between 9 and 22- break on abl (9) and bcr (22)
• Forms bcrabl fusion gene on chromosome 22.
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.
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.
what methods of testing are used on a fresh tumour
Fresh tumour – FISH or G-banding (culture – 1-20 days)