techniques of cytogentic analysis Flashcards
what 3 methods are used to analyse the whole genome
G banding
Next generation sequencing
Microarrays
what 3 techniques are used for targeted testing
Qf- PCR
MLPA
FISH
define convention genetics
look at full chromosome under microscope- must be in metaphase.
Molecular genetics
looks at specific parts of the human genome.
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.
what stain is commonly used in G banding
Trypsin or Leishmans stain
what is FISH used to analyse a gene or whole chromosome
both.
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 Aneuploidy 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
Yes
High copy no. of CCL3L1 = susceptibility to HIV
Low copy no. of FCGR3B = susceptibility to inflammatory autoimmune disorders
define MLPA
Multiplex Ligation-dependent Probe Amplification
what are 4 steps involved in carried out MLPA.
1- denature
2- hybridisation
3- ligate
4- Amplify.
How does PCR detect different sequences in MLPA apart
different size
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
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
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.
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.
