genome analysis methods Flashcards
what determines the type of approach you will take?
not knowing what gene you are focusing on
when is whole genome analysis used/ not used?
it is used for dosage changes or sequencing, but not used when know the gene you are focusing on
what does sequencing and dosage changes comprise?
sequencing - looking at the whole genome sequence and genes within it by using WGS or exome
dosage changes - number of chromosomal copies using karyotyping or aCGH
why dont we use whole genome analysis for a known gene?
other methods have advantages in precision, cost and speed
what do you do if you a) know the gene and mutation or b) know the gene but not the mutation?
a) specific assay - PCR and OLA
b) gene sequencing through PCR and sequencing
what could you use for single gene variants, known genes and unknown genes?
conventional, DNA sequencing, PCR and next generation
what would you use karyotyping, FISH or aCGH for?
chromosome number, copy number variants, structural changes
what is the issue with cytogenetics?
no single one of the genome analysis methods can deal with all types of pathology
what is analysis of a single locus?
there is a large amount of tangled bases within DNA - we need to determine one single gene and isolate one region of interest to find a single nucleotide change in this - there are 3x10^9 base pairs
what is the hybridisation principle?
base pairing allows design of reagents to determine sequences. two DNA or RNA molecules will anneal and form a duplex if their complementary base pairs align to the Watson Crick base pairing rules. Mismatched pairs will destabilise the duplex. A DNA probe is manufactured and if it does not match due to incomplete base pairing then the hybrid is unstable allowing discrimination between very similar targets
what is PCR?
it is amplification of DNA in vitro - in vitro synthesis of large amount of DNA copies from small starting quantities specifying the target
what are oligonucleotides?
they are small synthetic primers that define the boundaries of synthesis
what are deoxyribonucleotides?
they are the monomors from which DNA polymerase synthesises DNA
what is the first step of PCR?
separate the DNA using heat at 94 degrees - denaturation
how many molecules will 30 cycles give you?
one billion
what can you use PCR in?
viral DNA in blood cycle, forensics and fingerprinting
what are stages 2-4 of PCR?
primer annealing at 55 degrees, primer extension at 72 degrees, repeating
what are the characteristics of PCR?
extreme sensitivity, 2^30 = 10^9, exponential process as the target region doubles in number every cycle
what will a three base pair DNA deletion result in?
deletion of a single AA
what is the most common cause of CF?
around 75% show the elimination of a single AA in CFTR - F508del - easy to recognise as changes the size of the PCR product by 3bps
what is simple PCR based mutation assay?
analysis using PCR and gel electrophoresis - altered product size
what is an application of simple PCR based mutation assay?
prenatal CF diagnosis with results in 24 to 48 hours - can distinguish between homo and hetero cases
what is allele specific mutation detection?
it is distinguishing between two alleles that may only differ by a single base pair - distinguish between a know disease causing point mutation and normal allele - only interrogates this one mutation
what is oligonucleotide ligation assay?
when the allele specific oligonucleotides are designed so that their 3’ end base pairs pair with the variable nucleotide - the pairing cannot occur if it is not perfectly matched and therefore can distinguish two alleles - it is aimed at one specific know mutation
what are the advantages of OLA?
it is rapid, easy to do within a few hours
what is done if the identity of the disease causing mutation is unknown?
sequence the DNA of the region of interest - could be one, several or all genes
what is the issue with polymorphism?
there is a great deal of variation between all genomes, and therefore you must be able to distinguish between harmless and pathogenic changes
in which direction is DNA synthesised?
5-3’ - new nucleotides are added to 3’ end
what is dATP?
the substrate for a residue going onto a chain in DNA sanger sequencing - makes a synthetic nucleotide that is a dideoxy that is a chain terminator
what are the characteristics of sanger DNA sequencing?
one PCR product only exons targeted - protein coding regions only around 500 bps one sequence large genome must be broken into parts
why is multiple gene sequencing not easy and how would you do this?
need a highly parallel sequencing approach - difficult and expensive
clonal and NGS
look at multiple target regions simultaneously
what is a disadvantage of NGS?
not clinically interpretable as only 2% are exons
implications in terms of data storage and analysis
what is a clinical exome?
it is 25Mbp (8000 genes) compared to an exome which is 60Mbp - cheaper and smaller - do not want to sequence whole thing
what does hybridisation occur between?
to the reagent that is made of synthetic oligonucleotides that match all known exons
what is the enriched target exposed to?
the enriched target contains all known exons and is exposed to NGS
why do we not always use exomes?
there are too many variations - 10-20,000 protein changing variations from the reference genome
too many DNA variants will be returned so need to decide which ones are causative
how would you define a set of genes of interest?
exome based diagnostic panels
good for single base changes
not so good for large variants or copy number analysis
what is a gene panel?
focusing on only the genes that you know are involved with the disease
what is a large scale genome pathology?
when several million base pairs have changed - rearrangements of the genome, copy number change that is not by 2 - chromosome number or structure
how would you test for copy number variants?
cytogenetic analysis:
conventional - metaphase karytoyping or live cells
molecular cytogenetics - all cell-cycle stages
in situ - FISH - meta and interphase
DNA - array CGH and QF-PCR
what method required live cells?
G banding - it uses metaphase chromosomes
what is molecular cytogenetics?
where you visualise a particular DNA sequence in a chromosome on a microscope slide
what whole genome methods are there to detect CNV?
G banding, aCGH and NGS
what methods are targeted in identifying CNV?
QF-PCR, FISH, MLPA
what are the stages of mitosis?
interphase, prophase, metaphase, anaphase, telophase, cytokinesis
why are metaphase chromosomes ideal?
they are condensed so visible
which drug can be added to arrest cell in metaphase?
colchicine
what is karyotyping?
it is a cell culture and G banding with over 5m bps as cannot see smaller than that. It is slow and expensive with variable resolution
what is FISH?
fluorescence in situ hybridisation - where a piece of DNA and a target sequence are paired and lit up - exploits specificity of DNA to see where the region sits on the chromosome
what is the process of FISH?
labelling, denaturation, hybridisation and visualisation
what does interphase FISH use?
centromeric probes
where is FISH commonly used?
DiGeorge Syndrome
2mbp deletion of chromosome 22q11.2
green is control
therefore more green shows up
what is aCGH?
array comparative genomic hybridisation - standard modern replacement for karyotyping. It is a whole genome copy number analysis that is cheap with high resolution
what is MLPA?
it is multiplex ligation dependent probe amplification and is a targeted method of analysis
what is QF-PCR?
it is quantitative fluorescent PCR that is a targeted, cheap and rapid method of analysis with prenatal applications
how do we find copy number sequencing?
it is a form of whole genome sequencing - you divide the genome into ‘bins’ and determine how many DNA sequences are in each segment of the chromosome
deletion - relative copy number drop
the cost and resolution is similar to aCGH but there is poor sample quality as the DNA may be degraded
why are DNA methods good for dosage?
they are cheaper, easier and have higher resolution
what is needed for genome rearrangements?
cytogenetic analysis
what are the potential disadvantgaes of cytogenetic analysis in genomic rearrangement?
not good at detecting balanced translocations where the DNA number is the same
copy number may give a normal result but function may be impaired
one single method does not work for all pathologies
