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
