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ems genetics > genome analysis methods > Flashcards

genome analysis methods Flashcards

1
Q

what determines the type of approach you will take?

A

not knowing what gene you are focusing on

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2
Q

when is whole genome analysis used/ not used?

A

it is used for dosage changes or sequencing, but not used when know the gene you are focusing on

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3
Q

what does sequencing and dosage changes comprise?

A

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

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4
Q

why dont we use whole genome analysis for a known gene?

A

other methods have advantages in precision, cost and speed

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5
Q

what do you do if you a) know the gene and mutation or b) know the gene but not the mutation?

A

a) specific assay - PCR and OLA

b) gene sequencing through PCR and sequencing

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6
Q

what could you use for single gene variants, known genes and unknown genes?

A

conventional, DNA sequencing, PCR and next generation

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7
Q

what would you use karyotyping, FISH or aCGH for?

A

chromosome number, copy number variants, structural changes

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8
Q

what is the issue with cytogenetics?

A

no single one of the genome analysis methods can deal with all types of pathology

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9
Q

what is analysis of a single locus?

A

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

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10
Q

what is the hybridisation principle?

A

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

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11
Q

what is PCR?

A

it is amplification of DNA in vitro - in vitro synthesis of large amount of DNA copies from small starting quantities specifying the target

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12
Q

what are oligonucleotides?

A

they are small synthetic primers that define the boundaries of synthesis

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13
Q

what are deoxyribonucleotides?

A

they are the monomors from which DNA polymerase synthesises DNA

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14
Q

what is the first step of PCR?

A

separate the DNA using heat at 94 degrees - denaturation

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15
Q

how many molecules will 30 cycles give you?

A

one billion

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16
Q

what can you use PCR in?

A

viral DNA in blood cycle, forensics and fingerprinting

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17
Q

what are stages 2-4 of PCR?

A

primer annealing at 55 degrees, primer extension at 72 degrees, repeating

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18
Q

what are the characteristics of PCR?

A

extreme sensitivity, 2^30 = 10^9, exponential process as the target region doubles in number every cycle

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19
Q

what will a three base pair DNA deletion result in?

A

deletion of a single AA

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20
Q

what is the most common cause of CF?

A

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

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21
Q

what is simple PCR based mutation assay?

A

analysis using PCR and gel electrophoresis - altered product size

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22
Q

what is an application of simple PCR based mutation assay?

A

prenatal CF diagnosis with results in 24 to 48 hours - can distinguish between homo and hetero cases

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23
Q

what is allele specific mutation detection?

A

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

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24
Q

what is oligonucleotide ligation assay?

A

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

25
Q

what are the advantages of OLA?

A

it is rapid, easy to do within a few hours

26
Q

what is done if the identity of the disease causing mutation is unknown?

A

sequence the DNA of the region of interest - could be one, several or all genes

27
Q

what is the issue with polymorphism?

A

there is a great deal of variation between all genomes, and therefore you must be able to distinguish between harmless and pathogenic changes

28
Q

in which direction is DNA synthesised?

A

5-3’ - new nucleotides are added to 3’ end

29
Q

what is dATP?

A

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

30
Q

what are the characteristics of sanger DNA sequencing?

A 
one PCR product 
only exons targeted - protein coding regions 
only around 500 bps 
one sequence 
large genome must be broken into parts
31
Q

why is multiple gene sequencing not easy and how would you do this?

A

need a highly parallel sequencing approach - difficult and expensive
clonal and NGS
look at multiple target regions simultaneously

32
Q

what is a disadvantage of NGS?

A

not clinically interpretable as only 2% are exons

implications in terms of data storage and analysis

33
Q

what is a clinical exome?

A

it is 25Mbp (8000 genes) compared to an exome which is 60Mbp - cheaper and smaller - do not want to sequence whole thing

34
Q

what does hybridisation occur between?

A

to the reagent that is made of synthetic oligonucleotides that match all known exons

35
Q

what is the enriched target exposed to?

A

the enriched target contains all known exons and is exposed to NGS

36
Q

why do we not always use exomes?

A

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

37
Q

how would you define a set of genes of interest?

A

exome based diagnostic panels
good for single base changes
not so good for large variants or copy number analysis

38
Q

what is a gene panel?

A

focusing on only the genes that you know are involved with the disease

39
Q

what is a large scale genome pathology?

A

when several million base pairs have changed - rearrangements of the genome, copy number change that is not by 2 - chromosome number or structure

40
Q

how would you test for copy number variants?

A

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

41
Q

what method required live cells?

A

G banding - it uses metaphase chromosomes

42
Q

what is molecular cytogenetics?

A

where you visualise a particular DNA sequence in a chromosome on a microscope slide

43
Q

what whole genome methods are there to detect CNV?

A

G banding, aCGH and NGS

44
Q

what methods are targeted in identifying CNV?

A

QF-PCR, FISH, MLPA

45
Q

what are the stages of mitosis?

A

interphase, prophase, metaphase, anaphase, telophase, cytokinesis

46
Q

why are metaphase chromosomes ideal?

A

they are condensed so visible

47
Q

which drug can be added to arrest cell in metaphase?

A

colchicine

48
Q

what is karyotyping?

A

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

49
Q

what is FISH?

A

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

50
Q

what is the process of FISH?

A

labelling, denaturation, hybridisation and visualisation

51
Q

what does interphase FISH use?

A

centromeric probes

52
Q

where is FISH commonly used?

A

DiGeorge Syndrome
2mbp deletion of chromosome 22q11.2
green is control
therefore more green shows up

53
Q

what is aCGH?

A

array comparative genomic hybridisation - standard modern replacement for karyotyping. It is a whole genome copy number analysis that is cheap with high resolution

54
Q

what is MLPA?

A

it is multiplex ligation dependent probe amplification and is a targeted method of analysis

55
Q

what is QF-PCR?

A

it is quantitative fluorescent PCR that is a targeted, cheap and rapid method of analysis with prenatal applications

56
Q

how do we find copy number sequencing?

A

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

57
Q

why are DNA methods good for dosage?

A

they are cheaper, easier and have higher resolution

58
Q

what is needed for genome rearrangements?

A

cytogenetic analysis

59
Q

what are the potential disadvantgaes of cytogenetic analysis in genomic rearrangement?

A

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

ems genetics (19 decks)

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