ZS 2: genetische testen Flashcards
Whole exome sequencing
Gene chips (= Oligonucleotide microarrays): >Divesed for scanning mutations in certain genes of high interest
> designed to hybridize to just one gene in a test DNA sample.
> there are often thousands
of different oligonucleotides
> the oligonucleotide sequences are
chosen to comprise a series of overlapping sequences that collectively
cover all the known functionally important parts of the gene of interest:
1.the coding sequence
2.exon–intron boundaries
and any other known
functionally important sequence.
> The method relies on a high hybridization
stringency so that only perfect base matching is tolerated: if the test
sample has a mutation, oligonucleotide probes spanning the mutation
site can detect the abnormality.
-NGS (= Massive parallel DNA sequencing)
(je kent dit al)
-targeted sequencing
> Exome sequencing
involves first capturing exons from the DNA of affected individuals,
and then sequencing the captured DNA.
desired sequences are captured from a genomic DNA sample by a DNA hybridization method and submitted for DNA sequencing (biotine binden op gesynthetiseerde oligonucleotiden die target gen zullen binden . Streptavidine op een magnetische bead toevoegen. Binding biotine streptavidine. Vervolgens DNA sample (gefragmeteerd) toevoegen, en mbv de beads afzonderen
Whole genome sequencing
-NGS (massive parallel DNA sequencing )
In standard dideoxy sequencing:
> individual DNA
sequences of interest must first be purified
>then sequenced, one after another.
> then the different lengths
separated by gel electrophoresis
By contrast, massively parallel
DNA sequencing (often called next-generation
sequencing) is indiscriminate: all of the different
DNA fragments in a complex starting DNA sample
can be simultaneously sequenced without any need
for gel electrophoresis. That allows a vastly greater
sequencing output.
Gene panels
Targeted gene sequencing panels (NGS techniek) are useful tools for analyzing specific mutations in a given sample. Focused panels contain a select set of genes or gene regions that have known or suspected associations with the disease or phenotype under study.
Geef de Massieve parallele DNA sequencing technieken
Whole exome sequencing
Whole genome sequencing
Gen panel sequencing
RNA sequencing
Variant interpretatie
-Interpreting sequence variants and the problem of variants of
uncertain clinical significance:
> tijdens het scannen voor gekende mutaties (in genoom/exon), zullen er nieuwe variaties ontdekt worden.
veel van deze varianten zijn niet pathogenetisch
*genen die loss of function vertonen, grote deleties, frameshifts, nonsense mutaties, zijn waarschijnlijk pathogenisch.
*in tegenstelling, puntmutaties zijn moeilijker te beoordelen.
-Mutation interpretation and databases:
Checking mutation or SNP databases is important to determine whether a variant
has previously been reported and what information might be available.
If the variant has previously
been reported and is common (with a frequency of more than
0.01), it is unlikely to be associated with a rare single-gene disorder (but
many rare nonpathogenic variants will not be in the database).
-Evolutionary conservation is another important factor. A substitution
is unlikely to be pathogenic if it changes the amino acid to one that is
the normal (wild-type) amino acid at an equivalent position in a clearly
orthologous protein from another species. Conversely, if the normal
amino acid is very highly conserved across a range of species, a mutation
producing a nonconservative amino acid change at this position
becomes highly significant.
Scanning for possible pathogenic changes in cytosine
methylation patterns: Altered cytosine methylation patterns represent another type of smallscale
DNA change that can contribute to pathogenesis.
gebruik bisulfite sequencing.
Bisulfiet sequencing
Methylated and unmethylated cytosines can be distinguished by making
the DNA single stranded and treating it with sodium bisulfite (Na2SO3).
Under controlled conditions, the unmethylated cytosines are deaminated
to produce uracils, but 5-methylcytosines remain unchanged. After
treatment ,the relevant region can be amplified
by PCR, during which newly created uracils are read and propagated as
thymines.
New DNA strands are synthesized without incorporating methyl
groups so that any retained methylated cytosines in the template DNA
are propagated as unmethylated cytosines. That allows different ways of
distinguishing the methylated cytosines from the original unmethylated
cytosines.
ARMS
Amplification refractory mutation system
- goed voor de detectie van SNV
- PCR amplificatie , afhankelijk van correcte baseparing van 3’ nucleotide op primer -> afhankelijk van taq polymerase werking
boek info:
If the 3ʹ end
nucleotide of a PCR primer is not correctly base paired, PCR amplification
is not possible when using a DNA polymerase such as TaqI polymerase
that lacks a proofreading activity. The allele-specific primer has a terminal 3ʹ
nucleotide that base pairs correctly with either the normal or the mutant
nucleotide.
That is, the primer is designed so that the 3ʹ end nucleotide is
placed opposite the mutation site
(die mag er niet voor of na stoppen, want anders zal polymerase gewoon zijn werk doen?)
OLA
Oligonucleotide ligation assay:
- goed voor de detectie van SNV
- hier kan je ook target DNA met een probe vinden
- > goed voor kleinere mutaties
- hoeveelheid pcr product is gebaseerd op werking van ligase
Boek info
Like the ARMS method, this method
depends on binding by an allele-specific oligonucleotide (ASO) in which
the specificity depends on correct base pairing at the terminal nucleotide
at the 3ʹ end (which is intended to base pair with the mutant nucleotide
or its normal equivalent).
When the ASO and another oligonucleotide are
simultaneously annealed to adjacent sequences on a DNA template, they
can be ligated together only if the oligonucleotides perfectly match the
template at the junction. That is, ligation is impossible if, at the junction,
the end nucleotide of either oligonucleotide is incorrectly base-paired
Pyrosequencing
https://www.youtube.com/watch?v=wY8to-_zAEo&ab_channel=QuickBiochemistryBasics
When DNA is synthesized
from deoxynucleoside triphosphate (dNTP) precursors, the
bond between the α and β phosphates of the dNTP is cleaved, so that a
dNMP (containing the α phosphate) is incorporated into the growing DNA
chain. That leaves a pyrophosphate (PPi) residue made up of the β and γ phosphates, which is normally discarded.
In pyrosequencing, sequential
enzyme reactions detect the pyrophosphate that is released when a
nucleotide is successfully incorporated into a growing DNA chain. Pyrosequencing can be used to discriminate between normal
and mutant sequence variants because individual dNTPs needed for DNA
synthesis are provided in separate reactions in a defined order (Figure
11.16B). It has been particularly helpful in detecting mutations in lowlevel
mosaicism, and in heterogeneous tumor samples.
Sanger sequenving
In single-gene disorders in
which the causative mutations are limited to one gene, or one of a few
genes, Sanger (dideoxy) sequencing remains the foremost approach for
mutation scanning. Here mutation scanning usually involves amplifying
sequences from individual exons of a single gene, plus short regions of
intron sequence adjacent to exons, and sequencing the amplified DNA
Geef variant detectie technieken voor kleine varianten
- Sanger sequencing
- whole exome sequencing
- whole genome sequencing
- g
Bespreek QF-PCR
- Kwantitatieve Fluorescentie
- Ideale test voor aneuplodie
- door polymorfe , fluorescente merkers van specifieke chromosoom te gebruiken als primers in multiplex PCR
Boek info:
hoe?
-fluorescent labeled primers worden gebruikt in multiplex PCR (meerdere polymorfe merkers op chromosomen betrokken in de aneuploidie simultaan amplificeren)
vb : voor trisomy 21 kan je AMEL merker gebruiken. Dit is een hoog polymorfe short tandem repeat, te vinden op chromosoom 21.
(dus : vb 1 ouder heeft 10 repeats voor de AMEL merker, terwijl andere ouder 15 heeft -> kind kan 10/30 signaal uitzenden)
Je kan dan 2 gelijke signalen, of 1 grote ontvangen voor de merker (= normaal)
Of je kan 2 ongelijke signalen ontvangen, of 3 signalen (= aneuploidie)
a 2:1 ratio for the two
length variants for each of marker.
uninformative markers show only one length variant, which is presumably due to three alleles of
identical lengths. (C)
*indien er toevallig bij de trisomie, merkers met exact dezelfde repeats aanwezig zijn, zal er slechts 1 pcr product worden gevormd, waardoor het vals negatief kan uitkomen.
Chromosomale microarray analyse : bespreek aCGH
aCGH: Array comparative genome hybridization (aCGH)
-Detecteert CNV (deleties/inserties) in het genoom
-hogere resolutie dan karyotypering
(kan duplicaties/deleties kleiner dan 5-10 MB detecteren)
Boek info:
Hoe werkt het?
Je neemt genomische DNA uit patient, en vergelijkt het met controle groep.
De genen in het genoom zijn gelabled
then mixed and hybridized to a panel of unlabeled DNA probes
By comparing the
hybridization patterns of component DNA sequences in the control and
test samples, we can screen for changes in copy number over quite large
regions of DNA.
aCGH, it is used in detecting:
>chromosomal imbalances and large-scale
changes in cancer
*using tumor samples referenced against a control
of lymphocyte DNA from the same individual).
> in a pediatric setting to identify suspected gene imbalances in children
with developmental disabilities or congenital anomalies
Take:
>two genomic DNA populations
>test sample
> a normal control
collectively representing the genome
All are labeled with different fluorophores?
then mixed and hybridized to a panel of unlabeled DNA probes
By comparing the
hybridization patterns of component DNA sequences in the control and
test samples, we can screen for changes in copy number over quite large
regions of DNA.
Chromosomale microarray analyse : bespreek SNP microarray
Single nucleotide polymorphism (SNP) microarrays:
- detecteert SNP in het genoom (= gen per gen op elk allel)
- kunnen zo ook deleties en inserties oppikken, maar aCGH is nauwkeuriger (boek zegt dit, mr prof zegt beiden zijn hetzelfde)
Boek info
> comprise panels
of oligonucleotides that are shorter in length than those used in aCGH, that are for each SNP locus.
(because they are designed to hybridize to individual alleles only).
> different oligonucleotides will be present that are
designed to hybridize to individual alleles at that locus.
> In contrast with aCGH, SNP arrays do not directly compare a patient’s test
sample with a control sample but a database of SNP array results from control individuals.
> Like aCGH,
SNP arrays can detect gains and losses of sequences across the genome.
Deletions can be identified because (loss of heterozygosity!! ) the
SNPs in the deleted area should show just a single allele. For duplications, the ratios of alleles will vary: in regions of partial trisomy, loci in which both alleles are evident might show skewed allele ratios and might appear as AAB (twice the signal for allele A compared
with B) or ABB, instead of AB.
> In practice, assuming DNA gains and
losses using SNP microarrays is less secure than in aCGH, and aCGH is
the more widely used method for this purpose.
Geef de moeilijkheden van microarray analyse
-In pediatric applications, it is important to obtain
follow-up parental DNA samples to establish whether any gain or loss
is present in a healthy parent and is therefore likely to be a benign copy
number variant. (anders = denovo mutatie in nakomeling)
-For de novo copy number variants the interpretation of the likely effect can sometimes be difficult to predict, simply because of
lack of data; a small percentage of tests will therefore report unclassified variants.
-Any genome scan can reveal incidental (secondary) findings—discoveries
made as a result of genetic testing that are unrelated to the medical reason for ordering the test but which can have important health
consequences.
- That would happen only occasionally in traditional (chromosome
banding) karyotyping - in aCGH incidental findings are more
frequent because of its higher resolution (vb: tijdens de pediatirshce scan voor neurodegeneratieve ziekte, kan een adult-onset kanker worden ontdekt)
Chromosoom FISH
fluorescence in situ hybridization:
- grote deleties en duplicaties
- translocaties
- je neemt chromosomen op een slide, je denatureert ze , en doet er fluorescente dye op.
*verschilt van gewone banding, in dat het nauwkeuriger is (banding duidt gewoon regios rijk aan een bepaalde NZ aan, terwijl deze specifieke sequenties kan aanduiden)
Boek info
> often used to confirm regions of chromosome duplication/deletion that have been suggested by other screening methods,
such as array CGH.
> used to screen for the amplification of
specific oncogenes that are associated with particular types of cancer,
such as amplification of the MYCN gene in neuroblastoma
> Another major application is in detecting translocations, notably acquired
translocations that are common in cancer.
*Recurrent translocations are
associated with certain types of cancer; often, the translocation involves
breakages in specific genes, producing hybrid genes that are inappropriately
expressed.
Stappen:
>fix chromosome preparations on microscopic slides
>treat the slides
to denature the DNA
> hybridize fluorescently labeled probes of interest
to the denatured DNA.
The locations of the fluorescent signals are
recorded against a background stain that binds to all DNA sequences (vb chromosomen hebben blauwe kleur dankzij dapi)
Chromosoom banding methode
- chromosoom is gecondenseerd: metafase vastzetten
- chromossom banding: denaturatie , vervolgens ook dye voor DNA (er zijn er die graag op AT rijke regios binden, en er zijn er die op GC rijke regios binden)
- > differentiele binding over hele chromosoom
vb: G-banding
gimsa bindt graag op AT -> AT regios kleuren donker, GT rijke regios kleuren licht.
Hoe pak je fenotypes die wél of niet een pathogenische CNV in het genoom indiceren?
As long as the clinical phenotype does not immediately suggest a defined
location for a pathogenic copy number variant, array CGH is the preferred
scanning option
When the phenotype does suggest
the chromosomal location
> large-scale deletions/ duplications of a specific chromosomal -> FISH
> small changes, such as expansions of unstable oligonucleotide
- > Southern hybridization
- > MLPA assays
MLPA
https://www.youtube.com/watch?v=hWNDldp00uM&ab_channel=MoleculaireDiagnostiek
Multiplex ligation-dependent probe amplification:
Wat?
>method that can detect copy
number changes over a broad range of DNA lengths.
(kan ook voor SNV gebruikt worden)
> It is most frequently
used to scan for intragenic deletions and duplications by monitoring the
copy number of individual exons within genes
(as in Duchenne muscular dystrophy, for example)
> It may also
serve as a general method of scanning for copy number changes
Hoe? (zie video)
De probe bestaat uit 2 delen. Elk deel bevat een primer bindende regio.
Als er geen mismatch is, wanneer de probes binden op de target, dan zal ligase ze covalent verbinden.
Hierna, zal er PCR amplificatie plaatsvinden, waarbij het signaal van de probe sterker wordt bij elke cyclus.
(Indien mismatch, geen ligase binding, geen PCR amplificatie, geen signaal- > onderzoek is gebaseerd op vermogen van ligase, om enkel non-mismatch NZ te ligeren)
(dit zoals OLA)
> uses pairs of short single-stranded sequences (= probes) that are designed to bind to specific exons or other sequences (= target sequences) whose relative copy number we wish to determine.
> Each pair of probes is designed to hybridize collectively to a
continuous target DNA sequence; that is, when they bind to the target
DNA, the pair of probes align immediately next to each other. The gap
between them can then be sealed using DNA ligase to give a single probe
that is complementary to the target
.
TP-PCR
Triplet repeat-primed PCR
> primer (which hybridizes to a sequence
flanking the oligonucleotide repeat array, plus a
primer that can hybridize to target sequences outside it (kan op de grens tussen in en outside)
Because of the tandem repetition, the internal primer
can hybridize to multiple possible binding sites within
the array, producing a series of peaks of increasing
size.
Increased sizes ( = increased repeats) are apparent in patients when referenced against controls
Southern blot
-Very large oligonucleotide expansion assays
Hoe?
-digest DNA with restriction
endonucleases and separate fragments
by size on an agarose gel
-denature DNA and transfer to membrane
-hybridize labeled probe to DNA
fragments bound to membrane
-wash to remove unhybridized
probe and apply X-ray film
-develop film to reveal autoradiograph
of labeled target DNA
The labeled probe detects restriction fragments
from the region of interest: changes in the
DNA in that region => changes in size of the detected DNA fragments.
Southern blot hybridization assays have often been
used to detect abnormally long arrays of tandem
repeats in myotonic dystrophy and facioscapulohumeral
dystrophy. In type 2 myotonic dystrophy, the
arrays can expand to as many as 5000 repeats
Bespreek southernblot vs PCR voor de detectie van expansies
Standard PCR assays are often used to follow modest
expansions in oligonucleotide repeats, including
those associated with CAG codon expansions in various
neurodegenerative disorders such as Huntington
disease.
For larger expansions, Southern blot hybridization
assays have been used
However ,modified PCR reaction has become the preferred method (vb: Triplet repeat primed pcr assays)
Geef de testen om grote varianten te tracen
-karyotypering
-chromosoom FISH
-aCGH
-SNP array
-southern blotting
QF-PCR
-MLPA
-TP-PCR
Geef de testen om kleine varianten te tracen
-sanger sequencing
-NGS
>whole genome/exome
>gen panel
>RNA sequencing
- bisulfiet sequencing
- pyrosyequencing
- ARMS
- OLA
- Q-PCR
- Digitale PCR
Digital PCR
Digital PCR is a new approach to nucleic acid detection and quantification that offers an alternate method to conventional real-time quantitative PCR for absolute quantification and rare allele detection.
https://www.youtube.com/watch?v=Qqdmw3wvMFo&ab_channel=StillaTechnologies
Geef technieken die translocaties opsporen
- WGS
- karyotypering
*wgs is duur
Geef het verschil tussen OLA en MLPA
Ola: vaak gebruikt voor SNP
MLPA: vaak gebruikt voor CNV, die je al weet kent, en wilt bevestigen.
je kan meerdere CNV tegelijk doen en
Je kan de verschillende probes van elkaar onderscheiden door de lengte van de 5’ aanhangsel (inerte molecule) in lengte te laten varieren -> scheiding in pcr
Boek info:
OLA:
The assay (OLA) depends on correctly hybridizing
two oligonucleotides to adjacent sequences
on the same template DNA: an allele-specific
oligonucleotide (ASO), whose specificity depends
on the terminal nucleotide at its 3ʹ end, and
a common oligonucleotide (which base pairs
with different alleles). The ASO is attached at
its 5ʹ end to an inert molecule whose size can
vary—equivalent to a length (L) of n or n + 2
nucleotides in this example—and the common
oligonucleotide has a fluorophore at its 3ʹ end.
Ligation is possible only if nucleotides at the
junction of the aligned oligonucleotides are
correctly base paired; when that happens, the
3ʹ-terminal hydroxyl group of the ASO becomes
covalently linked to the 5ʹ-terminal phosphate
of the common oligonucleotide. The resulting
larger molecule can be size-separated by capillary
electrophoresis on a suitable DNA sequencing
machine. By having different sizes for the inert
molecule attached to the 5ʹ end of the ASO,
different alleles can be detected simultaneously.
Ligation is not possible when an ASO is not
perfectly base paired at its 3ʹ end.
MLPA:
Note that
multiplexing allows multiple mutations at different
sites to be simultaneously assayed by adjusting the
size of the inert molecule attached to the ASOs.
