exam 2 Flashcards
normalized melting curves use
melting temp or dyes that fluoresce in the presence of dsDNA
unlabeled probe genotyping
used when greater detail or exact genotyping is needed
snapback primer
unlabeled probes on the 5’ end of a primer
snapback or unlabed primers are used for
common disease variants
subtyping viruses
epidemiology
factor V leiden genotyping
small amplicons, unlabeled probes, or snapback primers
gilbert syndrome
increased drug toxicity
TA repeats in promoter or UGT1A1
small amplicon genotyping is used
methylation analysis is used to
dx imprinting disorders
give degree of methylation
(unmethylated CG are converted to TA in pretreatment bc methylation is lost in PCR)
competitive PCR
used to detect exomic delections, trisomies, sex chromosome abnormalities
epigenetics
the study of mitotically heritable changes of a phenotype that do not result from changes in the genetic code
epigenetics are mediated by
postranscriptional histone modifications
histone variants
atp-dependent chromatin
small and noncoding RNAs
DNA methylation
DNA methyhlation is
methyl group on 5’ position of cytosines in the context of a GpG that interferes with binding of transcriptional activators
CpG
75% methylated
CpG islands
mostly actively transcribed
1-4kb
methylated islands are on inactivated chromosomes
theory of methylation
is essential for mammalian embryogenesis
origin of disease hypothesis
environmental influences
imprinting
there is altered methylation patterns in
cancer
neurodegenerative disorders
metabolic disorders
autoimmune disorders like lupus
increase of methylation in promoter regions of tumor suppressors and signal transduction genes
APC
methylation of DNA repair
MGMT
MLH1
BRCA1
methylation of detox genes
GSTP1
methylated cell cycle regulators
p15
p16
RB
angiogenesis
THBS1
VHL
apoptosis
caspases
p14
DAPK
clinical application of methylation
determine ratio of meth:unmeth CpGs
- independent of starting material and expression levels
- can be done on almost any specimen
changes in methylation
precede genetic changes
hypermethylation is seen prior to dysplastic changes
DNA methylation as a prognostic indicator
ID tumor growth and recurrence
predict survival time
stratify tumor to aid in therapy decision
predict response to chemo
methylation content has cons like
no info on location of DNA meth on genome
genome wide analysis of methylation
-measures mRNA expression after treating cells w/ a methylation inhibitor
-requires cell culture
-leads to expression of epigenetically silenced genes
-target and non target genes activated
other methods for genome wide methylation analysis
NGS
anti-methyl cytosine antibodies
purification of methylated DNA w/ MBD proteins
analysis of individual CpGs requires
PCR of MS digested DNA
MS multiplex ligation dependent probe amplification (MS-MLPA)
Methylation‐specific PCR (A) consists of two primers binding to sites with one or more CpGs. When the correct methylation pattern is present, in this case complete methylation, an amplicon is formed that can be detected on a gel.
true
Maxam Gilbert Sequencing
a chain breakage at specific NTs are then separated by gel electrophoresis and detected by autoradiography read 5’ to 3’
dimethyl sulfate
breaks at a G
Formic Acid
breaks at a G+A
Hydrazine
T and C
Hydrazine and salt
C
sanger sequencing is
modified DNA replication
growing chains are terminated by ddNTPs
why can’t ddNTPs bind
the 3’OH group is missing
sequencing ladder
fragments electrophoresed in 4 sample tubes and 4 lanes
Cycle sequencing utilizes
chain termination in a thermal cycler, uses one tube/capillary
a dye primer
heat stable DNA pol
cycle sequencing labels
fluorescein and rhodamine
each letter has its own color which corresponds to peaks and are read on an electrophoretogram
dye primer is on
5’
dye terminator is on
3’ end or ddNTPs
pyrosequencing is based on
light generated through release of a ppi on NT addition
pyrosequencing equation
PPi + APS -> ATP
ATP + luciferase -> oxyluciferin + light
Next Gen Sequencing requires
a library
custom linkers
a solid surface/interface
NGS steps
- shear high MW DNA
- polish ends
- ligate DNA adapters
- produce fractions and quantitate
- amplify on flow cell surface
- denature to ssDNA
- hybridize seq primers to the ssDNA
- sequence or hybrid capture
issues w/ PCR
preferential amplification/jackpotting
false + due to substitution errors
bias in high and low C/G fragments
hybrid capture
fragments from WGS library are selected w probes that match target
can purify w/ magnets
multiplex PCR
- design overlapping primers from gene of interest
- group primers according to G/C content/Tm
- amplify
- create library by ligation
- sequence
single nucleotide RT sequencing/SMRT
phi 29 DNA pol
very processive
phospholinked florescent dNTPs
false negatives are often due to
lack of coverage
false positives are due to
variants in one strand
variant at end of strand
poor coverage or matching
gene has paralogue
increased sensitivity variant algorithims
application of NGS technology
WGS
req database for spec mutations
exome sequencing
still need to consider regulatory region mutations*
targeted sequencing
isolate a subset of genes
allows ID of rare variants
plasma DNA seq
ID fetal and tumor genetic material circulating in blood
transcripteomics
total mRNA req
gives view of transcriptional profile
epigenetics
methylation sequencing
CHIP seq
bisulfite seq
whole genome seq
finds SNVs, deltetions, amplifications, translocations, inversions, indels, etc.
exome sequencing
validates WGS
ref-seq
over expression metrics SNVs gene fusions
ribosomal profiling
IDs rate of protein synthesis for predicting protein abundance
translation control
shows protected mRNA fragments
what does WGS analysis yield?
single nucleotide variants
amplifications
deletions
translocations
inversions
indels
current limitations of WGS
order of variants on parental xsome is not retained
difficulty mapping repetitive sequences
incr error rates
thousands of cells needed which excludes many cell types
WGSs clinical application
1st line of dx in sick children
dx of developmental delay
ID de novo mt/recessive alleles
goals of the non-invasive prenatal test
reduce exposure of fetus to risk
reduce false pos
enable incr detection
easy testing available to all preg ppl
NIFTY – non invasive fetal trisomy test
based on free fetal DNA in mom circulation
good for detecting aneuploidy but not enough resolution to detect smaller variants causing disease
cancer profiling
focus on a select group of known cancer causing genes
problems:
tumor material
% NL DNA vs tumor DNA
formalin or parafin fixed
WGS in susceptibility testing
culture dependent
no reliance on primers
ascertain resistance by phenotype
whole meta-genome sequencing in susceptibility testing
culture independent
determines relative abundance of resistance in a pop
can’t ID specific gene to a sub species or strain
can ID AMR genes that are divergent
culture based analysis of S. aureus
spp ID in 4 hr
MALDI-TOF confirmation at 12 hr
further susceptibility testing w disks another 12-18 hrs
sequence vs culture based AMR analysis
sequencing is faster
