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fall 2022 > exam 2 > Flashcards

exam 2 Flashcards

1
Q

normalized melting curves use

A

melting temp or dyes that fluoresce in the presence of dsDNA

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2
Q

unlabeled probe genotyping

A

used when greater detail or exact genotyping is needed

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3
Q

snapback primer

A

unlabeled probes on the 5’ end of a primer

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4
Q

snapback or unlabed primers are used for

A

common disease variants
subtyping viruses
epidemiology

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5
Q

factor V leiden genotyping

A

small amplicons, unlabeled probes, or snapback primers

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6
Q

gilbert syndrome

A

increased drug toxicity
TA repeats in promoter or UGT1A1
small amplicon genotyping is used

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7
Q

methylation analysis is used to

A

dx imprinting disorders
give degree of methylation
(unmethylated CG are converted to TA in pretreatment bc methylation is lost in PCR)

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8
Q

competitive PCR

A

used to detect exomic delections, trisomies, sex chromosome abnormalities

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9
Q

epigenetics

A

the study of mitotically heritable changes of a phenotype that do not result from changes in the genetic code

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10
Q

epigenetics are mediated by

A

postranscriptional histone modifications
histone variants
atp-dependent chromatin
small and noncoding RNAs
DNA methylation

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11
Q

DNA methyhlation is

A

methyl group on 5’ position of cytosines in the context of a GpG that interferes with binding of transcriptional activators

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12
Q

CpG

A

75% methylated

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13
Q

CpG islands

A

mostly actively transcribed
1-4kb
methylated islands are on inactivated chromosomes

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14
Q

theory of methylation

A

is essential for mammalian embryogenesis
origin of disease hypothesis
environmental influences
imprinting

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15
Q

there is altered methylation patterns in

A

cancer
neurodegenerative disorders
metabolic disorders
autoimmune disorders like lupus

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16
Q

increase of methylation in promoter regions of tumor suppressors and signal transduction genes

A

APC

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17
Q

methylation of DNA repair

A

MGMT
MLH1
BRCA1

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18
Q

methylation of detox genes

A

GSTP1

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19
Q

methylated cell cycle regulators

A

p15
p16
RB

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20
Q

angiogenesis

A

THBS1
VHL

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21
Q

apoptosis

A

caspases
p14
DAPK

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22
Q

clinical application of methylation

A

determine ratio of meth:unmeth CpGs
- independent of starting material and expression levels
- can be done on almost any specimen

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23
Q

changes in methylation

A

precede genetic changes
hypermethylation is seen prior to dysplastic changes

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24
Q

DNA methylation as a prognostic indicator

A

ID tumor growth and recurrence
predict survival time
stratify tumor to aid in therapy decision
predict response to chemo

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25
Q

methylation content has cons like

A

no info on location of DNA meth on genome

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26
Q

genome wide analysis of methylation

A

-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

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27
Q

other methods for genome wide methylation analysis

A

NGS
anti-methyl cytosine antibodies
purification of methylated DNA w/ MBD proteins

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28
Q

analysis of individual CpGs requires

A

PCR of MS digested DNA
MS multiplex ligation dependent probe amplification (MS-MLPA)

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29
Q

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.

A

true

30
Q

Maxam Gilbert Sequencing

A

a chain breakage at specific NTs are then separated by gel electrophoresis and detected by autoradiography read 5’ to 3’

31
Q

dimethyl sulfate

A

breaks at a G

32
Q

Formic Acid

A

breaks at a G+A

33
Q

Hydrazine

A

T and C

34
Q

Hydrazine and salt

A

C

35
Q

sanger sequencing is

A

modified DNA replication
growing chains are terminated by ddNTPs

36
Q

why can’t ddNTPs bind

A

the 3’OH group is missing

37
Q

sequencing ladder

A

fragments electrophoresed in 4 sample tubes and 4 lanes

38
Q

Cycle sequencing utilizes

A

chain termination in a thermal cycler, uses one tube/capillary
a dye primer
heat stable DNA pol

39
Q

cycle sequencing labels

A

fluorescein and rhodamine
each letter has its own color which corresponds to peaks and are read on an electrophoretogram

40
Q

dye primer is on

A

5’

41
Q

dye terminator is on

A

3’ end or ddNTPs

42
Q

pyrosequencing is based on

A

light generated through release of a ppi on NT addition

43
Q

pyrosequencing equation

A

PPi + APS -> ATP
ATP + luciferase -> oxyluciferin + light

44
Q

Next Gen Sequencing requires

A

a library
custom linkers
a solid surface/interface

45
Q

NGS steps

A
  1. shear high MW DNA
  2. polish ends
  3. ligate DNA adapters
  4. produce fractions and quantitate
  5. amplify on flow cell surface
  6. denature to ssDNA
  7. hybridize seq primers to the ssDNA
  8. sequence or hybrid capture
46
Q

issues w/ PCR

A

preferential amplification/jackpotting
false + due to substitution errors
bias in high and low C/G fragments

47
Q

hybrid capture

A

fragments from WGS library are selected w probes that match target
can purify w/ magnets

48
Q

multiplex PCR

A
  1. design overlapping primers from gene of interest
  2. group primers according to G/C content/Tm
  3. amplify
  4. create library by ligation
  5. sequence
49
Q

single nucleotide RT sequencing/SMRT

A

phi 29 DNA pol
very processive
phospholinked florescent dNTPs

50
Q

false negatives are often due to

A

lack of coverage

51
Q

false positives are due to

A

variants in one strand
variant at end of strand
poor coverage or matching
gene has paralogue
increased sensitivity variant algorithims

52
Q

application of NGS technology

A

WGS
req database for spec mutations
exome sequencing
still need to consider regulatory region mutations*

53
Q

targeted sequencing

A

isolate a subset of genes
allows ID of rare variants

54
Q

plasma DNA seq

A

ID fetal and tumor genetic material circulating in blood

55
Q

transcripteomics

A

total mRNA req
gives view of transcriptional profile

56
Q

epigenetics

A

methylation sequencing
CHIP seq
bisulfite seq

57
Q

whole genome seq

A

finds SNVs, deltetions, amplifications, translocations, inversions, indels, etc.

58
Q

exome sequencing

A

validates WGS

59
Q

ref-seq

A

over expression metrics SNVs gene fusions

60
Q

ribosomal profiling

A

IDs rate of protein synthesis for predicting protein abundance
translation control
shows protected mRNA fragments

61
Q

what does WGS analysis yield?

A

single nucleotide variants
amplifications
deletions
translocations
inversions
indels

62
Q

current limitations of WGS

A

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

63
Q

WGSs clinical application

A

1st line of dx in sick children
dx of developmental delay
ID de novo mt/recessive alleles

64
Q

goals of the non-invasive prenatal test

A

reduce exposure of fetus to risk
reduce false pos
enable incr detection
easy testing available to all preg ppl

65
Q

NIFTY – non invasive fetal trisomy test

A

based on free fetal DNA in mom circulation
good for detecting aneuploidy but not enough resolution to detect smaller variants causing disease

66
Q

cancer profiling

A

focus on a select group of known cancer causing genes
problems:
tumor material
% NL DNA vs tumor DNA
formalin or parafin fixed

67
Q

WGS in susceptibility testing

A

culture dependent
no reliance on primers
ascertain resistance by phenotype

68
Q

whole meta-genome sequencing in susceptibility testing

A

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

69
Q

culture based analysis of S. aureus

A

spp ID in 4 hr
MALDI-TOF confirmation at 12 hr
further susceptibility testing w disks another 12-18 hrs

70
Q

sequence vs culture based AMR analysis

A

sequencing is faster

fall 2022 (6 decks)

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