Brainscape's Knowledge GenomeTM


Top Flashcards (Certified)
All Flashcards

TMS exam > 8 - diagnosis and treatment of epigenetic alterations > Flashcards

8 - diagnosis and treatment of epigenetic alterations Flashcards

(58 cards)

Start Studying
1
Q

tissue and cell type profiling

A

bisorphite sequencing –> looks at histone modification and methylation of whole tissues

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

DNMTi

A

DNA methylation transferase inhibition

removes hypermethylation of tumour suppressor genes

cancer treatment

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

5-azacytidine

A

DNMTi
successful epigenetic therapy
DNMT enzyme binds to 5-azacytidine and decreases its activity

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

disadvantages of DNMTi therapy alone

A

lacks specificity
resistance common
unsuccessful in solid tumours

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

function of HDAC inhibitors

A

inhibit histone deacetylation
hyper-acetylation –> promotion of tumour suppressor genes
promote cell cycle arrest and apoptosis

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

examples of HDAC inhibitors

A

vorinostat
belinostat
romidepsin

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

combination treatments

A

combine HDACi with DNMTi

combine DNMTi with cytotoxic drugs

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

combination of HDACi with DNMTi

A

increased expression of silenced genes and antitumour apoptotic response

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

combination of DNMTi with cytotoxic drugs aim

A

resensitize cancers to the standard agents

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

discovery using next generation sequencing about chromatin cancer mutations

A

more than 50% of human cancers harbour mutations in enzymes involved in chromatin organisation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

cancer cell immune evasion using epigenetics

A
  • tumour cells use epigenetic process to escape chemotherapy and host immune surveillance
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

challenges to cancer epigenetic research:

A
  • order of events of abnormal gene silencing in cells
  • role of chromatin modifications in mediating these processes and targeting DNA methylation changes
  • maintenance of gene silencing in cancer cells
  • epigenetic biomarkers have low sensitivity –> need for multi-gene panels
  • epigenetic therapies lack specificity –> adverse side effects and of target effects
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

illumina 450K methylation array

A

epigenome interrogation technique
covers DNA methylation sites at single-nucleotide resolution
almost genome wide

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

disadvantages of bisulfite sequencing

A

difficult to optomise

base calling software –< cant cope with lack of ‘C’ signal
over-exaggerates at methylated sites

doesnt provide information about methylation sites of individual alleles

cant accurately quantify methylation (semi-quantitative)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

‘gold-standard’ for gene-specific methylation analysis

A

sequencing of cloned bisulfite PCR amplicons

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

pros of sequencing of cloned bisulfite PCR amplicons

A
  • PCR production cloned into plasmid vectors and clones sequenced
  • Highly quantitative
  • Gives molecule-specific information
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

cons of sequencing of cloned bisulfite PCR amplicons

A

time consuming

expensive

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

process of target-specific bisulfite-Pyrosequencing

A

Real-time sequencing method for analysis of short-medium length DNA sequences

o Nucleotides added sequentially to reaction
o Pyrophosphate released during nucleotide incorporation detected as light produced by enzyme substrate cascade system

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

pros of target-specific bisulfite-Pyrosequencing

A 
o	Fast and reliable
o	Cost-effective 
o	Targets specific regions of genome 
o	Provides single nucleotide read-out 
o	Reproducible
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

cons of target-specific bisulfite-Pyrosequencing

A

o Not high throughput

o Small fragments (max 100bp)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

features of whole-genome bisulfite sequencing

A
  • Whole genome coverage
  • Costly
  • Time-consuming –> need to resequence genome many times
  • Requires extensive bioinformatics
  • Limited scalability per run
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

method of whole-genome bisulfite sequencing

A

Measures single-base cytosine methylation levels (measures ratio of methylated molecules rather than enrichment)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

RRBS

A

Reduced representation bisulfite sequencing

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

pros/cons of RRBS

Study These Flashcards
A

pros:

  • Reduces sequencing costs
  • Increases read-depth at CpG-rich regions
  • Provides single nucleotide read out

Cons:

  • Short fragments can create mapping problems
  • Biased towards repeat sequences, CpG sequences
  • Can miss under-methylated regions
25
Target enrichment: | --> ‘sure select xt human methyl-seq’
The first comprehensive methylation discovery system probes independent of methylation state determines methylation state of all methyl sites in region
26
pros and cons of target enrichment
Pros: o reduced sequencing costs o targeting CpG rich regions of genome shores and DMRs o can create custom targets ``` cons: o library preparation is time-consuming o 3ug DNA required o not truly genome wide o automation not supported ```
27
process of methylated DNA immuno-precipitation (MeDIP)
o monoclonal antibody specific for 5meC | o immunoprecipitation of single stranded molecules containing one or more methylated cytosine
28
pros and cons of methylated DNA immuno-precipitation (MeDIP)
pros o reduces sequencing costs (5Gb required) o no bias to one specific sequence o can be adapted for different cytosine residues e.g. 5-hmc cons o don’t get base pair resolution (max res. 150bp) o cant target specific regions e.g. gene body shores etc. o hard to detect under-methylated regions o methylation levels hard to determine
29
pacific biosciences single-molecule DNA:
sequencing base modification directly - use polymerase kinetics - kinetic characteristics  time between two successive base incorporations are altered by the presence of a modification  increased space between fluorescence pulse interpulse duration
30
SMRT stands for
single molecule real time sequencing
31
pros and cons fo SMRT
Pros: - longer reads --> allows phasing of halotypes, repeat regions etc. - sequences base modifications directly - sequence information and base modification cons: - need good quality ds DNA - 5ug required - X250 coverage needed for hmC and h-hMc - Technology still being optomised
32
use of chromatin modification assays
- Histone marks along with other assays of open chromatin are presently the only reliable indicators of locations and activities of regulatory elements
33
open chromatin
euchromatin histones bound loosely to DNA binding sites accessible for transcription factors --> active
34
closed chromatin
heterochromatin | histones packaged tightly
35
process of chromatin immunoprecipitation (ChIP)
Antibody attached to a DNA binding protein is used to capture DNA bound to the protein in a living cell o DNA and protein cross-linked in the cell using formaldehyde o Crosslinked chromatin sheared yielding short fragments of DNA and protein complexes o Antibodies capture fragments containing DNA binding protein o DNA ‘released’ and analysed --> PCR - Creates pool of sequences highly enriched in binding sites for a particular protein o Requires availability of excellent antibodies that can detect the protein in its in vivo context
36
ATAC-SEQ stands for
(Assay for Transposase-Accessible Chromatin using Sequencing)
37
how does ATAC-SEQ assay work
- Transposons incorporate preferentially into genomic regions free of nucleosomes - Enrichment of sequences from certain loci in the genome indicates absence of DNA-binding proteins of nucleosome in the region - Regions of the genome where DNA was accessible during the experiment will contain significantly more sequencing reads = Peak
38
steps involved in ATAC-SEQ assay
1. Cell preparation 2. Transposase reaction 3. Library amplification 4. Sequencing on an illumina platform 5. Bioinformatic analysis
39
3 scales of chromatin structure
primary secondary tertiary
40
what does primary chromatin structure encompass
``` DNA methylation sequence features DNA-bound factors nucleosome positions modification DNA accessiblity ```
41
what does secondary chromatin structure encompass
local structures formed by nucleosome-nucleosome interactions
42
what does tertiary chromatin structure encompass
promoter-enhancer 3D contacts | mega-base scale domains
43
where are majority of GWAS variants found
regulatory regions (promoters/enhancers) not in protein-coding regions
44
original hypothesis
SNP caused by change in amino acid sequence | causes change in protein product
45
alternative hypothesis
SNP caused by DNA methylation | causes changes in gene expression
46
use of mQTL
used to identify functional consequences of genetic risk factors results overlapped with GWAS results
47
why can GWAS not identify the specific causal variant
GWAS identifies regions there could be two distinct causal variants in the same LD block
48
3 categories of laboratory techniques to interrogate the epigenome
- DNA methylation analysis - analysis of DNA/protein interaction - chromatin accessibility and conformation assays
49
methods of DNA methylation analysis
- bisulfite conversion (most common) - high resolution melt (HRM) analysis - methylation DNA immunoprecipitation (MeDIP)
50
which techniques is analysis of DNA/protein interaction
chromatin immunoprecipitation (ChIP)
51
general process of bisulfite conversion
identifies 5mC converts unmethylated cytosines to uracil 5mC is not converted --> identified through PCR quantifies levels of methylation
52
COBRA
Combined bisulfite restriction analysis - type of bisulfite conversion - Useful for analysing specific regions of DNA for methylation - Requires restriction enzymes
53
High resolution melt (HRM) analysis
- Qualitative analysis of DNA fragment’s melt curve following bisulfite conversion and PCR amplification
54
method of HRM analysis
PCR target region to amplify increase temperature of amplicon (DNA strands separate) obtain melt temperature and melt curve profile compare to standard DNA samples to determine relative levels of methylation
55
advantages of HRM analysis
Simple, cost-effective, fast
56
Methylation DNA immunoprecipitation (MeDIP)
- Genome-wide - DNA isolated from cells and sheared using sonication (application of sounds energy) - Antibodies specific to DNA fragments isolate methylated regions (5mC) - Methylated DNA identified using high-resolution DNA microarrays or next gen sequencing - Allows quantification of enriched methylated DNA fragments
57
limitation of ChIP
requires a large number of cells for reliable measurements
58
use of nuclease enzymes in chromatin accessibility assays
euchromatin (open) is digested nucleases are unable to digest heterochromatin (closed) therefore DNA is left available for examination

TMS exam (22 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2025 Bold Learning Solutions. Terms and Conditions