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CMB2000 > L-16 functional genomics > Flashcards

L-16 functional genomics Flashcards

1
Q

What do functional genomic experiments describe?

A

gene functions and interactions

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2
Q

What are the different types of study

A

Protein/DNA interactions
DNA methylation
Gene expression
Protein-protein interactions
Loss-of-function

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3
Q

What is a microarray?

A

a laboratory tool that analyses large amounts of genes or proteins simultaneously

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4
Q

What do we measure using microarrays

A

Hybridisation

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5
Q

Where are the samples to probes located on in microarrays

A

Sample to probes on array

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6
Q

What probe does cDNA have in expression experiment

A

complementary to coding sequence of known genes

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7
Q

What probe does protein-bound DNA have in ChIP experiment

A

Whole genome

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8
Q

What probe does whole genome have in SNP experiment

A

Known SNPs

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9
Q

Can direct sequencing can substitute for hybridisation

A

Yes

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10
Q

What probe does whole genome have in methylation experiment

A

Known CpG islands

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11
Q

What probe does whole genome have in CGH experiment

A

Whole genome

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12
Q

What is high-throughput sequencing

A

next generation sequencing

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13
Q

What principle is used in microarray and in RNA sequencing

A

Hybridisation in microarray Direct sequencing in RNA

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14
Q

Steps involved in microarrays

A

extracting RNA -> cDNA -> biotin labelled cRNA -> random fragmentations -> fragmented biotin labelled cDNA -> cRNA + GeneCHip hyberdisation -> Wash away non-specific
binders and stain with
streptavidin-phycoerythrin ->

Scan array with laser,
detect fluorescence with
CCD, read image into
computer

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15
Q

What resolution is used in microarray and in RNA sequencing

A

several 100 bp in microarray single base in RNA

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16
Q

What throughput is used in microarray and in RNA sequencing

A

high in microarray high RNA

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17
Q

is the whole genome is used in microarray and in RNA sequencing

A

yes in microarray sometimes in RNA

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18
Q

What level of background noise is there in microarray and in RNA sequencing

A

high in microarray low in RNA

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19
Q

Are microarray and RNA sequencing capable of map transcription and DGE

A

Yes microarray Yes in RNA

20
Q

What is the dynamic range in microarray and RNA seq.

A

Up to a few hundredfold in microarray and >8000 fold

21
Q

Comapre distinguishng of isoform in microarray and RNA seq

A

Limited in microarray and yes in RNA seq

22
Q

Comapre distinguishng of alleic expression in microarray and RNA seq

A

Limited in microarray and yes in RNA seq

23
Q

Comapre amount of RNA amount required in microarray and RNA seq

A

high in microarray and low in RNA seq

24
Q

Compare cost for large genomesin microarray and RNA seq

A

high in microarray and relatively low in RNA seq

25
Q

Which company is dominant in terms of high throughput suquencing

A

Illumina

26
Q

What are the grounds for competitionin high throughput seuqncing

A

driven down cost, increased throughput

27
Q

Illumina Sequencing

A

fragments of DNA (library) bound to solid surface (flow cells) -> bridge PCR = clonal clusers -> sequencing in cucles -> modified nucelotides w fluorescent group blocked extentiion = reversible termination

28
Q

How do we sequence RNA

A

High througput sequencing technologies to get info about RNA content -> mRNA -> cDNA -> used for sequnecing library gen. -> allws quantification, profiling and discovery of RNA

29
Q

How does RNA-seq work?

A

Sequences in final library are derived from RNA population in
sample
* Presence is proportional to original sample
* More abundant RNA species will be present more frequently in library
* ‘Random’ priming is attempt to remove bias
* Or not introduce it
* Actual randomness debatable

30
Q

What are RNA-seq considerations

A

Big data sets require expert processing
* Expression data can be noisy
* Careful experimental design important
* Easy for confounding factors to dominate
* Good practise same as for any statistical approach

31
Q

What are other functional genomics approaches

A

Big data sets require expert processing
* Expression data can be noisy
* Careful experimental design important
* Easy for confounding factors to dominate
* Good practise same as for any statistical approach

32
Q

WHat is ChIP-Seq

A

Cross-link proteins to DNA?

33
Q

What occurs in ChIP-Seq

A

Isolate DNA and shear
* Sonication for ‘random’ shearing
* Immunoprecipitate protein of
interest
* Reverse cross-linking
* Purify DNA
* Sequence

34
Q

What is ATAC-Seq

A

Assay for Transposase-Accessible Chromatin

35
Q

What is ATAC-seq similar to

A

Similar to older DNAse-Seq
* Relies on transposase Tn5
* High activity transposase
* Highly efficient cutting of exposed DNA
* Ligation of adapters to ends
* Adapter ligated fragments isolated, amplified and sequenced

36
Q

What is bisulphite sequencing

A

Bisulphite treatment is used to determine the methylation state of
DNA
* Methylated cytosine protected from deamination
* Unmethylated cytosine converted to thymine (via uracil)

37
Q

What occurs in bisulphite sequencing

A

Sequencing of bisulphite treated, along with untreated samples
identifies sites of methylation
* High depth sequencing can provide quantitative estimates of
methylation
* Hypo- and hyper-methylated regions can then be identified
(hyper = txn. silencing)

38
Q

Reduced Representation bisulphite sequencing (RRBS) - what causes this?

A

Human genome has 28m CpG sites and Assaying all of these in a single experiment problematic

39
Q

What enzyme does RRBS utilise to enrich for CpG?

A

Msp1

40
Q

What does Msp1 result in?

A

fragments which begin/end with CpG

41
Q

How much of the genome needs to be sequenced for RRBS?

A

1%

42
Q

High throughput sequencing produces ______ amounts of data

A

large

43
Q

Data can be ____ and complex

A

Noisy

44
Q

Computational approaches needed to make most of data for both

A

processing and analysis

45
Q
A

CMB2000 (10 decks)

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