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AP609 - 2 > Functional genomics > Flashcards

Functional genomics Flashcards

1
Q

Describe Gene Ontology?

A

Study off
§ Molecular Function (MF)
§ Biological process (BP)
§ Cellular component (CC)

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2
Q

What does gene ontology allow you to do with the information?

A

Allows genome comparison.
§ Facilitates gene grouping into:
§ Pathways.
§ systems.

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3
Q

Describe Molecular Function (MF)?

A

What a gene product can do without specifying where or when
§ Broad: “enzyme”
§ Narrower: “adenylate cyclase”

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4
Q

Describe Biological Process (BP)?

A

§ >1 distinct steps, time, transformation
§ Broad: ”signal transduction”
§ Narrower: “cAMP biosynthesis.”

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5
Q

Describe Cellular Component (CC) in gene ontology?

A

§ Part of some larger object (e.g. ribosome).

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6
Q

Explain how youd use gene ontology to determine why an E.coli strain would be antibiotic resitant and what two ways could this resistance occur?

A

Sequence the ecoli and compare the genome to another strain and see if resistance is due to:

  1. Single gene resistance specific for that antibiotic
    2.Acculmination of many genes that lead to this resistance
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7
Q

What is the workflow of using Functional genomics to determine genomic causes of disease?

A

§ Start with as much information as you can gather: genome.

§ Identify things that change (statistical significance): e.g. transcriptome.

§ Look at everything.
§ Look at what should be relevant.

§ Generate a hypothesis: Change in gene X results in phenotype Y.

§ Evaluate the hypothesis: Play with the function of gene X.
§ Increase gene expression.
§ Decrease gene expression.
§ Remove the gene or introduce the gene.
§ Mutate the gene.

§ Define the function (or start again, including more components).

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8
Q

Explain Comparative Genomics?

A

Do gene sets maintain function as you look through species?

§ Looks at genomes, gene sets, or genes as a whole.
§ No. of chromosomes / size.
§ Total number of genes (per chromosome).
§ Their (relative) locations (e.g. synteny).
§ Their sequence divergence (evolution).

Operates across large groupings.
§ Strains within a species.
§ Species within a genus.

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9
Q

What is the goal of comparative genomics for genes?

A

Aligns sequences for comparison.

Calculates common ancestors based on
known mutation rate models.

§ Identifies: Common regions
Functional domains
Unique features

Describes phylogenetic relationships.
§ Evolution.
§ Ebola transmission.

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10
Q

What are introns and exons

A

DNA (or RNA) that code for proteins are called exons. Introns are noncoding sections

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11
Q

Explain Gene Level
(phylogenetics & phylogenomics)

A

Aligns sequences for comparison.
§ Calculates common ancestors based on
known mutation rate models

X axis is time and Y axis in divergence

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12
Q

What does Identity in comparative genomics refer to?

A

% of common
characters.

Identical sequences in all species

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13
Q

What does Homologous genes:
in comparative genomics refer to?

A

Largely comparable sequences.
§ Descendants of a common
ancestor.
§ Sequence divergence.

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14
Q

What does Identity in comparative genomics refer to?

A
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15
Q

What does orthologues :
in comparative genomics refer to?

A

Orthologs are genes in different species that evolved from a common ancestral gene by speciation, and, in general, orthologs retain the same function during the course of evolution.

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16
Q

What does Orthologues in comparative genomics refer to?

A

Homologous genes caused by speciation.
§ Usually similar function.

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17
Q

What does Paralogues in comparative genomics refer to?

A

One of a set of homologous genes that have diverged from each other as a consequence of genetic duplication.

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18
Q

What does Paralogues in comparative genomics refer to?

A

Multiple copies of the same gene- Duplication event within a genome
§ Divergent sequence.
§ Different function

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19
Q

What does Xenologues in comparative genomics refer to?

A

§ Homologous genes transferred between species
(e.g. plasmids).
§ Usually very similar

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20
Q

What does Analogues in comparative genomics refer to?

A

Same (similar) function.
§ Unrelated sequence / structure.
§ Not from common ancestor.

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21
Q

Explain the difference between Forward and Reverse Genetics?

A

Forward : phenotype to gene
You have a known phenotype
induce mutatitions through radiation or insertion
look a individuals without phenotype
find which gene is responsible
Confirm function(s) by
complementation / overexpression.

Reverse: Gene to phenotype
Inhibit many (all) genes one at a
time
Test for phenotype / biomarker(s).
- Find putative responsible gene(s).
- Confirm function by reversing the
effect.

22
Q

Explain how you would knock out genes in mice?

A

1.Produce a targeting pDNA vector.
§ Contains target gene of interest.
§ Key exon disrupted e.g. exon 1 or 2.
§ Introduce selection gene e.g.
neomycin resistance.

2.§ Introduce into embryonic stem cells
by electroporation.

3 Culture with neomycin (G418).

4 Kill off wild type cells.

5 Confirm by PCR.

6 Mature animals and breed so that mice express the genes you have added and are not chimeras half normal cells / your added.

Aim homozygous

23
Q

Explain how you would Knocking out genes in yeasts?

A

Design a 50bp primer 5’ specific for gene of interest and a gene for drug resistance and cause a double strand break and your mutant gene will incorporate by homologous recombination

§ Highly efficient recombination.
§ Replace gene of interest with KanR.
§ Select for kanamycin resistance.
§ Observe phenotype.

24
Q

Explain Knocking out genes in bacteria:
Recombineering?

A

Design 5’ tailed primers and select a 50bp homology to target.

Location of homology creates gene deletion and an insertion of your gene with Drugr and primers

25
Q

What are the two approaches to Recombineering in bacteria: dsDNA systems?

A

lambda (upsidesownY) Red - 3 proteins

Gam protein: inhibits Recombinase BCD and dsDNA Exonuclease function
§ Increases efficiency

§ Exo protein:
§ dsDNA Exonuclease 5’à3’ ( creates 3 prime overhangs for hybridisation
§ Required for recombination

§ Beta protein:
§ ssDNA annealing
§ Recombinases for ds and ssDN

RecET : 2 proteins
RecE protein - mimics Exo
§ RecT protein - mimics Beta

26
Q

Explain in vivo cloning in recombinant bacteria?

A

These techniques involve transformation of exogenous DNA fragments into E. coli, which are ligated in vivo into desired sequences by the action of recombinase enzymes.

Gap repair or linear fragment joining.

No endogenous recombinase
needed.
§ Active cell replication needed.

Low efficiency: ~104 / 108 viable
cells

27
Q

Explain Recombineering in bacteria with ssDNA?

A

Add very high levels of oligos that align with the lagging strand.

§ Uses 40-70 nt synthetic oligos

Use of only beta protein to create overhangs.

Single point mutations or clusters
(knockout)

§ High efficiency: 105/108 viable cells.

28
Q

Describe Gene trapping in eukaryotes?

A

Transposon events add genes into embroyotic stem cells.
Not repeat regions.
Not essential genes.

Uses neomycin Resitant -polyA

Enhancer traps. - cloned upstream of enhancer before exon 1&2 looking at function of enhancer

Promoter traps - Cloned in 1st intron space close to promoter researchers to identify and study promoters that control the expression of nearby genes.

Poly(A) traps. - involves the insertion of a sequence followed by a polyadenylation signal allows researchers to identify and study genes that use this polyadenylation signal.

29
Q

Yeast gene trapping

A

Use random inserting transposon In the gene trapping method, a yeast strain is constructed that contains a Ty element that has been modified to carry a reporter gene, such as a gene encoding a fluorescent protein.

Use gene specific reverse primer,
Ty1 specific forward primer.

PCR amplicon generated only at
the disrupted gene. as you used TY1 specific primers.

30
Q

What is the difference between knockout and knowdown?

A

Knockout is the deletion of a gene

Knockdown is the repression of expression of a gene

31
Q

Explain siRNA library screens (knockdown)

A

Add complimentary 4-10 siRNA per gene through virus or chemically.

Induce siRNA-induced silencing through incorporation ( targeting Mrna to lower protein expression)

Rank in order effect on phenotype expression of each gene you do this too.

Assumes protein knockdown.
§ Assumes no epigenetic effects.
§ Screen for phenotypic
biomarkers.

32
Q

Explain CRISPR-CAS9 genome editing
screens (indel mutation)?

A

Cas9 induces single/double strand breaks

Use single guide RNAs (sgRNA) complimentary to target DNA to cause breaks in the DNA that during repair causes mutations resulting in knockouts.

Engineered sgRNA can cleave
mammalian genes.

33
Q

Explain the difference between Homologous repair, Non homologous end joining and Microhomology mediated end joining in double strand break repair?

A

A homologous repair usually has a repair template and thus can be repaired error free and can be large flanking sequences.

Non- homologous likely won’t have a repair template thus error-prone and usually cause deletions - knockout - may require additional testing to see if you’ve gotten the correct sequence

microhomology - uses short regions of microhomology (short sequence similarities) at the broken ends of the DNA to join the two ends together making a shorter DNA sequence

34
Q

What is the gold standard for Knockouts and Knockdrowns

A

siRNa - knockdowns that effect mRNA and reduce protein expression

sgRNA that cause deletions which are repaired however mutated leading to knockdowns

35
Q

What is the function of Trancriptional Analysis?

A

Measurment of RNA transcrips ( how many copies)

36
Q

What is the most common RNA type in cells

A

rRNA = 90%

37
Q

Describe the steps in Transcriptional analysis?

A

Requires purification of RNA.
§ Requires elimination of DNA. (Dnase)
§ Requires labelling approach.
§ Requires quantification approach

38
Q

What are important things to consider in transcriptional analysis ?

A

What genes are being used?
§ Do these change?
§ By stimulus or intervention (e.g. KO)?
§ Over time (development, aging)?
§ By location (tissue, cell type)?
§ By organism (mouse vs. man)?
§ Do they go up or down?
§ Differential expression (DE).

Mutations impact assay
performance.
§ Splice variants (known / unknown). - ignored usually
§ RNA can be edited. - ignored
§ RNA is not protein

39
Q

Describe the 3 abundance classes in transcriptional analysis?

A

Superabundant
§ 15-90% of mRNA mass
§ <10 structural gene transcripts
§ >5000 molecules per cell per sequence

Abundant
§ 50-75% of mRNA mass
§ ~200-1000 structural gene transcripts (5% of diversity)
§ 500-2500 molecules per cell per sequence

Rare/complex
§ <25% of mRNA mass; individual sequences <0.01%
§ 95% of mRNA diversity
§ 1-10 molecules per cell per sequence

40
Q

What technology is used for direct anylysis of RNA?

A

Oxford nonpore

41
Q

what is an oligo dt primer and how is it used in trancriptional analysis?

A

Oligo dT primers are oligonucleotides that contain a segment of repeating deoxythymidines (dT). The dT anneal to the polyadenosine (polyA) tails of messenger RNA (mRNA), guiding the synthesis of complementary DNA (cDNA).

This binds to everything with a poly A tail and is used in the 3-5 prime to reverse transcript RNA for quantification.

This is called DNA archiving and involves large DNA analysis of many genes.

42
Q

What are the 3 methods for transcriptional analysis?

A
  1. Reverse transcriptase primer specific for RNA which creates cDNA
  2. Oligo dt primer with repeating deoxythymidines and a polyA tail that creates CDA but for all genes with poly a tail ( all mRNA)
  3. Use of random hexamer as a primer and reverse transcribe any and all RNA

all these lead to cdna which gets cloned / PCR / sequencing using NSG

RNA is usually treated with DNAse breaking all DNA leaving on CRNA

43
Q

What is delta ct ?

A

Delta Ct: The difference between the Ct level of the gene of interest and that of an endogenous control gene; aka the normalised expression level of the gene of interest. Expressed as Ct (of gene of interest) minus Ct (of endogenous control)

44
Q

What is delta ct?

A

Delta Ct: The difference between the Ct level of the gene of interest and that of an endogenous control gene; aka the normalised expression level of the gene of interest. Expressed as Ct (of gene of interest) minus Ct (of endogenous control)

45
Q

Define ct?

A

Delta Ct: The difference between the Ct level of the gene of interest and that of an endogenous control gene; aka the normalised expression level of the gene of interest. Expressed as Ct (of gene of interest) minus Ct (of endogenous control)

46
Q

Define delta delta ct?

A

Delta Delta Ct: The difference in delta Ct for a gene of interest between a treated sample and a control sample; aka the normalised expression level of a gene of interest in a treated sample expressed as a fold difference to that normalised level in a control sample

47
Q

What does David do?

A

Database for Annotation, Visualisation and Integrated Discovery

Identify enriched biological themes,
§ Discover enriched functional-related gene groups

Visualize genes on BioCarta & KEGG pathway maps - can see pathways effected

48
Q

What does GSEA do?

A

Calculate a peak enrichment score –
assess p value by swapping labels and
looking for enrichment

Gene Set Enrichment Analysis
§ Test significance of an (a priori specified) group of genes e.g.
WNT/wingless pathway genes

Rank gene from most to least expressed in one group.
* Define a set of genes:
* Positional (chromosomal region)
* Pathway (WNT pathway genes)

The genes might:
§ Belong to a known pathway or
§ Be the top genes from a related experiment.
§ The set might be significant even if individual genes are not.

49
Q

What does KEGG do?

A

The Kyoto Encyclopaedia of Genes and
Genomes

Searchable pathways for
§ Molecular interactions
§ Reaction networks for metabolism,
§ Various cellular processes and human
diseases

49
Q

What does KEGG do?

A

The Kyoto Encyclopaedia of Genes and
Genomes

Searchable pathways for
§ Molecular interactions
§ Reaction networks for metabolism,
§ Various cellular processes and human
diseases

50
Q

What does KEGG do?

A

The Kyoto Encyclopaedia of Genes and
Genomes

Searchable pathways for
§ Molecular interactions
§ Reaction networks for metabolism,
§ Various cellular processes and human
diseases

AP609 - 2 (7 decks)

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