Functional genomics: discovering gene function

Question 1

What is genetic linkage?

Answer 1

Tendency of nearby DNA sequences on a chromosome to be inherited together during meiosis. Closer = lower probability of recombination/recombination frequency.

Genes far apart are independently assorted and not linked

Question 2

What is forward/classical genetics?

Answer 2

Phenotype-driven approach which asks the question: what gene contains the mutation responsible for this phenotype?

Question 3

Describe the protocol for forward genetics

Answer 3

1. Identify alternative phenotype/mutant line in a model organism
2. Cross mutant with WT to follow its inheritance
3. Map the gene thought to be involved using molecular markers/family studies
4. Clone the genome/rough locus
5. Identify clones spanning the mapped region and analyse for candidate genes
6. Confirm hypothesis by rescuing mutant with WT gene

Question 4

What is reverse genetics?

Answer 4

Asks the question: what phenotype is caused by a loss of function mutation in a novel gene which has been discovered in a sequence databsed or by quantitative trait loci analysis?

Question 5

What is a novel gene?

Answer 5

A gene with no known function or only predicted function based on homology with other characterised genes

Question 6

What is a quantitative trait locus (QTL)?

Answer 6

A genetic region which influences the phenotypic variation of a complex trait through genetic/epigenetic interactions

Question 7

Describe the protocol for reverse genetics

Answer 7

1. Identify novel gene sequence via high throughput sequencing
2. Generate loss of function mutant
3. Define gene function - possibly with homology search

Question 8

What is comparative genomics?

Answer 8

Use of cross-species comparisons to identify genes/proteins and predict their function

Question 9

What is genetic synteny?

Answer 9

Segments of chromosomes from different species which share the same linear organisation of genes. Syntetic genes have a common origin, so function is often retained.

Question 10

List 3 methods of targeted mutagenesis

Answer 10

1. Homologous recombination
2. CRISPR/Cas9
3. RNA interference

Question 11

What is homologous recombination for targeted mutagenesis?

Answer 11

Direct knockout of genes:
1. Clone mutant gene
2. Introduce gene into a vector
3. When introduced into an organism, homologous recombination replaces cloned gene with WT

Question 12

What does Cas9 require to function and what are the individual roles of these factors?

Answer 12

2 guiding RNAs:
* crRNA - 20b sequence complementary to target DNA (protospacer) and another region complementary to tracrRNA
* tracrRNA - general Cas9 function
* Protospacer adjustment motif - NGG

Question 13

What can the two Cas9 guiding RNAs be fused to form?

Answer 13

Short guide RNA (sgRNA)

Question 14

What does the sgRNA/Cas9 complex do?

Answer 14

Cuts at specific locations in the genome, as defined by the guiding RNA, to give blunt double-stranded DNA breaks

Question 15

Will Cas9 cut at the same place twice? Why?

Answer 15

No!!!! Since the DNA sequence has been changed, the sgRNA is no longer complementary to it.

Question 16

How can DNA be repaired after cutting with sgRNA/Cas9?

Answer 16

• Non-homologous end joining
• Homology-dependent repair

Question 17

What is non-homologous end joining? What does this result in?

Answer 17

Ligation of dsDNA breaks without a template. Results in indel mutations.

Question 18

What is homology-dependent repair? What is this used for?

Answer 18

Using the sequence of an unaffected homologous chromosome as a template to fill the gap in the cut DNA. Used for precise gene editing.

Question 19

What is RNA interference? How is it used in gene expression studies?

Answer 19

A natural mechanism used to degrade foreign RNA. Used to control gene expression by reducing the amount of a transcript produced - used in knockdown studies where gene knockouts are lethal.

Question 20

Describe the process of RNA interference

Answer 20

1. dsRNA cut into short fragments ~20bp to produce short interfering RNA (siRNA)
2. siRNA recruited by RISC (RNA induced silencing complex) and converted to single strands
3. siRNA strands recognise and hybridise to target mRNA which is cleaved

Question 21

Which two ways can RNA interference be used to disrupt gene function?

Answer 21

1. Make dsRNA in vitro; inject into cells
2. Introduce transgene with reverse repeat into genome so RNA forms self-complementary stem-and-loop when transcribed

Question 22

What is systems biology?

Answer 22

Combining biology/technology etc with an aim to link all genes and proteins into a functional network used for predictive purposes. Largely based on the -OMICs.