Everything

enzymatic

terminators

Heterochromatin

15%

P(0) = e^(-m) m:voverage

1,743

14000 (13,986)

Affymetrix

Modifier screens

Eye shape, narrowness of the eyes

genetic interactions

yeast

80%

Yeast-2-hybrid

2D-PAGE

“faux” reads

The length L such that 50% of the sequence lies in contigs of at least L.

Homologous recombination

 RNA interference?

longer read lengths

SOLiD

Ion Torrent

plasmids

BioGrid

EMS (ethylmethanesulfonate) (only the abbreviation EMS was given as answer)

causes point mutations, which are changes at a single nucleotide position. Mutations may be nonsense (introduce a premature stop codon) or mis-sense (cause an amino acid replacement). They may also be in non-coding sequence, affecting splicing signals or regulatory elements that control gene expression. This approach allows for many different mutations within gene regions, but these are difficult to map.

bacterial artificial chromosome

plasmids

molecular weight (kilo dalton)

Serial Analysis of Gene Expression

1.8 Mb

 RNA interference

1/3 (or ~33%)

 of the genome.

PacBio

SOLiD but by ligation

Protein Microarrays (In vitro)

Pull-down Assays (In vitro)

RNAi (shRNA/siRNA) Screens

CRISPR-Cas9-Based Screens

Synthetic lethal screening aims to identify gene pairs where the loss of either gene alone is viable, but the loss of both leads to cell death. 

Frederick Sanger and colleagues in 1977

original Sanger sequencing method (1977)

Ion Torrent Sequencing

Nanopore Sequencing (Oxford)

DNA -> mRNA -> protein -> interactions with internal/external environment -> phenotype

(Expressed Sequence Tags) EST Sequenzing was the first gnome-wide method. mRNA is reverse-transcribed into cDNA, then a large number of cDNAs are sequenced.

Pro: gives an estimate of absolute mRNA abundance (if cDNA library is random); very useful for gene discovery (annotating expressed regions of genomes and intron/exon boundaries). Con: expensive, time-consuming, requires large-scale sequencing; much of the sequencing is redundant (ESTs from highly-expressed genes are sequenced many times); must sequence 100,000's of ESTs to get a good representation of genes expressed at low levels; the ESTs only reveal gene expression levels in the particular tissue or sample that was used for mRNA preparation; genes expressed in specific tissues, cells, developmental stages, etc. may be missed.

C) SAGE

SAGE stands for Serial Analysis of Gene Expression. It is a genome-wide method for measuring gene expression levels. Unlike microarrays, which rely on pre-designed probes, SAGE does not require prior knowledge of the genes—it is unbiased and sequence-based.

1. Extract mRNA → reverse transcribe to cDNA.

2. Use restriction enzymes to cut cDNA at specific sites (e.g., NlaIII at “CATG”).

3. Isolate tags near the 3’ end of transcripts.

4. Add adapters, then link tags together (ditags).

5. PCR-amplify ditags, then ligate them into long chains.

6. Sequence many tags in one go.

7. Map tags back to the genome to identify the originating gene.

False it measures relative abundance

B) It is efficient at detecting low-abundance transcripts.

relative gene expression level

B) 25 bp

Advantage: More efficient; allows many transcripts to be analyzed in one sequencing reaction via tag concatenation.

Disadvantage: Requires a well-annotated genome to match short tags accurately.

 C) Affymetrix GeneChips

C) Cuts cDNA at recognition sites to create tags

It might be expressed at very low levels or only in a tissue or condition not sampled.

False

B. Observe phenotype → Identify gene

A genetic screen is a technique used in forward genetics to identify and study genes involved in a particular biological process or phenotype.

It involves the following steps:

1. Mutagenesis – Organisms (like Drosophila, C. elegans, bacteria, or plants) are treated with a mutagen (e.g., X-rays, EMS, or transposons) to introduce random mutations into their genomes.

2. Screening – The offspring of these mutagenized organisms are then screened for specific phenotypes of interest, such as defects in development, metabolism, or behavior.

3. Identification – Once mutants with the desired phenotype are identified, researchers then work to map and identify the mutated gene(s) responsible.

The goal of a genetic screen is often to find all of the genes involved in a particular trait or process, making it a powerful tool for dissecting complex genetic pathways.

a-ii, b-i, c-iii

EMS Mutagenesis – Pros:

• High mutation rate → Efficient gene disruption

• Random → Unbiased gene discovery

• Simple & cheap

• Can reveal partial/essential gene functions

EMS Mutagenesis – Cons:

• Random → Not gene-specific

• Many mutations → Hard to find causal one

• No marker → Mapping is slow

• Bias for G/C → A/T mutations

C. Saturation mutagenesis with phenotype screening

Reverse Genetics in the Post-Genomic Era – Why Useful?

• Known genes, unknown function: Genomes are sequenced → many genes identified, but their functions aren’t.

• Targeted approach: Reverse genetics allows direct testing of gene function by altering specific genes.

• Tools available: Technologies like CRISPR, RNAi make gene targeting efficient.

• Faster insights: Speeds up linking genes to phenotypes.

False

screening

Transposable element excision in Drosophila allows precise identification and disruption of genes, making it easier to link genotype to phenotype.

B. RNAi – dsRNA feeding

The wHy method increases deletion frequency by using two transposons—one P-element and one piggyBac—flanking the target gene. When both elements excise, larger deletions spanning the gene are more likely, compared to standard single P-element excision, which typically causes smaller or local changes.

Comparison

• Both allow targeted genome modifications

• HR is more precise but less efficient

• CRISPR is faster, easier, and more versatile

Homologous Recombination (HR)

• Precise, uses a DNA template for targeted edits

• Low efficiency, especially in higher eukaryotes

• Slow and labor-intensive

• Often used in yeast and mice

CRISPR-Cas9

• High efficiency and speed

• Uses guide RNA to target DNA, can induce DSBs

• Can cause insertions, deletions, or precise edits (with repair template)

• Widely applicable across species

True

The parental DNA is digested with DpnI during the QuikChange mutagenesis method because DpnI specifically digests methylated DNA (the parental plasmid), leaving the newly synthesized, mutated DNA (which is not methylated) intact. This ensures that only the mutated DNA is retained, while the original (unmutated) plasmid is removed. This step helps enrich the sample with the correct, newly generated mutations.

C. GFP

The functional conclusion from mutating the 3’ UTR of the Drosophila Adh gene was that the wild-type 3’ UTR sequence acts as a negative regulatory element. When this region was deleted, alcohol dehydrogenase (Adh) enzymatic activity in the flies increased twofold, suggesting that the 3’ UTR normally serves to regulate down the expression of the gene. Removing this regulatory sequence leads to an increase in gene expression.

B

B (meaning it cannot detect interactions of more than 2 proteins)

D

B

C

A genetic interaction refers to the functional relationship between genes, often observed when mutations in two genes lead to a different phenotype than expected based on the individual mutations. A physical interaction refers to direct molecular contact between two molecules, such as two proteins or a protein and DNA.

Because dominant mutations will show their phenotype even in the heterozygous state, making it impossible to determine whether two mutations complement each other based on offspring phenotype.

Synthetic lethality occurs when two mutations, each of which is non-lethal on its own, result in lethality when combined. It can be used to identify genes with redundant or parallel functions. For example, in yeast, knocking out two genes involved in the same essential pathway might lead to cell death.

Advantage: It allows the purification of protein complexes, identifying both direct and indirect interactions.

Limitation: It cannot distinguish between direct binding partners and those that are part of a complex via intermediates.

1. 
   

   • Perform mutagenesis on Bar mutant flies.

   • Screen for offspring with either more extreme (narrower) or less extreme (wider) eye phenotypes.

   • Narrower eyes = potential enhancer mutations.

   • Wider/more wild-type eyes = potential suppressor mutations.

   • Further genetic mapping and complementation testing would identify the genes involved.

   
   

1. 
   

   • Y2H is high-throughput and detects direct interactions but may miss complex or context-dependent ones.

   • Co-IP detects protein complexes in their native cellular context but has lower throughput and requires good antibodies.

   • Y2H is preferred for large-scale screens; Co-IP is preferred for confirming interactions in the native cell environment.

1. 
   

   • Interacting genes often participate in similar processes.

   • By clustering genes with similar interaction profiles, functional modules can be identified.

   • Genes with unknown function can be assigned to pathways based on their interaction partners.

C

H. influenzae, yeast, Drosophila, Arabidopsis, human

False – it requires a known reference genome.

C

A - 3

B - 1

C - 2

C

Paired-end reads involve sequencing both ends of a DNA fragment. They help with genome assembly by providing information about the distance between reads and can resolve repetitive regions better.

C

RNA-seq is sequencing of cDNA to quantify gene expression. It maps millions of reads back to the genome to measure transcript abundance.

D

(Typing of SNPs (Single Nucleotide Polymorphisms) means detecting which version (allele) of a specific SNP is present in a sample.)

False – NGS typically has higher error rates.

• Short read lengths (50–250 bp typically)

• Higher error rates than Sanger sequencing

• Slow base-by-base calling process

C

 C

C

True

False

False

True

pH

Single Molecule Real Time (SMRT)

field

Zero-Mode Waveguide (ZMW)

→ Advantage: Extremely long reads, portability, and low cost

→ Disadvantage: High error rate (15–30%) and requires optimization for sample types

They help with de novo genome assembly by spanning repetitive regions and resolving complex genomic structures.

It detects the release of a proton, which causes a measurable pH change during base incorporation.

Replacing the protein nanopore with a solid-state nano-channel or DNA transistor, which may offer faster and cheaper sequencing once technical challenges are solved.

Heat-shock proteins (HSPs) are a group of molecular chaperones that help protect cells from stress, particularly from elevated temperatures, but also from other stressors like toxins, oxidative stress, or infection.

• FAULX reads are simulated reads, not real sequencing data.

• No sequencing errors, biases, or gaps — they are “perfect” reads.

Roche 454 sequencing

•  contig is a continuous stretch of DNA sequence assembled from overlapping sequencing reads.

• It represents a single contiguous sequence with no gaps.

A scaffold is a portion of the genome sequence reconstructed from end-sequenced whole-genome shotgun clones. Scaffolds are composed of contigs and gaps.