alex maria

Question 1

Phenotypic approach:

Answer 1

Look for phenotypic mutant and isolate genes - (complementation, biochemical assays, sequence comparison)
Make more mutants to characterise it (BEST APPROACH)
Isolate mutants, map mutations to genetic map, map mutations to physical map, chromosome walking

Question 2

Candidate gene approach

Answer 2

Clone gene implicated in system and make a series of mutants from it
Problems: need some knowledge of genes function and sequence, may miss secondary characteristics

Question 3

Cloning candidates

Answer 3

1. Genome sequenced: go to database, take sequence, design primers, amplify gene OR extract RNA, make cDNA
2. CLOSE RELATIVES: Find homologue protein sequence, use as a probe for genomic library and form BAC tile path
3. NO SEQUENCE OR CLOSE RELATIVES: Align multiple related organism protein sequences + your organism. Look for high conserved protein regions and back translate (produce set of all possible codon + make pool of primers), use primers to amplify genomic regions, isolate clones and sequence

Question 4

Gene annotation

Answer 4

1. Compare genomic sequence to cDNA to identify 5’ and 3’ UTR and introns
2. Identify ORFs using consensus sequences
3. RT-PCR to produce cDNA and compare to gDNA - wont’ obtain a full transcript unless you use RACE

Question 5

RACE = rapid amplification of cDNA ends

Answer 5

Start PCR from within transcript and extend to 5’ or 3’ end

Amplify with reverse transcriptase

Question 6

3’ RACE:

Answer 6

Use GSP (gene specific primer) to a region of known sequence within your gene, nearest 3’ end and second primer to polyA tail –> amplify –> 3’ sequence

Question 7

5’ RACE:

Answer 7

Use GSP to a known internal region near 5’ end and ss cDNA hybrid
Add homopolymeric tail
2nd GSP and primer –> amplify 5’ end

Question 8

Northern blot

Answer 8

Analyse splice variants of gene in diff tissues under diff contitions
Extract DNA: denature to remove secondary structure, run on gel, separate, blot to membrane, hybridise with probe, expose to xray

Question 9

qPCR

Answer 9

Measures amplification reaction in early stages when it is still linear
cDNA + nucleotides + primers + SYBR green or Taqman

Question 10

Cp value

Answer 10

Cycle number at which cDNA is first detected

Highly expressed transcripts, lower Cp value

Question 11

Taqman

Answer 11

Gene specific probe binds transcript + 2 fluorescent molecules
Measure loss of fluoresces as measure of expression levels

Question 12

Microarray

Answer 12

1.Amplified DNA molecules into glass slide in known order
2. RNA from 2 diff individuals OR from mutant/WT OR individuals from diff conditions, label RNA with diff label for each sample
3. Hybridise to glass slide
4. If gene is more highly expressed –> greater fluorescence
PROBLEMS: Competitive, not always definitive

Question 13

RNA seq

Answer 13

Transcript levels using NGS
RNA _ adaptors of known sequence + slide –> NGS –>amplify
Align to reference or de novo

Question 14

Nuclear run on

Answer 14

Nuclei from organism differering in allele of a gene –> lyse cells on ice to get nuclei
Transcription that has already begun will be allowed to proceed, BUT NO NEW ROUNDS
Hybridise radioactively labelled transcripts to non-labelled DNA probes

Question 15

CHiP

Answer 15

Antibody to RNAP II
Clearer than nuclear run on
Cells + formaldehyde to attach proteins bound to DNA –>extract DNA –>use endonucleases to shear it (regions bound by protein protected)
Add primary antibody to protein of interest (RNAP) + antibody binding beads that will drag down protein and attached DNA
Wash away other proteins and DNA
Add proteinase K (reverses covalent links and extracts DNA)

Question 16

Reporter assay

Answer 16

Promoter of gene and attach to remoter gene (lacZ, luciferase)
Inject into embryo/cell and wherever gene is being expressed-> reporter gene

Question 17

TAP-tag

Answer 17

Used to study protein-protein interactions using 2 rounds of enrichment to remove unspecific proteins

Question 18

Yeast mating

Answer 18

a (ste2 receptor) and alpha (ste3)
Release pheromones
Mating type has diff cell surface receptor
Reception of pheromone –> G protein –> kinase cascade

Question 19

Yeast sexual cycle

Answer 19

Pheromone binds –> alpha subunit –> GDP–>GTP –> beta and gamma subunits released from receptor –> signalling cascade

Question 20

GEF

Answer 20

guanine nucleotide exchange factor: catalyses GDP to GTP exchange

Question 21

GAP

Answer 21

GTPase activating protein: Catalyses GTP to GDP

Question 22

GDI

Answer 22

Guanine nucleotide dissociation inhibitors

Question 23

Homothallamic

Answer 23

E.g. yeast
Mate with themselves
Swi5 - segregated during division in mother and daughter cell
Ash 1: segregated into ONLY daughter cell by She1
Ash1 inhibits Swi 5
Swi5 allows cell type switiching by allowing activation of HO(nuclease)
HO is dominant to ho (renders cells heterothallic)