Devo Lect 3 - techniques

Question 1

Gene cloning

Answer 1

Copying genes for study, from cDNA library or genomic

Question 2

Promoter region

Answer 2

RNA poly and TF bind there, control on/off genes, KEY for developmental bio

Question 3

DNA hybridization

Answer 3

complementary binding; southern blots; visualize presence of genes (probes); look for conserved across organisms

Question 4

DNA sequencing

Answer 4

dideoxy nucleotides: fluoresce and terminate

Question 5

northern blot

Answer 5

temporal and spatial (when and where) gene expression patterns; gel, transfer, probe

Question 6

in situ hybridization

Answer 6

temporal and spatial (when and where) gene expression patterns; probe put on tissues, bind to complimentary DNA

Question 7

PCR

Answer 7

amplifies segment of DNA; 95, 37, 72 - denature, anneal, taq polymerase

Question 8

RTPCR

Answer 8

Real time or reverse transcriptase PCR - makes DNA from RNA, then PCR - shows genes that are even barely expressed (sensitive)

Question 9

QPCR

Answer 9

Quantifies the amount of DNA; primer fluoresces so we can measure it by that

Question 10

Expressing a gene to study

Answer 10

in xenopus oocytes, tissue culture, bacteria, transgenic organism and study effects

Question 11

Knock out

Answer 11

insert some DNA to recombine (homologous sequence), get heterozygote, then breed to get homozygous knockout and study effects

Question 12

Antisense DNA

Answer 12

insert little bits of complementary sequences which bind to mRNA, can’t be translated; not used that much

Question 13

RNAi

Answer 13

use a very little bit of dsRNA, stimulates machinery of cell to cut up the RNA of choice

Question 14

Western Blot

Answer 14

where proteins are expressed; need labeled antibody

Question 15

immunofluoresence microscopy

Answer 15

fluorescent antibody so you can see proteins

Question 16

Glofish

Answer 16

Transgenic zebrafish; designed as environmental detector; expresses GFP, RFP, etc. in muscles; insert muscle-specific promoter in front of GFP

Question 17

Bioreactor purposes

Answer 17

A living system to grow large amounts of something, either to isolate something (ie. insulin, vitamins) or a useful protein (ie golden rice)

Question 18

Glofish reproductive rate study

Answer 18

Slower reproduction; due possibly to colour; also due to energy expenditure to express GFP at high levels

Question 19

Aquabounty atlantic salmon

Answer 19

Has chinook salmon growth hormone with promoter from Ocean pout - growth hormone is active all year rather than periodically in wild type

Question 20

Concern about transgenic salmon

Answer 20

Possibly high growth factor in the flesh, and does this have an effect on humans; Process could disrupt normal genes; allergic reactions; competitive advantage over wild type fish?