Devo Lect 3 - techniques Flashcards
(20 cards)
Gene cloning
Copying genes for study, from cDNA library or genomic
Promoter region
RNA poly and TF bind there, control on/off genes, KEY for developmental bio
DNA hybridization
complementary binding; southern blots; visualize presence of genes (probes); look for conserved across organisms
DNA sequencing
dideoxy nucleotides: fluoresce and terminate
northern blot
temporal and spatial (when and where) gene expression patterns; gel, transfer, probe
in situ hybridization
temporal and spatial (when and where) gene expression patterns; probe put on tissues, bind to complimentary DNA
PCR
amplifies segment of DNA; 95, 37, 72 - denature, anneal, taq polymerase
RTPCR
Real time or reverse transcriptase PCR - makes DNA from RNA, then PCR - shows genes that are even barely expressed (sensitive)
QPCR
Quantifies the amount of DNA; primer fluoresces so we can measure it by that
Expressing a gene to study
in xenopus oocytes, tissue culture, bacteria, transgenic organism and study effects
Knock out
insert some DNA to recombine (homologous sequence), get heterozygote, then breed to get homozygous knockout and study effects
Antisense DNA
insert little bits of complementary sequences which bind to mRNA, can’t be translated; not used that much
RNAi
use a very little bit of dsRNA, stimulates machinery of cell to cut up the RNA of choice
Western Blot
where proteins are expressed; need labeled antibody
immunofluoresence microscopy
fluorescent antibody so you can see proteins
Glofish
Transgenic zebrafish; designed as environmental detector; expresses GFP, RFP, etc. in muscles; insert muscle-specific promoter in front of GFP
Bioreactor purposes
A living system to grow large amounts of something, either to isolate something (ie. insulin, vitamins) or a useful protein (ie golden rice)
Glofish reproductive rate study
Slower reproduction; due possibly to colour; also due to energy expenditure to express GFP at high levels
Aquabounty atlantic salmon
Has chinook salmon growth hormone with promoter from Ocean pout - growth hormone is active all year rather than periodically in wild type
Concern about transgenic salmon
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?
