Exam 2: Ch 5 Part 2 Flashcards
Southern blotting
hybridization technique to detect a single restriction fragment out of a complex mix of fragments
gel electrophoresis + complementary probe
Northern blotting
expression of a single gene linked back to corresponding mRNA
detect amount of specific RNA in a sample
denatured RNA –> gel electrophoresis –> complementary probe
in situ hybridization
detect mRNA encoded by a particular gene in a tissue sample or embryo
DNA microarray/DNA chip
monitor expression of thousands of genes simultaneously
organized array of thousands of individual gene specific sequences on a microscope slide
DNA microarray technique
uses PCR or multiple DNA oligonulceotides to attach to microscope slide
medical uses of E. coli expression systems
produce low-abundance proteins like insulin and growth hormone
vector containing gene for protein and the lac promoter
medical use of DNA microarray analysis
distinguish tumors with a poor prognosis from a good prognosis
transfection
cloning genes into eukaryotic expression vectors and introduced into animal cells for study
two types of transfection
transient
stable (transformation like in E. coli)
transient transfection
plasmid vector with virus replication origin infects mammalian cells and has a strong promoter recognized by RNA polymerase
Foreign gene not integrated into cell genome: not replicated
Gene product produced for a few days
retroviral expression system
after cell infection, cloned gene is reverse-transcribed into DNA then transported to nucleus and integrated into host genome
reporter protein: green fluorescent protein
promoter of gene of interest also attached to GFP (promoter-fusion)
when gene expressed, green fluroesces
1st step in IDing cause for inherited human disease
identify affected gene and its encoded protein
monogenic disease
human disease resulting from a mutation in one specific gene
autosomal dominant (Huntington’s), autosomal recessive (cystic fibrosis), X-linked recessive (Duchenne muscular distrophy)
genetic heterogenecity
mutations in one of multiple different genes cause the same disease
ex. retinitis pigmentosa (degeneration of retina)
polygenic disease
alleles of multiple genes contribute to occurrence and severity of the disease
GWAS
examine a large number of DNA markers in populations without disease vs. with disease to find disease causing mutations
3 gene-inactivation techniques
replace a normal gene with other sequences
introduce an allele whose encoded protein inhibits functioning of normal protein
promote destruction of mRNA expressed from a gene
disrupting yeast cells with homologous recombination
PCR generates a disruption construct that is transfected into yeast cells
method has shown that 4500/6000 yeast genes are not required for viability
transcription can be controlled in a gene ligated to a regulated promoter
in yeast, a promoter GAL1 is active in cells grown on galactose, but not glucose
an essential gene ligated to GAL1 is put in a shuttle vector into haploid yeast where the essential gene was mutated
this yeast grows on galactose (b/c normal copy of essential gene), but not on glucose b/c GAL1
gene knockout
altered gene
gene knockout mice
DNA with disrupted allele of target gene introduced to embryonic stem cells and grown and selected for
ES cells heterozygous for the knockout mutation are injected into a wild-type mouse blastocyte
mating produces homozygotes with mutation
site-specific DNA recombination site
loxP site in mice and Cre enzyme to catalyze recombination
expression of Cre controlled by a cell-type specific promoter
only specific tissue has gene knockout
dominant-negative allele
genetically dominant: produce a mutant phenotype in cells with a wild-type copy
produce a loss of function mutation
transgene
randomly inserted dominant-negative gene controlled by a regulated promoter
RNA interference
RNAi is the easiest method to inhibit function of specific genes by destroying mRNA
RNAi in roundworms
dsRNA blocks expression of its corresponding mRNA but not mRNAs with a diff sequence
RNA endonucleas Dicer RNAi –> small inhibitory RNA (siRNA)
RISC
protein complex that cause cleavage of mRNA
cleaves mRNA-siRNA hybrid
Stable Transfection
Transient transfection of gene of interest + antibiotic resistance gene like Neo
Gene integrated into genome RARELY
Treat all cells with antibiotic, only Neo stably transfected cells survive
