Exam 2: Ch 5 Part 2 Flashcards Preview

Cellular and Molecular Biology > Exam 2: Ch 5 Part 2 > Flashcards

Flashcards in Exam 2: Ch 5 Part 2 Deck (29):

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



cloning genes into eukaryotic expression vectors and introduced into animal cells for study


two types of transfection


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



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



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)



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