Exam 2: Ch 5 Part 2 Flashcards Preview

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Flashcards in Exam 2: Ch 5 Part 2 Deck (29):
1

Southern blotting

hybridization technique to detect a single restriction fragment out of a complex mix of fragments

gel electrophoresis + complementary probe

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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

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in situ hybridization

detect mRNA encoded by a particular gene in a tissue sample or embryo

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DNA microarray/DNA chip

monitor expression of thousands of genes simultaneously

organized array of thousands of individual gene specific sequences on a microscope slide

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DNA microarray technique

uses PCR or multiple DNA oligonulceotides to attach to microscope slide

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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

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medical use of DNA microarray analysis

distinguish tumors with a poor prognosis from a good prognosis

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transfection

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

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two types of transfection

transient

stable (transformation like in E. coli)

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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

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retroviral expression system

after cell infection, cloned gene is reverse-transcribed into DNA then transported to nucleus and integrated into host genome

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reporter protein: green fluorescent protein

promoter of gene of interest also attached to GFP (promoter-fusion)

when gene expressed, green fluroesces

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1st step in IDing cause for inherited human disease

identify affected gene and its encoded protein

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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)

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genetic heterogenecity

mutations in one of multiple different genes cause the same disease

ex. retinitis pigmentosa (degeneration of retina)

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polygenic disease

alleles of multiple genes contribute to occurrence and severity of the disease

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GWAS

examine a large number of DNA markers in populations without disease vs. with disease to find disease causing mutations

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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

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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

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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

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gene knockout

altered gene

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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

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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

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dominant-negative allele

genetically dominant: produce a mutant phenotype in cells with a wild-type copy

produce a loss of function mutation

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transgene

randomly inserted dominant-negative gene controlled by a regulated promoter

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RNA interference

RNAi is the easiest method to inhibit function of specific genes by destroying mRNA

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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)

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RISC

protein complex that cause cleavage of mRNA

cleaves mRNA-siRNA hybrid

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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