Exam 2: Ch 5 Part 2

Question 1

Southern blotting

Answer 1

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

gel electrophoresis + complementary probe

Question 2

Northern blotting

Answer 2

expression of a single gene linked back to corresponding mRNA

detect amount of specific RNA in a sample

denatured RNA –> gel electrophoresis –> complementary probe

Question 3

in situ hybridization

Answer 3

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

Question 4

DNA microarray/DNA chip

Answer 4

monitor expression of thousands of genes simultaneously

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

Question 5

DNA microarray technique

Answer 5

uses PCR or multiple DNA oligonulceotides to attach to microscope slide

Question 6

medical uses of E. coli expression systems

Answer 6

produce low-abundance proteins like insulin and growth hormone

vector containing gene for protein and the lac promoter

Question 7

medical use of DNA microarray analysis

Answer 7

distinguish tumors with a poor prognosis from a good prognosis

Question 8

transfection

Answer 8

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

Question 9

two types of transfection

Answer 9

transient

stable (transformation like in E. coli)

Question 10

transient transfection

Answer 10

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

Question 11

retroviral expression system

Answer 11

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

Question 12

reporter protein: green fluorescent protein

Answer 12

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

when gene expressed, green fluroesces

Question 13

1st step in IDing cause for inherited human disease

Answer 13

identify affected gene and its encoded protein

Question 14

monogenic disease

Answer 14

human disease resulting from a mutation in one specific gene

autosomal dominant (Huntington’s), autosomal recessive (cystic fibrosis), X-linked recessive (Duchenne muscular distrophy)

Question 15

genetic heterogenecity

Answer 15

mutations in one of multiple different genes cause the same disease

ex. retinitis pigmentosa (degeneration of retina)

Question 16

polygenic disease

Answer 16

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

Question 17

GWAS

Answer 17

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

Question 18

3 gene-inactivation techniques

Answer 18

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

Question 19

disrupting yeast cells with homologous recombination

Answer 19

PCR generates a disruption construct that is transfected into yeast cells

method has shown that 4500/6000 yeast genes are not required for viability

Question 20

transcription can be controlled in a gene ligated to a regulated promoter

Answer 20

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

Question 21

gene knockout

Answer 21

altered gene

Question 22

gene knockout mice

Answer 22

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

Question 23

site-specific DNA recombination site

Answer 23

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

Question 24

dominant-negative allele

Answer 24

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

produce a loss of function mutation

Question 25

transgene

Answer 25

randomly inserted dominant-negative gene controlled by a regulated promoter

Question 26

RNA interference

Answer 26

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

Question 27

RNAi in roundworms

Answer 27

dsRNA blocks expression of its corresponding mRNA but not mRNAs with a diff sequence

RNA endonucleas Dicer RNAi –> small inhibitory RNA (siRNA)

Question 28

RISC

Answer 28

protein complex that cause cleavage of mRNA

cleaves mRNA-siRNA hybrid

Question 29

Stable Transfection

Answer 29

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