MCB 317 Final Exam Questions with CORRECT
Answers
Requirements for transcriptional analysis of TXN control regions
A. A way to make mutants(systematic)
B.A way to assay for transcription(Quantitative)
Linker-scanner mutations
are substitution mutations
Length of mutant = same length as original clone
identifies individual elements within control region
Site-directed Mutagenesis
Use of Oligos to Synthesize Mutant Alleles
Mutate at a single place(SNP)
Isolate DNA->Denature->add SNP->Extend with Polymerase
In vitro Txn Assay
told us Promoters are Sufficient for TxnPromoter Elements Conserved Among Eukaryotes
No Individual Element found at All Promoters
quantify radioactive RNA and determine transcription efficiency
Do Promoter Elements function in vivo similarly to the way the function in vitro?
Transfection and Electroporation
Transient Transfection Assay
,promoters are not sufficent for transcription invivo
RNA poly 2 needs promoter and inhancer
Properties of Enhancers
Enhancers= short regions (typically ~ 200 bp) of densely packed consensus elements
different combinations of elements found in other enhancers
Some elements found in both promoters and enhancers
all enhancers function independent of orientation and distance
enhancers work with virtually any promoter
enhancers can act over a large distance to allow for regulation even when there are mutations
RT-PCR
reverse transcriptase-make dna copy of mRNA
A Qualitative Test for Whether an mRNA is present
SYBR green is a quantative measure of Double stranded DNA is present. measure rna expression
qPCR machine
Can quantify the level of a given RNA in a sample by measuring the number of cycles it takes to
produce a "threshold" level of PCR product(in real time)
The threshold level is the Ct value; which is a value in the linear range of amplification on a
logarithmic plot
Response elements
Heat shock, Glucocorticoid, serum, phurbol ester
How did RNA poly get named 1,2,3
, They were given their numbers by the way they eluted off a column
Positive controls
tell you the integrity of the assay is maintained, make sure your experiment works
Negative control
sets the "baseline" or defines the "signal-to-noise" ratio
Basal Factors
General factors, Required for txn of all RNAPII genes, only give basal level of transcription
Order of Addition Assay
extract + gene 1 +gene 2
extract + gene 1 then add gene 2 all the factors in extract bind to gene 1 to get RNA product 1
only
GENE 1 bound stably interacted with the Transcription factors
Footprinting
DNAase I Footprinting
DNAase 1 is an endonuclease, nicks at 5 prime phosphate and 3 prime hydroxyl and then falls
off, not processive , cuts only one strand, only allow nicking each dna molecule once or twice
then run on sequence gel, and radioactively label one strand, DNAase can nut cut where protein
is seating at
Gel Mobility Shift
Answers
Requirements for transcriptional analysis of TXN control regions
A. A way to make mutants(systematic)
B.A way to assay for transcription(Quantitative)
Linker-scanner mutations
are substitution mutations
Length of mutant = same length as original clone
identifies individual elements within control region
Site-directed Mutagenesis
Use of Oligos to Synthesize Mutant Alleles
Mutate at a single place(SNP)
Isolate DNA->Denature->add SNP->Extend with Polymerase
In vitro Txn Assay
told us Promoters are Sufficient for TxnPromoter Elements Conserved Among Eukaryotes
No Individual Element found at All Promoters
quantify radioactive RNA and determine transcription efficiency
Do Promoter Elements function in vivo similarly to the way the function in vitro?
Transfection and Electroporation
Transient Transfection Assay
,promoters are not sufficent for transcription invivo
RNA poly 2 needs promoter and inhancer
Properties of Enhancers
Enhancers= short regions (typically ~ 200 bp) of densely packed consensus elements
different combinations of elements found in other enhancers
Some elements found in both promoters and enhancers
all enhancers function independent of orientation and distance
enhancers work with virtually any promoter
enhancers can act over a large distance to allow for regulation even when there are mutations
RT-PCR
reverse transcriptase-make dna copy of mRNA
A Qualitative Test for Whether an mRNA is present
SYBR green is a quantative measure of Double stranded DNA is present. measure rna expression
qPCR machine
Can quantify the level of a given RNA in a sample by measuring the number of cycles it takes to
produce a "threshold" level of PCR product(in real time)
The threshold level is the Ct value; which is a value in the linear range of amplification on a
logarithmic plot
Response elements
Heat shock, Glucocorticoid, serum, phurbol ester
How did RNA poly get named 1,2,3
, They were given their numbers by the way they eluted off a column
Positive controls
tell you the integrity of the assay is maintained, make sure your experiment works
Negative control
sets the "baseline" or defines the "signal-to-noise" ratio
Basal Factors
General factors, Required for txn of all RNAPII genes, only give basal level of transcription
Order of Addition Assay
extract + gene 1 +gene 2
extract + gene 1 then add gene 2 all the factors in extract bind to gene 1 to get RNA product 1
only
GENE 1 bound stably interacted with the Transcription factors
Footprinting
DNAase I Footprinting
DNAase 1 is an endonuclease, nicks at 5 prime phosphate and 3 prime hydroxyl and then falls
off, not processive , cuts only one strand, only allow nicking each dna molecule once or twice
then run on sequence gel, and radioactively label one strand, DNAase can nut cut where protein
is seating at
Gel Mobility Shift
