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What does quantitative PCR determine?
How much of a target (RNA/DNA) is in a sample
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For each PCR sample, one uses 3-4 tubes, each with the same amount of _______ RNA template but different amounts of sample
competitive
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The competitor RNA template wants to be as similar to the target ______ _____ as possible.
mRNA sequence
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Competitor RNA Templates generally synthesize a truncated form of the mRNA of interest in which sequences from the _____ of the mRNA are removed
middle
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Truncation makes it possible to differentiate the ______ and _______ products.
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Development of ultra-sensitive, non-radiolabel nucleic acid detection methods allow for ______ ________ of PCR.
rapid quantitation
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Real-time PCR Methods are All based on ________ _______ of dye-tagged nucleic acids, dsDNA dyes, and fluorescence-quenching.
fluorescence detection
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Real time PCR _____ and ______ run in the same instrument and tubes.
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TaqMan labes are attatched to the _____ primer
Forward
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_____ detection has both a fluorescent and a quenching label attached to it.
TaqMan
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____ detection has only a fluorescent marker.
LUX
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_____ ______is the least sensitive & semi-quantitative but yields MW and can run multiple samples per blot.
Northern blotting
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Of the Methods to Quantitate mRNA _____ ______ has Moderate to high sensitivity and allows for multiple mRNA species quantitation
Solution Hybridization (RNase Protection)
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The most sensitive approach to Quantitate mRNA are ______ ___ & ____-____ _____
- Competitive PCR
- real-time PCR
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Medical related applications of the PCR technique include _______ individuals for drug metabolizing enzymes
Genotyping
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Medical related applications of the PCR technique include detecting the presence of ______ ____, e.g. hepatitis C
viral DNA
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Medical related applications of the PCR technique include Quantitating the amount of ______ ____, e.g. HIV
viral load
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Medical related applications of the PCR technique include _______ testing
genetic
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It is now easier to sequence ______ than sequence ______ a complete reversal over the past 20 years.
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DNA sequencing factories can sequence over _______ bases per day
1,000,000
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This was the original method for DNA sequencing work.
Maxam & Gilbert Method
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Maxam & Gilbert Method Uses chemical degradation of the _____ ______.
ORIGINAL DNA
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Maxam & Gilbert Method is generally used for: (3)
- (1) Confirmation of a Sanger sequence
- (2) Analysis of DNA modifications, e.g. methylation
- (3) Only good for DNA swquences of <200 bases
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Methylation of N7 with dimethyl sulfate breaks the DNA bond at
G
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Piperidine formate at pH 2 weakens glycosidic bonds between
A+G
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Hydrazine opens pyrimidine rings, which recyclize in 5 member rings susceptible to removal of _____
C+T
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In the presence of 1.5 M NaCl, only ______ reacts appreciably with hydrazine
Cytosine
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1.2 N NaOH at 90 results in strong cleavage at ___ and weaker cleavage at ___
A>C
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90 piperadine is used to cleave the _____ _____ _____ of DNA at the sites of chemical modification
sugar-phosphate chain
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Maxam & Gilbert Strategy involves 2 steps. What are they?
- (1) Radilabel target DNA at only one end
- (2) Carry out 5 base-specific cleavage reactions
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In the Maxam-Gilbert Sequencing Gel, Aas are red 5' to 3' starting at the _____
bottom
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_____ is the most widely used DNA sequencing method.
Sanger Sequencing Method
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Sanger Sequencing Method Uses a _______ to terminate chain elongation at a specific nucleotide.
dideoxynucleotide
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dideoxynucleotide is missing a _____, making it a chain terminator
3' hydroxyl group
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Sanger Sequencing Method Uses a single stranded template DNA and attaches an _____ _____
oligonucleotide primer
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In Sanger Sequencing, new complimentary strand synthesis terminate when a _____ ______ is incorporated.
Terminating dideoxynucleotide
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In Sanger Synthesis, fragments of radio labeled DNA are denatured and seperated by _______
electrophoresis
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In Sanger Sequencing, the original template is the _____ of the sequence generated
compliment
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AS replicated DNA strands get longer in ______ sequencing, compression of the space between NTs occurs
Sanger sequencing
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_______ allows for different fluorescent probes to be matched with differnet dyes, distinguishng them.
Sequencer Output
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Therapeutically, we can alter amino acids in a therapeutic protein to enhance its ______ or ______ properties.
- pharmacodynamic,
- pharmacokinetic
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_____ _____ _____ _____ allows us to investigate the importance of a specific amino acid in a protein
Site Specific DNA Mutation
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_____ _____ _____Allows one to insert a small sequence of altered bases into a sequence.
Oligonucleotide-mediated Mutagenesis
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Oligonucleotide-mediated Mutagenesis Requires three reagents:
- (1) M13 bacteriophage (single strand) containing sequence to be mutated
- (2) Mutagenic oligonucleotide
- (3) Universal primer
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Oligonucleotide-mediated Mutagenesis requires DNA insertion into single stranded _____ _____
M13 bacteriophage (fasza)
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A _____ _____ _____ is comlementarily stranded to the DNA insert
mutagenic oligo-nucleotide
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A _____ _____ is used to ensure complementary stranding of M13 bacteriophage
Universal primer
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in order for a complimentary strand of M13 to form from the universal primer, 3 things must be added to the mixture
- (1) DNA polymerase
- (2) dNTP
- (3) DNA ligase
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DNA strands containing a Mutagenic Oligonucleotide are able to be used ifor _____ of E coli
transfection
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____ _____ _____ is limited to the mutation of just a few nucleotides at a time.
PCR-mediated Mutagenesis
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In PCR-mediated Mutagenesis, Need to design a primer that has the appropriate ______ located within it.
mismatch
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In PCR-mediated Mutagenesis, As the PCR products are formed, the mutated primers are incorporated into the final ______ DNA.
amplified
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If the primer on the 3' end of the DNA segment of interest is mutated, all products will have the _____ _____ _____ incorporated into it.
altered base sequence
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in PCR Mutagenesis, strands are heated to ____ _____
separate them
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in PCR mutagenesis, once strands are cooled _____ are added
primers
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in PCR mediated Mutagenesis II, primers that ajoin mutations to create blunt end nucleases are called _____ _____
design primers
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by digesting cleaved design primers with endonuclease and DNA ligase we are able to move mutation from the _____ to the _____
end, middle
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