GEN #9 PCR
Card Set Information
GEN #9 PCR
HUSOP Gen EXAM2 PCR
Questions from genomics PCR lecture
What does quantitative PCR determine?
How much of a target (RNA/DNA) is in a sample
For each PCR sample, one uses 3-4 tubes, each with the same amount of _______ RNA template but different amounts of sample
The competitor RNA template wants to be as similar to the target ______ _____ as possible.
Competitor RNA Templates generally synthesize a truncated form of the mRNA of interest in which sequences from the _____ of the mRNA are removed
Truncation makes it possible to differentiate the ______ and _______ products.
Development of ultra-sensitive, non-radiolabel nucleic acid detection methods allow for ______ ________ of PCR.
Real-time PCR Methods are All based on ________ _______ of dye-tagged nucleic acids, dsDNA dyes, and fluorescence-quenching.
Real time PCR _____ and ______ run in the same instrument and tubes.
TaqMan labes are attatched to the _____ primer
_____ detection has both a fluorescent and a quenching label attached to it.
____ detection has only a fluorescent marker.
_____ ______is the least sensitive & semi-quantitative but yields MW and can run multiple samples per blot.
Of the Methods to Quantitate mRNA _____ ______ has Moderate to high sensitivity and allows for multiple mRNA species quantitation
Solution Hybridization (RNase Protection)
The most sensitive approach to Quantitate mRNA are ______ ___ & ____-____ _____
Medical related applications of the PCR technique include _______ individuals for drug metabolizing enzymes
Medical related applications of the PCR technique include detecting the presence of ______ ____, e.g. hepatitis C
Medical related applications of the PCR technique include Quantitating the amount of ______ ____, e.g. HIV
Medical related applications of the PCR technique include _______ testing
It is now easier to sequence ______ than sequence ______ a complete reversal over the past 20 years.
DNA sequencing factories can sequence over _______ bases per day
This was the original method for DNA sequencing work.
Maxam & Gilbert Method
Maxam & Gilbert Method Uses chemical degradation of the _____ ______.
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
Methylation of N7 with dimethyl sulfate breaks the DNA bond at
Piperidine formate at pH 2 weakens glycosidic bonds between
Hydrazine opens pyrimidine rings, which recyclize in 5 member rings susceptible to removal of _____
In the presence of 1.5 M NaCl, only ______ reacts appreciably with hydrazine
1.2 N NaOH at 90 results in strong cleavage at ___ and weaker cleavage at ___
90 piperadine is used to cleave the _____ _____ _____ of DNA at the sites of chemical modification
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
In the Maxam-Gilbert Sequencing Gel, Aas are red 5' to 3' starting at the _____
_____ is the most widely used DNA sequencing method.
Sanger Sequencing Method
Sanger Sequencing Method Uses a _______ to terminate chain elongation at a specific nucleotide.
dideoxynucleotide is missing a _____, making it a chain terminator
3' hydroxyl group
Sanger Sequencing Method Uses a single stranded template DNA and attaches an _____ _____
In Sanger Sequencing, new complimentary strand synthesis terminate when a _____ ______ is incorporated.
In Sanger Synthesis, fragments of radio labeled DNA are denatured and seperated by _______
In Sanger Sequencing, the original template is the _____ of the sequence generated
AS replicated DNA strands get longer in ______ sequencing, compression of the space between NTs occurs
_______ allows for different fluorescent probes to be matched with differnet dyes, distinguishng them.
Therapeutically, we can alter amino acids in a therapeutic protein to enhance its ______ or ______ properties.
_____ _____ _____ _____ allows us to investigate the importance of a specific amino acid in a protein
Site Specific DNA Mutation
_____ _____ _____Allows one to insert a small sequence of altered bases into a sequence.
Oligonucleotide-mediated Mutagenesis Requires three reagents:
(1) M13 bacteriophage (single strand) containing sequence to be mutated
(2) Mutagenic oligonucleotide
(3) Universal primer
Oligonucleotide-mediated Mutagenesis requires DNA insertion into single stranded _____ _____
M13 bacteriophage (fasza)
A _____ _____ _____ is comlementarily stranded to the DNA insert
A _____ _____ is used to ensure complementary stranding of M13 bacteriophage
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
(3) DNA ligase
DNA strands containing a Mutagenic Oligonucleotide are able to be used ifor _____ of E coli
____ _____ _____ is limited to the mutation of just a few nucleotides at a time.
In PCR-mediated Mutagenesis, Need to design a primer that has the appropriate ______ located within it.
In PCR-mediated Mutagenesis, As the PCR products are formed, the mutated primers are incorporated into the final ______ DNA.
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
in PCR Mutagenesis, strands are heated to ____ _____
in PCR mutagenesis, once strands are cooled _____ are added
in PCR mediated Mutagenesis II, primers that ajoin mutations to create blunt end nucleases are called _____ _____
by digesting cleaved design primers with endonuclease and DNA ligase we are able to move mutation from the _____ to the _____