GEN #9 PCR

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HUSOP2014
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134968
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GEN #9 PCR
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2012-03-02 08:25:51
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HUSOP Gen EXAM2 PCR
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Questions from genomics PCR lecture
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  1. What does quantitative PCR determine?
    How much of a target (RNA/DNA) is in a sample
  2. For each PCR sample, one uses 3-4 tubes, each with the same amount of _______ RNA template but different amounts of sample
    competitive
  3. The competitor RNA template wants to be as similar to the target ______ _____ as possible.
    mRNA sequence
  4. Competitor RNA Templates generally synthesize a truncated form of the mRNA of interest in which sequences from the _____ of the mRNA are removed
    middle
  5. Truncation makes it possible to differentiate the ______ and _______ products.
    • target,
    • competitor
  6. Development of ultra-sensitive, non-radiolabel nucleic acid detection methods allow for ______ ________ of PCR.
    rapid quantitation
  7. Real-time PCR Methods are All based on ________ _______ of dye-tagged nucleic acids, dsDNA dyes, and fluorescence-quenching.
    fluorescence detection
  8. Real time PCR _____ and ______ run in the same instrument and tubes.
    • reaction
    • detection
  9. TaqMan labes are attatched to the _____ primer
    Forward
  10. _____ detection has both a fluorescent and a quenching label attached to it.
    TaqMan
  11. ____ detection has only a fluorescent marker.
    LUX
  12. _____ ______is the least sensitive & semi-quantitative but yields MW and can run multiple samples per blot.
    Northern blotting
  13. Of the Methods to Quantitate mRNA _____ ______ has Moderate to high sensitivity and allows for multiple mRNA species quantitation
    Solution Hybridization (RNase Protection)
  14. The most sensitive approach to Quantitate mRNA are ______ ___ & ____-____ _____
    • Competitive PCR
    • real-time PCR
  15. Medical related applications of the PCR technique include _______ individuals for drug metabolizing enzymes
    Genotyping
  16. Medical related applications of the PCR technique include detecting the presence of ______ ____, e.g. hepatitis C
    viral DNA
  17. Medical related applications of the PCR technique include Quantitating the amount of ______ ____, e.g. HIV
    viral load
  18. Medical related applications of the PCR technique include _______ testing
    genetic
  19. It is now easier to sequence ______ than sequence ______ a complete reversal over the past 20 years.
    • DNA
    • protein,
  20. DNA sequencing factories can sequence over _______ bases per day
    1,000,000
  21. This was the original method for DNA sequencing work.
    Maxam & Gilbert Method
  22. Maxam & Gilbert Method Uses chemical degradation of the _____ ______.
    ORIGINAL DNA
  23. 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
  24. Methylation of N7 with dimethyl sulfate breaks the DNA bond at
    G
  25. Piperidine formate at pH 2 weakens glycosidic bonds between
    A+G
  26. Hydrazine opens pyrimidine rings, which recyclize in 5 member rings susceptible to removal of _____
    C+T
  27. In the presence of 1.5 M NaCl, only ______ reacts appreciably with hydrazine
    Cytosine
  28. 1.2 N NaOH at 90 results in strong cleavage at ___ and weaker cleavage at ___
    A>C
  29. 90 piperadine is used to cleave the _____ _____ _____ of DNA at the sites of chemical modification
    sugar-phosphate chain
  30. 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
  31. In the Maxam-Gilbert Sequencing Gel, Aas are red 5' to 3' starting at the _____
    bottom
  32. _____ is the most widely used DNA sequencing method.
    Sanger Sequencing Method
  33. Sanger Sequencing Method Uses a _______ to terminate chain elongation at a specific nucleotide.
    dideoxynucleotide
  34. dideoxynucleotide is missing a _____, making it a chain terminator
    3' hydroxyl group
  35. Sanger Sequencing Method Uses a single stranded template DNA and attaches an _____ _____
    oligonucleotide primer
  36. In Sanger Sequencing, new complimentary strand synthesis terminate when a _____ ______ is incorporated.
    Terminating dideoxynucleotide
  37. In Sanger Synthesis, fragments of radio labeled DNA are denatured and seperated by _______
    electrophoresis
  38. In Sanger Sequencing, the original template is the _____ of the sequence generated
    compliment
  39. AS replicated DNA strands get longer in ______ sequencing, compression of the space between NTs occurs
    Sanger sequencing
  40. _______ allows for different fluorescent probes to be matched with differnet dyes, distinguishng them.
    Sequencer Output
  41. Therapeutically, we can alter amino acids in a therapeutic protein to enhance its ______ or ______ properties.
    • pharmacodynamic,
    • pharmacokinetic
  42. _____ _____ _____ _____ allows us to investigate the importance of a specific amino acid in a protein
    Site Specific DNA Mutation
  43. _____ _____ _____Allows one to insert a small sequence of altered bases into a sequence.
    Oligonucleotide-mediated Mutagenesis
  44. Oligonucleotide-mediated Mutagenesis Requires three reagents:
    • (1) M13 bacteriophage (single strand) containing sequence to be mutated
    • (2) Mutagenic oligonucleotide
    • (3) Universal primer
  45. Oligonucleotide-mediated Mutagenesis requires DNA insertion into single stranded _____ _____
    M13 bacteriophage (fasza)
  46. A _____ _____ _____ is comlementarily stranded to the DNA insert
    mutagenic oligo-nucleotide
  47. A _____ _____ is used to ensure complementary stranding of M13 bacteriophage
    Universal primer
  48. 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
  49. DNA strands containing a Mutagenic Oligonucleotide are able to be used ifor _____ of E coli
    transfection
  50. ____ _____ _____ is limited to the mutation of just a few nucleotides at a time.
    PCR-mediated Mutagenesis
  51. In PCR-mediated Mutagenesis, Need to design a primer that has the appropriate ______ located within it.
    mismatch
  52. In PCR-mediated Mutagenesis, As the PCR products are formed, the mutated primers are incorporated into the final ______ DNA.
    amplified
  53. 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
  54. in PCR Mutagenesis, strands are heated to ____ _____
    separate them
  55. in PCR mutagenesis, once strands are cooled _____ are added
    primers
  56. in PCR mediated Mutagenesis II, primers that ajoin mutations to create blunt end nucleases are called _____ _____
    design primers
  57. 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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