DNA replication and pcr.txt

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Author:
rincrocci
ID:
119421
Filename:
DNA replication and pcr.txt
Updated:
2011-11-28 17:18:33
Tags:
dna
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Description:
replication
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  1. DNA composition
    • Base unit is called NUCLEOTIDE, which is composed of the following:
    • 1. nitrogenous base: adenine, guanine, cytosine, thymine
    • 2. phosphate
    • 3. deoxyribose sugar
  2. Chargoff's rule
    • no. of A=T
    • no. of G=C
    • (no. of A&T's does NOT equal G&C's)
  3. Linus Pauling
    Proposed that weak Hydrogen bonds hold nitrogenous bases together
  4. Rosalind Franklin & Maurice Wilkins
    X-ray diffraction to suggest DNA had 2 similar parts parallel to each other and helical shape
  5. Watson & Crick
    • used Franklin & Wilkins info to determine the following:
    • 1. DNA was a DOUBLE Helix
    • 2. Nucleotides held together through PHOSPHODIESTER bond (b/w 5' phosphate and 3' deoxyribose)
    • 3. (Pauling) 2 helicies held together by Hydrogen bonds (A2T, C3G)
  6. DNA Replication facts and requirements
    • Semi-conservative
    • Bidirectional
    • Always occurs in 5-3' direction
    • Requires the following:
    • Helicase
    • DNA gyrase (topoisomerase)
    • Primase
    • DNA Polymerase III
    • DNA Polymerase I: exonuclease activity, excises RNA and replaces w/DNA bps
    • DNA Ligase
    • single stranded binding protein
  7. DNA Replication - 4 steps
    • 4 steps:
    • 1. Helicase splits DNA strands, RNA primers put down by primase for DNA Pol III to recog
    • 2. DNA Pol III produces leading and lagging (Okazaki) strands
    • 3. DNA Pol I removes RNA @ 5' end of fragment and fills gaps w/complementary bases
    • 4. DNA ligase connects adjacent fragments
  8. DNA gyrase
    • aka topoisomerase
    • STABILIZES DNA when helicase unwinds it
  9. single stranded binding protein
    Helps keep DNA accessible for replication machinery
  10. Dehydration synthesis
    Loss of water when a phosphate and deoxyribose connect
  11. PCR facts and ingredients
    • polymerase chain reaction
    • created by Kary Mullis
    • Recipe:
    • 1. DNA template
    • 2. Enzyme - DNA polymerase (Taq)
    • 3. Primers - forward and reverse (act as Primase for Taq)
    • 4. dNTPs - nitrogenous bases
    • 5. MgCl2 - help detect polymerase fxn
    • 6. H20 - rxn buffer
  12. PCR instructions
    • 1. Heat DNA @ 96 (denature-separate strands)
    • 2. Cool @ 60 (allow for primers to Anneal)
    • 3. Annealing & extension @ 72
    • repeat for 20-35 cycles
  13. Agarose gel electrophoresis types
    • 1. EtBr - glows white or pink when bound w/dsDNA...TOXIC
    • 2. SYBR green - similar to EtBr, but not as good...non-toxic, non-specific binding and not quantitative
    • 3. 5' Nuclease Assay - fluorescence of released reporter binds to ssDNA, very specific and quantitative

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