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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
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Chargoff's rule
- no. of A=T
- no. of G=C
- (no. of A&T's does NOT equal G&C's)
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Linus Pauling
Proposed that weak Hydrogen bonds hold nitrogenous bases together
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Rosalind Franklin & Maurice Wilkins
X-ray diffraction to suggest DNA had 2 similar parts parallel to each other and helical shape
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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)
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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
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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
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DNA gyrase
- aka topoisomerase
- STABILIZES DNA when helicase unwinds it
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single stranded binding protein
Helps keep DNA accessible for replication machinery
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Dehydration synthesis
Loss of water when a phosphate and deoxyribose connect
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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
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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
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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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