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Nucleotides and
Nucleic Acids
Cellular Processes
DNA
RNA (mRNA)
Proteins
Lipids Carbohydrates
replication
transcription
translation
Nucleotides – molecules containing a pentose (5 carbon sugar),a phosphate groups, and a nitrogen-containing base.
Nucleoside – a nucleotide without the phosphate group
Nucleic Acid – a polynucleotide where the nucleotides arelinked by phosphodiester bonds.
Nucleotides and Nucleic Acids
Deoxyribonucleotides found in DNA
dA dG dT dC
Nucleotides arelinked by
phosphodiesterbonds
DNA is double strandedBases form a specific hydrogen bond pattern
DNA is a Double-Helix5’
5’ 3’
3’
MajorGroove
MinorGroove
Amplification of DNA using the Polymerase Chain Reaction
(PCR)
Polymerization of nucleotides by DNA polymerase
Phosphodiester bonds are formed by a nucleophilic attack of a free 3’-OH on the 5’--PO4 of the incoming dNTP.
The enzyme DNA polymerase.
A DNA template to guide synthesis.
A primer, a short nucleotide segment complementary to the template that can provide a free 3’-OH for synthesis.
Requirements for DNA replication
Thermus aquaticus (Taq) DNA polymerase
DNA polymerase isolated from a bacterium found in hot springs
Can withstand the high temperatures in PCR needed to denature double stranded DNA templates
Can replicate 1000 base pairs of DNA in 10 seconds at 72°C
Has low replication fidelity; error rate of 1 in 9,000 nucleotides
A variety of thermostable polymerases that have a greater fidelity are now available from several companies.
Thermal cycling during PCR
Initial denaturation
Cycle 1 Cycle 2 Cycle 3 Cycle 30
Extension
Annealing
Theoretical accumulation of PCR products during the first 20 cycles of a PCR with a
single genomic DNA template
Fill in the following table
Cycle number # of single strands of undefined length
# of single strands of defined length
# of copies of double strand target
0 2 0 1
1
2
3
4
5
6
…
20
Theoretical accumulation of PCR products during the first 20 cycles of a PCR with a
single genomic DNA template
Fill in the following table
Cycle number # of single strands of undefined length
# of single strands of defined length
# of copies of double strand target
0 2 0 1
1 4 0 2
2 6 2 4
3 8 8 8
4 10 22 16
5 12 52 32
6 14 114 64
…
20 42 2,097,110 220=1,048,576
Amplification of DNA using PCRReagents: Taq DNA polymerase
Amplification buffer Magnesium chloride (MgCl2)deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP)Template DNA (provided)5’ primer (10 M concentration)3’ primer (10 M concentration)H2O to 50 l volume
Thermocycle parameters: 1. 94ºC for 5 minutes2. 94ºC for 15 seconds3. 45ºC or 55ºC for 30 seconds4. 72ºC for 1 minute5. Go to step 2, repeat 29 times6. 4ºC forever
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