1. Nucleotides and Nucleic Acids

2. Cellular Processes DNA RNA (mRNA) Proteins LipidsCarbohydrates replication transcription translation

3. 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 are linked by phosphodiester bonds. Nucleotides and Nucleic Acids

4. Deoxyribonucleotides found in DNA dA dG dTdC

5. Nucleotides are linked by phosphodiester bonds

6. DNA is double stranded Bases form a specific hydrogen bond pattern

7. DNA is a Double-Helix 5’ 3’ Major Groove Minor Groove

8. Amplification of DNA using the Polymerase Chain Reaction (PCR)

9. Polymerization of nucleotides by DNA polymerase Phosphodiester bonds are formed by a nucleophilic attack of a free 3’- OH on the 5’-  -PO 4 of the incoming dNTP.

10. 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

11. 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.

12. Thermal cycling during PCR Initial denaturation Cycle 1Cycle 2Cycle 3 Cycle 30 Extension Annealing

16. 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 0201 1 2 3 4 5 6 … 20

17. 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 0201 1402 2624 3888 4102216 5125232 61411464 … 20422,097,1102 20 =1,048,576

18. Amplification of DNA using PCR Reagents: Taq DNA polymerase Amplification buffer Magnesium chloride (MgCl 2 ) deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP) Template DNA (provided) 5’ primer (10  M concentration) 3’ primer (10  M concentration) H 2 O to 50  l volume Thermocycle parameters: 1. 94ºC for 5 minutes 2. 94ºC for 15 seconds 3. 45ºC or 55ºC for 30 seconds 4. 72ºC for 1 minute 5. Go to step 2, repeat 29 times 6. 4ºC forever