Nucleotides and Nucleic Acids. Cellular Processes DNA RNA (mRNA) Proteins LipidsCarbohydrates replication transcription translation.

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Presentation transcript:

Nucleotides and Nucleic Acids

Cellular Processes DNA RNA (mRNA) Proteins LipidsCarbohydrates 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 are linked by phosphodiester bonds. Nucleotides and Nucleic Acids

Deoxyribonucleotides found in DNA dA dG dTdC

Nucleotides are linked by phosphodiester bonds

DNA is double stranded Bases form a specific hydrogen bond pattern

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

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’-  -PO 4 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 1Cycle 2Cycle 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 … 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 … 20422,097, =1,048,576

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