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