nucleic acids DNA and RNA are polymers of nucleotides

1
Nucleotide Structure

• nucleic acids (DNA and RNA) are polymers of
  nucleotides
• bases purines (A,G) or pyrimidines (C,T,U)
• thymine (DNA only) uracil (RNA only)
• nucleoside unphosphorylated (5)
• 1, 2 or 3 phosphates nucleotide
• DNA 2-deoxy nucleotides

2
(No Transcript)
3
The Central Dogma

• cells make exact copies of DNA
• RNA is transcribed from a DNA template
• proteins are translated from RNA template

4
DNA/RNA Synthesis

• the DNA strands are separated
• each strand serves as template
• complementary strands are synthesized (5'?3')
• yields 2 identical DNA molecules
• carried out by DNA polymerase

• RNA synthesis is similar
• RNA polymerase
• single template
• U/T and 2'-O
• processing

retrovirus/reverse transcriptase
5
RNA Processing

• ? addition of caps and tails to primary
  transcript
• some genes have regions of non-coding sequence
  (introns) which are removed
• messenger RNA (mRNA)

6
Translation
carried out by ribosomes
triplet code (tRNA)
7
The Central Dogma

• information about proteins contained in DNA and
  RNA
• sequencing DNA relatively simple
• cloning and manipulating genes is possible

8
Isolation of Nucleic Acids

• Goals
• removal of proteins
• DNA vs RNA
• isolate specific type of nucleic acid

• Types of Methods
• differential solubility
• adsorption methods
• density gradient centrifugation

• Types of DNA
• genomic (chromosomal)
• organellar (satellite)
• plasmid (extra-chromosomal)
• phage/viral (ds or ss)
• complementary (mRNA)

• General Features
• denaturing cell lysis (SDS, alkali, heating,
  chaotropic)
• ? enzyme treatments
• protease
• RNase (DNase-free)
• DNase (RNase-free)

9
High MW Genomic DNA Isolation

• Typical Procedure
• Cell Lysis
• 0.5 SDS proteinase K (55o several hours)
• Phenol Extraction
• gentle rocking several hours
• Ethanol Precipitation
• RNAse followed by proteinase K
• Repeat phenol extrac-tion and EtOH ppt

• Phenol Extraction
• mix sample with equal volume of sat. phenol soln
• retain aqueous phase
• optional chloroform/isoamyl alcohol extraction(s)

10
High MW Genomic DNA Isolation

• Typical Procedure
• Cell Lysis
• 0.5 SDS proteinase K (55o several hours)
• Phenol Extraction
• gentle rocking several hours
• Ethanol Precipitation
• RNAse followed by proteinase K
• Repeat Phenol Extrac-tion and EtOH ppt

• EtOH Precipitation
• 2-2.5 volumes EtOH, -20o
• high salt, pH 5-5.5
• centrifuge or spool out

11
Isolation of RNA Special Considerations

• RNAse inhibitors!
• extraction in guanidine salts
• phenol extractions at pH 5-6
• (pH 8 for DNA)
• treatment with RNase-free DNase
• selective precipitation of high MW forms (rRNA,
  mRNA) with LiCl
• oligo-dT column

12
Adsorption Methods

• nucleic acids selectively absorb to silica or
  diatomaceous earth in presence of certain
  chaotropic agents or salts

Plasmid Miniprep Protocol 1. Solubilize bacteria
in alkali solution 2. Neutralize with
Na-acetate 3. Centrifuge, discard pellet 4. Mix
supernatant with resin chaotropic agent 5. Wash
resin 6. Elute DNA with low salt buffer

• applications
• plasmid preps
• fragments after electrophoresis
• PCR templates

13
Density Gradient Centrifugation

• rate zonal/sucrose (size fractionation)
• electrophoresis more common
• isopycnic/CsCl (density)
• DNA 1.7 g/cm3
• protein 1.3 g/cm3
• RNA gt DNA
• ssDNA gt dsDNA
• GC content

14
CsCl Gradients

• Applications
• large scale preparations
• high purity
• RNA cushions
• satellite DNA

Cesium Chloride Gradients
15
Evaluation of Nucleic Acids

• spectrophotometrically
• quantity
• quality
• fluorescent dyes
• gel electrophoresis