Topic 1 2012 Basic Nucleic Acid Isolation, Manipulation and Analysis

Key points:-

DNA and RNA easily purified but as whole cellular populations, not specific sequences.
RNA preps. can have significant DNA contamination.
DNA in plasmids or phage can be purified selectively.
Oligonucleotides can be synthesized chemically but efficiency and purity decrease with length. Also, product has 5’ –OH after de-protection. Variants can be made with unnatural bases or variations on phosphodiester bond linkages.
Ligase requires at least one 5’-phosphate (and 3’-OH) and perfect complementarity of overhangs.
DNA polymerases require primer with 3’-OH annealed to template with perfect match at 3’ end.
For molecular biology most useful RNA polymerases are from phage (SP6, T7, T3), requiring only a binding sequence in dsDNA and no accessory proteins.
Most useful DNA polymerases from E.coli, other bacteria or phage with engineered variants; stability, processivity, accuracy, exonuclease activities are key characteristics that differ among specific DNA polymerases used.
DNA sequencing- basic principle of Sanger sequencing (key ingredients, modifications and how they improve sequencing, what limits length of sequence that can be read). Longer sequences generally from primer walking or shotgun sequencing.
Hybridization underlies majority of techniques for nucleic acid analysis and manipulation. Parameters affecting stringency. How to make specific probes (radioactive or not). Basic hybridization techniques (how to perform and for what purpose)- Southerns (also dot blots, colony lifts and plaque lifts), Northerns, chromosome in situs, RNA in situs.

Purification of DNA (pp 29-33)

All DNA is stable and has similar biochemical properties. This is convenient for purifying DNA in general but means special methods (cloning, PCR) must be devised for separating one type of DNA molecule from another.
Size of DNA, linear vs. circular, ds vs ss, affect some properties, allowing selective purification (as for plasmid DNA below or purification of DNA according to size by gel electrophoresis).

DNA purification is generally easy & can be accomplished in many ways. Choice depends on source and convenience.

Common sources:- Circular ds plasmid DNA in bacteria Chromosomal DNA from eukaryotic cells (using tissue culture cells or blood, or flies or insects in amber….) Viral, 'phage DNA in growth medium By mail from other researchers or research centers

Break open cell (Osmotic pressure, detergent, mechanical, enzymes)
Remove low molecular weight materials and other macromolecules (proteins, carbohydrates, RNA).

Proteinase K degrades protein Phenol extraction removes protein Dnase-free RNAse degrades RNA Ethanol precipitation removes low molecular wt. material

Alternatively, selectively pull out the DNA (& RNA) using an affinity matrix such as silica gel (Bind silica gel at high salt; elute with low salt [DNA, RNA]) or other specially formulated positively charged resins (manipulate pH & salt to regulate affinity for different nucleic acids).

For chromosomal DNA be gentle to avoid shearing.

For small circular plasmid DNA: Take advantage of unique topology of plasmid DNA SDS/alkaline lysis (denatures protein & DNA) Neutralize w. KAc (small topologically linked single strands of plasmid DNA renature but large chromosomal DNA & protein ppts). Phenol extract, Ethanol ppt. (or fish out DNA by silica affinity, as in Qiagen kits), treat w. RNase

Final product- absorption spectrum (A260 vs A280), run on gel for amount and purity.

Has all relevant DNA been recovered? (mitochondrial DNA, nuclear DNA, episomes)
How pure is it? (other macromolecules, DNA contamination of RNA prep)
How pure does it need to be? (cutting, sequencing, transfecting, micro-injecting, use in humans)

Purification