Electrophoresis polynucleotides

The contents of each tube are then subjected to electrophoresis m separate lanes on the same sheet of polyacrylamide gel and the DNAs located by autoradiography A typical electrophoresis gel of a DNA fragment containing 50 nucleotides will exhibit a pattern of 50 bands distributed among the four lanes with no overlaps Each band cor responds to a polynucleotide that is one nucleotide longer than the one that precedes it (which may be m a different lane) One then simply reads the nucleotide sequence according to the lane m which each succeeding band appears... 

Section 28 14 The nucleotide sequence of DNA can be determined by a technique m which a short section of single stranded DNA is allowed to produce its complement m the presence of dideoxy analogs of ATP TTP GTP and CTP DNA formation terminates when a dideoxy analog is incorporated into the growing polynucleotide chain A mixture of polynucleotides dif fermg from one another by an incremental nucleoside is produced and analyzed by electrophoresis From the observed sequence of the comple mentary chain the sequence of the original DNA is deduced... 

Based on its nature (aqueous solutions, physiological conditions, well-investigated labeling, and staining reactions) and the historical transition from slab-gel electrophoresis to CE, the main targets are biological and bioequivalent samples such as proteins, peptides, polynucleotides, oligonucleotides, and carbohydrates. 

An automatic sequencing instrument has been developed that uses the chain-terminator method. To avoid the use of radioactive labels, a different color fluorescent dye is attached to the primer in each of the four reactions used to synthesize the DNA fragments. The mixture of fragments from all four reactions is then analyzed using electrophoresis in a single lane. A fluorescent spot appears for each polynucleotide of increasing size. The 3 -terminal base for each spot can be determined by the color of the fluorescence. The detection system is computer controlled, and the acquisition of data is automated. A schematic representation... 

Fig. 2.11. In (a), 3 -end labelling is achieved using deoxynucleotidyl transferase and an ff-[32P] ribonucleotide triphosphate followed by elimination of the ribonucleotide residues. In (b), 5 -end labelling is carried out using polynucleotide kinase and y-[32P]ATP. Partial digestion with spleen phosphodiesterase removes nucleotides sequentially from the 5 -end of the oligonucleotide giving a mixed population of partially degraded molecules each labelled at the 3 -end. Venom phosphodiesterase removes nucleotides from the 3 -end similarly yielding a mixed population of shortened fragments. The products of the reaction are resolved by two-dimensional electrophoresis-homochromatography and the sequence deduced by the characteristic...

The deprotected oligonucleotide synthetic product is precipitated twice in ethanol, and a 0.5 fig/fd solution in water is prepared (concentration is measured from a UV absorption spectrum). One microliter of the oligo-deoxynucleotide solution is mixed with 2 fd of 10X PL, 5 fd of [y-32P]ATP (or [y-35S]ATP), 1 fd of T4 polynucleotide kinase, and 11 fd water. After incubation at 37 ° (for 45 min with [y-32P]ATP or for 2 hr with [y-35S]ATP), the reaction is stopped by the addition of 150 [A of 5 M ammonium acetate, pH 5.5, and 130 fd water and 10 fd of the yeast tRNA solution are added to the mixture before precipitation with 1 ml ethanol. After chilling at —70° for at least 15 min, the precipitate is collected by centrifugation (12,000 g, 15 min), redissolved, and submitted to two additional cycles of precipitation-redissolution. Finally, the precipitate is redissolved in 20 fd of gel loading mix and the mixture analyzed on a 8% acrylamide-7 Af urea slab gel in IX electrophoresis buffer, until the bromphenol blue has reached the middle of the gel. 

Baba Y, Matsuura T, Wakamoto K, Tsuhako M (1991a) Comparison of high-performance liquid chromatography with capillary gel electrophoresis in single base resolution of polynucleotides. J Chromatgr 558 273-284. 

Fig. 7.5. Some typical molecular weiglit-mobility (or sedimentation coefficient) relationstiips for electrophoresis of RNA in polyacrylamide gels, showing conformational effects a) set of single-stranded viral and ribosomal RNAs in 2.4% gel (Bishop et al. 1967). b) shows a typical separation of different species on a single gel, displayed as a densitometric trace c) native single-stranded RNAs ( ), formaldehyde treated RNAs (O). polyriboadenylic acid fractions with (A) and without (A) formaldehyde treatment, and polyribouridylic acid fractions ( ) treated with formaldehyde. These data show that the mobilities are little affected by single-stranded stacking, but substantially by partial base pairing, and that formaldehyde treatment is inadequate to reduce RNA to the conformation of a single-stranded unpaired polynucleotide (from Finder et al., 1974).

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