Post Electrophoretic Analysis Articles
Pouring Sequencing Gels
Denaturing PAGE gels for DNA sequencing generally employ 6-8 M urea as their denaturant and TBE as their buffer system. They are poured as described in the section on denaturing PAGE of DNA and RNA. After a 2-2.5 hour run, a 6% polyacrylamide sequencing gel will give 200-250 bases of readable sequence starting at or close to the end of the primer. A number of variations, enhancements and improvements to the basic PAGE gel have been developed to increase the number of bases which can be read from one gel.
The pattern of sample loading can be important. The best method is to load in a pattern such as GATCGTAC, in which each reaction is loaded twice, and each reaction borders on every other reaction once. At the top of the gel, where bands are compressed, this allows unambiguous assignment of order and thus base position. Loading in this pattern can extend readability by up to 50 bases, while at the same time diminishing errors in reading throughout the sequence.
Significantly more sequence information can be derived from a set of reactions by “double loading.” Portions of the samples are loaded into half the wells in the gel, and the gel is run for 1 – 2 hours. The power is turned off, and the remaining portions of the samples are loaded into the remaining lanes. Running the gel for 1 – 2 more hours gives runs of 2 and 4 hours, which will allow sequence to be read from the primer out to 350 – 400 bases.
The use of a wedge gel or a buffer gradient system can extend read length by up to 30%. Wedge gels, cast with spacers wider at the bottom of the gel, work well but are inconvenient to dry. Buffer gradient gels are more difficult to pour, but easier to handle after the run. Both options work by decreasing the electrical resistance in the bottom portion of the gel. Wedge gels have a wider cross sectional area at the bottom, while buffer gradient gels have a higher salt content in that area. The decreased resistance causes the voltage drop across the lower portion of the gel to be diminished. The DNA molecules in this region are subjected to less force, so they slow down relative to bands in the upper portion of the gel. The net effect is to “compress” the bands as they migrate into the bottom of the gel, allowing longer runs and more readable sequence per gel. A similar, although less marked effect may be obtained simply by filling the upper chamber with 0.5X TBE, and the lower with 2X TBE. The pre-run gradient will not be as continuous as one poured into the gel, and some information may be lost at particularly steep points in the gradient.
Pouring a Buffer Gradient Sequencing Gel
- Prepare 2 gel solutions, containing the desired concentration of Acrylamide/Bis-Acrylamide and Urea. One solution should contain TBE at 0.5X, and one at 2.5X (containing 10% sucrose). Add Bromophenol Blue to the 2.5X TBE solution to 0.001% (just sufficient to give a visibly blue tint). The volume of each solution should be 75% of the amount needed to completely fill the gel cassette.
- Add APS and TEMED to the two solutions to initiate polymerization.
- Draw 2 – 3 air bubbles through the pipette, to mix the two solutions at the interface.
- Fill the cassette with the solution in the pipette. Fill the remainder of the cassette with the 0.5X TBE gel solution.
- Allow to polymerize 2 hours, and run with 0.5X TBE in the upper buffer chamber, and 1X TBE in the lower chamber.
In some situations, the information read from a gel is of sufficient length, but insufficient quality. Substrates with a high G-C content will tend to retain enough secondary structure, even in 6M Urea at 55°C, to cause anomalous gel migrations. On the gel, this is observed as multiple bands in G or C lanes that are too close together for accurate reading. These regions are known as G-C compressions, and they can be very hard to resolve. Inclusion of a stronger denaturant in the gel alleviates most, if not all, GC compression problems. The denaturant of choice is formamide, which is strongly denaturing to DNA, uncharged, and easily miscible with high concentrations of urea in water. Gels with 6M urea and 40% formamide are typically formulated to resolve GC compressions. Formamide solutions must be made fresh, using deionized formamide.
Another source of difficulty in reading sequencing gels is the inclusion of glycerol in samples run on gels containing TBE. Glycerol, often used to stabilize the polymerase in the sequencing reactions, forms a complex with the borate in TBE. This creates a “salt wave” of altered conductivity, which migrates through the gel with the DNA, distorting a narrow range of DNA sizes. This problem is alleviated by using non-borate buffers, such as TTE, in gels used to run glycerol containing samples.
NEXT TOPIC: Automated Sequencers
- Using PAGE to Determine Nucleic Acid Molecular Weight
- SSCP Analysis
- Sanger Sequencing
- Sample Preparation for Native PAGE of DNA
- Sample Prep for Denaturing PAGE of DNA
- S1 Mapping
- Run Conditions in Denaturing PAGE
- RNA Mapping
- RNA Electrophoresis
- Ribonuclease Protection
- Restriction Digest Mapping
- Primer Extension
- Preparing Denaturing DNA & RNA Gels
- Preparation of Denaturing Agarose Gels
- Preparation of Agarose Gels
- Pouring Sequencing Gels
- PFGE and FIGE
- PCR Analysis: Yield and Kinetics
- PCR Analysis: An Examination
- Native PAGE of DNA
- Mobility Shift Assay
- Methylation & Uracil Interference Assays
- Maxam & Gilbert Sequencing
- Manual Sequencing
- In Gel Enzyme Reactions
- Heteroduplex Analysis
- Gel Preparation for Native PAGE of DNA
- Gel Electrophoresis of PCR Products
- DNase I Footprinting
- DNA/RNA Purification from Agarose Gels – Electroelution
- Differential Display
- Denaturing Polyacrylamide Gel Electrophoresis of DNA & RNA
- Conformational Analysis
- Automated Sequencers
- Analysis of DNA/Protein Interactions
- Agarose Gel Electrophoresis of DNA and RNA – Uses and Variations
- Agarose Gel Electrophoresis of DNA and RNA – An Introduction
- Sample Preparation
- Electrophoresis Buffers
- Blotting and Detection Buffers
- Gel Matrices
- Gel Accessories
- Ultra Pure Reagents