What causes DNA fragments to not separate in deoxyribonucleic acid (DNA) electrophoresis?

From the Guidelines

Bands may not separate in electrophoresis of DNA due to various factors, including suboptimal electrophoresis conditions, such as incorrect voltage, buffer concentration, or temperature, as well as technical errors or sample preparation issues. Several factors can contribute to this issue, such as using too high voltage which generates excessive heat that denatures DNA and causes smearing, improper buffer concentration that affects the electrical field, overloading wells with too much DNA sample, contamination with proteins or salts that interfere with migration, using an inappropriate agarose concentration for the DNA fragment size being analyzed, or allowing the gel to dry out during the run 1. Technical errors such as improper gel preparation with air bubbles, uneven gel casting, or incorrect loading technique can also prevent proper band separation. Additionally, DNA degradation prior to loading, expired reagents, or problems with the power supply delivering inconsistent current can result in poor resolution. The composition of the electrophoresis buffer, strength of electrophoresis, and duration of electrophoresis are crucial factors that affect DNA migration, and high temperature during electrophoresis may also impact the results 1. To achieve clear band separation, it's essential to optimize voltage settings, use fresh buffers at the correct concentration, select appropriate agarose percentage for your fragment size, and ensure proper sample preparation by removing contaminants.

Some studies suggest that the choice of restriction enzyme and electrophoretic conditions can also impact the resolution of DNA bands, particularly in the case of smaller expansions or somatically variable expansions 1. However, the most recent and highest quality study emphasizes the importance of optimizing electrophoresis conditions, such as voltage, buffer concentration, and temperature, to achieve clear band separation 1.

Key factors to consider when optimizing electrophoresis conditions include:

• Voltage: excessive heat generated by high voltage can denature DNA and cause smearing
• Buffer concentration: improper concentration can affect the electrical field and DNA migration
• Temperature: high temperature during electrophoresis can impact DNA migration and resolution
• Agarose concentration: inappropriate concentration can affect the separation of DNA fragments
• Sample preparation: removing contaminants and ensuring proper DNA preparation is crucial for clear band separation.

From the Research

Factors Affecting DNA Separation in Electrophoresis

• The separation of DNA fragments in electrophoresis can be influenced by several factors, including the concentration of the gel matrix and the degree of sample dilution 2.
• The use of a lower concentration non-gel matrix (0.4%) can allow for the complete separation of larger size fragments of lambda DNA/Hind III (125 bp-23 130 bp) 2.
• In contrast, higher concentration (1.6%) non-gel matrix can be used to separate smaller size fragments of pBR322-Hae III DNA (18 bp-587 bp) 2.
• The ratio of sample dilution can also impact the separation of DNA fragments, with a ratio of 10 allowing for the separation of two adjacent fragments (123 bp and 124 bp) of pBR322-Hae III DNA 2.

Common Issues in DNA Electrophoresis

• Contamination and degradation of DNA samples can occur due to their small volume (microL level) and can affect the separation of DNA fragments 2.
• The technique used for electrophoresis, such as agarose gel electrophoresis or capillary electrophoresis, can also impact the separation of DNA fragments 3, 4.
• The type of detector used, such as laser-induced fluorescence charge-coupled device, can also influence the detection and separation of DNA fragments 2.

Methods for Improving DNA Separation

• Using a non-gel sieving matrix, such as hydroxyethyl cellulose, can improve the separation efficiency and resolution of DNA fragments 2.
• Denaturing gradient gel electrophoresis (DGGE) can be used to separate PCR products of the same length from different bifidobacterial species 5.
• Two-dimensional (2D) gel electrophoresis can be used to analyze DNA replication intermediates and other cellular biochemical reactions 6.