Question:

The technique commonly used to resolve large-sized DNA fragments (>20 kb) following digestion with restriction enzymes is:

1. Polyacrylamide gel-electrophoresis

2. Capillary electrophoresis

3. Pulse field gel-electrophoresis

4. Preparative HPLC

Correct Answer:

3. Pulse field gel-electrophoresis

Detailed Explanation:

When it comes to separating large DNA fragments (greater than 20 kb in size), specialized techniques are required due to the physical constraints of traditional electrophoresis methods. The ideal method must allow for effective separation of these larger DNA pieces based on size, and Pulse Field Gel Electrophoresis (PFGE) is specifically designed for this purpose.

1. Polyacrylamide Gel-Electrophoresis (PAGE):

• Polyacrylamide gel-electrophoresis is commonly used for resolving smaller DNA fragments (up to around 1 kb).

• While this method works well for short sequences, it is not effective for larger fragments (>20 kb), because the gel matrix used in PAGE is too dense to allow proper resolution of such large DNA molecules.

2. Capillary Electrophoresis:

• Capillary electrophoresis is a high-throughput method typically used for analyzing smaller DNA fragments, such as those found in Sanger sequencing.

• This method does not provide the necessary resolution for separating large DNA fragments (>20 kb) because the capillaries used in the system are typically small and unable to handle very large molecules effectively.

3. Pulse Field Gel-Electrophoresis (PFGE):

• Pulse Field Gel Electrophoresis is a specialized technique developed for resolving large DNA fragments, typically ranging from 20 kb to several megabases (Mb).

• PFGE works by periodically changing the direction of the electric field, allowing the DNA to reorient itself in the gel. This helps large DNA molecules to move through the gel more efficiently and separate based on size.

• It is widely used in applications like genomic mapping, DNA fingerprinting, and chromosome separation.

4. Preparative HPLC:

• Preparative High-Performance Liquid Chromatography (HPLC) is a technique typically used for purifying and isolating individual compounds from a mixture, rather than resolving large DNA fragments.

• It is not suitable for separating large DNA fragments because it is more commonly applied to small molecules and does not provide the resolution required for large biomolecules like DNA.

Conclusion:

The most appropriate method for resolving large DNA fragments (>20 kb) is Pulse Field Gel Electrophoresis (PFGE). This technique is specifically designed to handle large molecules, enabling efficient separation and analysis. While other methods like polyacrylamide gel-electrophoresis and capillary electrophoresis are useful for smaller DNA fragments, PFGE is the gold standard for handling large DNA pieces.