Question

A human cDNA clone was isolated from a cDNA library using a homologous mouse gene as a probe. The clone was grown in \(E\) coll, miniprepped to isolate the \(c D N A\), and then amplificd by \(\mathrm{PCR}\) using primers designed to the sequence of the mouse gene. When the PCR reaction was run on an agarose gel, there were multiple weak bands present instead of one clear band. What are the possible reasons for this result? How can the experiment be improved so that one clear band is amplified next time?

Short answer

Answer: Multiple weak bands on the agarose gel could result from several issues such as low-quality or impure cDNA samples, contamination, unspecific primers, insufficient amounts of starting DNA or primers, and suboptimal PCR conditions. To obtain a single clear band next time, consider optimizing PCR conditions by adjusting primer concentration, temperature gradient, using high-quality reagents, designing specific primers, using a hot-start PCR technique to reduce non-specific amplification, purifying cDNA samples using column-based methods, and running a negative control.

Step-by-step solution

Understanding cDNA Cloning and Isolation

cDNA cloning involves reverse transcribing the mRNA (messenger RNA) into complementary DNA (cDNA) and isolating specific cDNA clones from the cDNA library using a homologous probe. cDNA clones insertion into the Escherichia coli (E. coli) allows them to replicate, forming a bacterial colony containing the desired clone. In our case, a mouse gene is used as the probe.

Identifying Possible Issues in cDNA Cloning

There could be potential issues, including low-quality or impure cDNA samples, contamination, or problems related to the homologous mouse gene probe.

Understanding PCR Amplification

The Polymerase Chain Reaction (PCR) is a technique used to amplify specific DNA sequences using specific primers designed for the target gene sequence. In this case, the primers are designed according to the sequence of the mouse gene.

Identifying Possible Issues in PCR Amplification

PCR can produce multiple weak bands on the agarose gel due to various factors, such as unspecific primers (which bind to other regions besides the target gene), insufficient amounts of starting DNA or primers, contamination, and suboptimal PCR conditions (incorrect annealing temperature, poor quality reagents, or incorrect primer concentrations).

Understanding Agarose Gel Electrophoresis

Agarose gel electrophoresis is a method used for separating DNA fragments based on their size. The separated fragments appear as bands on the gel when visualized under UV light.

Identifying Possible Issues in Agarose Gel Electrophoresis

Multiple bands could be due to artifacts in the agarose gel electrophoresis resulting from impurities in the preparation, incorrect staining, or improper embedding of the amplified insert.

Improving the Experiment to Obtain a Clear Band

To achieve one clear band in the PCR experiment next time, consider evaluating the following improvements: 1. Optimize the PCR conditions by adjusting primer concentration, temperature gradient, and using high-quality reagents. 2. Design specific primers based on the target gene sequence to avoid unspecific binding. 3. Opt for using a hot-start PCR technique to reduce non-specific amplification. 4. Use a column-based method for purifying the cDNA sample to avoid impurities and contamination. 5. Consider running a negative control and analyzing your results to rule out possible false positives.