Question

The right cuts. Suppose that a human genomic library is prepared by exhaustive digestion of human DNA with the EcoRI restriction enzyme. Fragments averaging about \(4 \mathrm{kb}\) in length would be generated. Is this procedure suitable for cloning large genes? Why or why not?

Short answer

This procedure is not suitable for cloning large genes due to fragmentation.

Step-by-step solution

Understand the Context

The problem involves creating a genomic library using EcoRI restriction enzyme, resulting in fragments averaging about 4 kilobases (kb) in length.

Analyze the Fragment Length

Consider the average length of human genes, which can be quite large. Human genes vary in size, but many are significantly larger than the 4 kb fragments produced in this scenario.

Determine Suitability for Large Genes

Since the average fragment size is 4 kb, large genes would be broken into smaller pieces, inhibiting the capture of entire large genes intact in a single clone.

Final Conclusion

Since large genes will be split into multiple small fragments, this method is not suitable for cloning large genes intact because a single clone will not include the entire gene.

Key concepts

EcoRI Restriction Enzyme

EcoRI is a type of restriction enzyme, which is a protein that cuts DNA at specific sequences. Restriction enzymes serve as molecular scissors in genetic engineering. They recognize, bind to, and cut DNA at specific recognition sites.

In the case of EcoRI, it recognizes a specific palindromic sequence generally six nucleotides long. The sequence for EcoRI is GAATTC. Whenever it encounters this sequence in a DNA strand, it cuts between the G and A nucleotides on both strands. This results in "sticky ends," or overhanging single-stranded ends, which are useful for ligating DNA fragments together.

EcoRI is commonly used in molecular biology for creating recombinant DNA. By cutting and then pasting together different DNA sequences, scientists can create new genetic combinations. Such applications include creating genomic libraries, which are collections of DNA fragments representing the entire genome of an organism. The sticky ends made by EcoRI facilitate the cloning of DNA fragments into plasmids or other vectors used in DNA cloning.

Gene Cloning

Gene cloning is a fundamental technique in genetic engineering. It involves making multiple copies of a specific gene. This is useful for research and various applications, such as gene therapy and the production of proteins like insulin.

The process generally begins with the isolation of DNA containing the target gene. The DNA may be fragmented using restriction enzymes like EcoRI. Then, these DNA fragments are inserted into a vector, such as a plasmid, which is a circular DNA molecule capable of replication within a host cell.

Once the DNA fragment is inserted and the vector is formed, the recombinant DNA is introduced into a host organism, commonly bacteria. The host cells replicate, creating numerous copies of the vector and hence the cloned gene.

This method allows scientists to study specific genes in detail. They can observe the gene's function, assess its expression, and utilize the protein it encodes. This is how important medicines, like insulin, are mass-produced by genetically engineered cells.

DNA Fragmentation

DNA fragmentation is the process of breaking down DNA molecules into smaller pieces. This can occur naturally as well as through artificial means designed for research purposes. In the laboratory, restriction enzymes are often used for controlled DNA fragmentation.

For instance, when creating a genomic library, DNA is often digested using a restriction enzyme like EcoRI. This enzyme cuts DNA at specific sequences, generating fragments of a more manageable size for sequencing and cloning.

The primary purpose of DNA fragmentation in genetic research is to allow scientists to manipulate specific sections of the genome. By focusing on smaller, manageable fragments, researchers can clone, sequence, or otherwise modify specific genes.

Fragmentation is crucial when dealing with large genomes, such as the human genome. Since human genes can be quite large, DNA fragmentation allows the analysis and experimentation on genes of interest by breaking them into smaller pieces that are easier to handle in the laboratory setting. However, fragmentation must be precise enough to ensure the pieces produced are suitable for the subsequent steps in a cloning experiment.