Question

a. There are three primary methods for obtaining donor DNA when doing genetic engineering. What are those methods (two-to six-word descriptions of each are acceptable)? b. You need to produce a protein from humans in \(E\). coli. You do not know the primary amino acid sequence. You suspect that introns are present. Which method will you use to obtain the donor DNA?

Short answer

a. Genomic DNA, cDNA from mRNA, Chemical Gene Synthesis. b. Use cDNA from mRNA.

Step-by-step solution

Identify the Methods for Obtaining Donor DNA

List the three primary methods used in genetic engineering to obtain donor DNA.

Step 1a: Isolation from Genomic DNA

Use genetic materials directly from the genomic DNA of an organism.

Step 1b: Synthesis from mRNA (cDNA)

Convert mRNA into complementary DNA (cDNA) by using reverse transcriptase.

Step 1c: Chemical Gene Synthesis

Chemically synthesize the DNA sequence in a laboratory setting without using a biological template.

Determine Appropriate Method for Protein Production in E. coli

Select the method suitable for producing a human protein in E. coli, considering the presence of introns in the human gene.

Step 2a: Use cDNA from mRNA

Choose to create complementary DNA (cDNA) from mRNA because cDNA does not contain introns, which are typically present in eukaryotic genes but not in prokaryotic organisms like E. coli.

Key concepts

genomic DNA isolation

Genomic DNA isolation is a method used to extract DNA directly from the cell's genome. This technique involves breaking open cells and nuclei to release the DNA. The process typically includes the following steps:
- Cell lysis: Using a buffer to break down cell walls and membranes, this releases the cellular contents including the DNA.
- DNA stabilization: Using a solution to protect DNA from being degraded by enzymes.
- Separation: Centrifuging the sample to separate the DNA from other cellular components.
- Purification: Removing proteins and other contaminants, often using a combination of chemical and enzymatic treatments.

Genomic DNA isolation is useful for obtaining a complete set of an organism's genes, but it comes with a challenge: eukaryotic genomic DNA typically has introns. These introns can create issues when transferring genes to prokaryotic organisms like E. coli, which cannot process introns efficiently.

cDNA synthesis

cDNA synthesis is the process of converting messenger RNA (mRNA) into complementary DNA (cDNA). This technique bypasses the problem of introns found in eukaryotic genes, making it ideal for expressing eukaryotic genes in prokaryotic organisms like E. coli. To create cDNA, the following steps are generally followed:
- Isolation of mRNA: Collect mRNA molecules from cells since these molecules represent gene sequences without introns.
- Reverse transcription: The enzyme reverse transcriptase is used to create cDNA from the isolated mRNA.
- Second-strand synthesis: DNA polymerase is used to create the complementary DNA strand, leading to double-stranded cDNA.

cDNA synthesis is beneficial when you suspect introns in the gene of interest. The final cDNA product can be easily cloned into expression vectors for protein production in E. coli.

chemical gene synthesis

Chemical gene synthesis is a technique for creating artificial genes in a laboratory setting without using biological templates. This method involves chemically synthesizing DNA sequences from scratch. Here's how it typically works:
- Sequence design: A DNA sequence is designed based on the desired gene.
- Oligonucleotide synthesis: Small, single-stranded DNA fragments (oligonucleotides) are chemically synthesized.
- Assembly: These oligonucleotides are then assembled together to form the full-length synthetic gene.
- Cloning and verification: The synthesized gene is cloned into a vector and sequenced to ensure its accuracy.

Chemical gene synthesis allows for the creation of custom DNA sequences, including those that do not naturally exist. It's particularly useful for producing genes with specific mutations or for synthetic biology applications.