In this article, you will learn about a fundamental technique of DNA amplification called Molecular Cloning. Let’s explore the biochemistry behind the procedure, as well as some important applications.
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Across the various disciplines of biology and biochemistry, the analysis of DNA serves an important role in understanding biological systems. However, DNA sequences can really only be analyzed at high concentrations, using methods like running gels or measuring light absorption. This presents difficulties for scientists interested in particular genes or sequences. After all, a human gene usually has, at most, a handful of copies inside a cell. Such low concentrations yield little sequence information to an interested scientist.
Thus, since DNA became a molecule of interest for scientists, techniques to replicate and produce large quantities of DNA sequences have remained crucial. Indeed, DNA amplification processes have a wide variety of important applications, such as genetic modification, industrial drug production, pathogen testing, genetic screening, and DNA fingerprinting.
To understand how each of these applications is performed, let’s take a look at one of the most important amplification techniques, molecular cloning. If you’d like to learn about another important technique, check out our article on PCR.
Molecular cloning takes advantage of the DNA replication that bacteria perform naturally. Specifically, it involves placing a desired sequence into live bacterial cells and allowing them to replicate multiple times. This can result in millions of bacteria that each have a desired DNA sequence.
In addition to a live strain of bacteria (usually E. coli), you need two important components to effectively use molecular cloning:
- Cloning Vector
- Restriction Endonuclease
A cloning vector is a short stretch of DNA that carries your desired sequence to be replicated. In nature, bacteria uptake small pieces of DNA, called plasmids, through a process called transduction. This involves the DNA transporting across the bacterial membrane and cell wall. Once inside, the bacteria can express genes on the plasmid, as well as replicate and pass the plasmid into daughter cells. Often, cloning vectors contain an antibiotic resistance gene useful for selecting bacteria that successfully transduced your sequence.
A restriction endonuclease is an enzyme that cuts DNA at certain sequences. The particular sequence to which a restriction enzyme binds and cuts is called its “recognition site”. Interestingly, these enzymes cut the two DNA strands at different points, creating a short stretch of unpaired nucleotides. Biochemists call these unpaired stretches “sticky ends” because they readily base pair with complementary sequences.
To understand how these components allow for DNA amplification, we need to take a closer look at the steps of molecular cloning.
First Step: Inserting your DNA Sequence
To effectively amplify DNA using molecular cloning, the first step involves inserting your desired sequence into your cloning vector. To do this, you need to select a cloning vector that contains a recognition site for your chosen endonuclease. You also need to engineer a very short DNA double-strand that includes your sequence of interest, as well as two of the same recognition sites upstream and downstream to your sequence. Thus, when your restriction enzyme cleaves the cloning vector and sequence of interest, the sticky ends can base-pair. Then, a DNA ligase can catalyze the reaction which joins the strands of the vector and sequence together.
Second Step: Getting your Plasmid into Bacteria
Second, you need your bacterial cells to uptake the vector-sequence plasmid you just made. Transduction usually has low efficiency; bacteria rarely uptake a foreign plasmid spontaneously. To increase the odds of your bacteria uptaking your plasmid, you can perform a “heat shock”. This involves keeping your bacteria culture cold for a long duration and then rapidly raising the temperature. In practice, an effective heat shock would be a quick dunk into warm-to-hot water after keeping your tube of bacteria on ice for a while. Heat shocks serve to increase the permeability of bacterial cell walls, which increases transduction efficiency. Still, even with a heat shock, relatively few bacteria in the culture will readily perform transduction.
Third Step: Isolating your Desired Bacteria
Third, you need to separate the bacteria that have your plasmid from those that don’t. This is where the antibiotic resistance gene on the cloning vector comes into play. Typically, the liquid bacterial culture is spread across a plate that contains an antibiotic and nutrients. This allows the bacteria that have your plasmid to survive and grow, while those without the plasmid won’t survive.
Fourth Step: Harvesting your DNA Sequence
Fourth, you need to grow up your resistant bacteria and harvest their plasmids. One method involves using a pipette tip to poke a bacterial colony on your antibiotic plate, which should have your plasmid, and placing the tip in a nutrient broth. This tremendously increases the population of bacteria with your plasmid, turning the transparent broth cloudy. Then, you can lyse the bacteria under alkaline conditions and purify your plasmid through centrifugation and column chromatography. This concentrates your desired fragment apart from the bacteria.
Applications of Molecular Cloning
Genetic Modification of Plants
One of the most impactful applications of molecular cloning concerns the development of genetically modified organisms in agriculture. With advancements in biotechnology, the agricultural industry has greatly invested in genetically altering crops to improve quality and increase yield. These genetically modified crops often have genetic resistance to pests and herbicides meant to kill weeds.
To genetically modify plants, agricultural scientists use the bacterial species Agrobacterium tumafaciens, a known plant pathogen. In nature, A. tumafaciens implants its plasmid into plant tissue, which induces the formation of tumors, caused by the T-DNA region of the plasmid. Using molecular cloning, scientists can construct and amplify versions of this plasmid where the T-DNA region is replaced with different genes. This then provides genes advantageous to the agriculture industry a way into plant tissue.
Production of Proteins
Another important application of molecular cloning involves the mass production of proteins used in pharmaceutical drugs. In such cases, the sequence implanted into the cloning vector includes a gene for a protein with some therapeutic value. When transduced into bacteria, the bacteria’s molecular machinery transcribes and translates the gene, synthesizing the protein of interest. When cloned over multiple generations, large quantities of the protein can be produced.
Insulin, a crucial drug for those with diabetes, is one such protein that scientists can create using molecular cloning.