Alkaline lysis

Alkaline lysis is the process of isolating plasmid deoxyribonucleic acid (DNA) in bacteria. It is a standard method used in molecular biology to isolate the plasmid without obtaining chromosomal DNA. The first alkaline lysis was performed by Birnom and Doly in 1979. Since then, slight modifications have been made to the procedure to get to today's most widely used approach. The steps of alkaline lysis can be summarized as the formation of a pellet, resuspension of the pellet in solution, cell lysis, neutralization, and centrifugation. Alkaline lysis takes advantage of the small and supercoiled physical composition of plasmid DNA compared to chromosomal DNA, along with its ability to reanneal double stranded DNA when ideal conditions are established. This allows for the isolation of plasmid DNA apart from chromosomal DNA and other cellular components.

What does it deliver?

Alkaline lysis is often an initial step in molecular processes. A proper completion of alkaline lysis yields a pure bacterial plasmid. A plasmid is a circular DNA molecule found naturally in bacteria that replicates independently from chromosomal DNA. Plasmids can also less commonly be found in the other two domains: Archaea and Eukarya. The plasmid contains genetic information that often provides an advantage, such as antibiotic resistance or virulence factors. In bacteria, plasmids can be passed through horizontal transmission via transduction, transformation, and conjugation or vertical transmission from parent to offspring. Plasmids are of interest to scientists because of their versatility and relatively simple manipulation. The plasmid acquired from alkaline lysis can be applied to numerous processes and treatments.

Method

Step 1: Formation of Pellet

Centrifuge sample containing bacteria to form a pellet of bacterial cells at the bottom of the tube. The centrifugation allows for the separation of the bacterial cells from the growth medium or excess debris in the sample. Dispose of the supernatant growth medium, leaving the pellet of cells at the bottom of the tube.

Step 2: Resuspension

Resuspend the pellet in solution containing: EDTA, Tris-HCl buffer, glucose, and RNase. Ethylenediaminetetraacetic acid (EDTA) is a chelating agent that binds metal ions, primarily divalent or trivalent ions. In this step, EDTA is added to bind up Calcium and Magnesium ions, which are naturally found in cells. In the presence of Magnesium or Calcium ions, the enzyme DNase cleaves double-stranded DNA. EDTA binds Magnesium and Calcium ions which prevents a DNase from degrading plasmid DNA. Tris Hydrochloride (HCl) is a buffer solution used to stabilize the pH and protect the integrity of the DNA. Tris-HCl is necessary due to the high pH environment that is established in order to lyse open the cells. Glucose is an osmolyte, a molecule that helps regulate osmotic stress. Glucose is added to prevent cells from lysing uncontrollably and damaging DNA. RNase are enzymes that degrade RNA. RNases are added to prevent contamination of the plasmid DNA with RNA present in the cell.

Step 3: Cell lysis

Sodium dodecyl sulfate detergent (SDS) and Sodium Hydroxide are used to lyse the cells. Sodium Hydroxide establishes an alkaline environment that disrupts the phospholipid bilayer and breaks hydrogen bonds between double-stranded chromosomal DNA, making it single-stranded. Due to the supercoiled nature of plasmid DNA, strands of the circular DNA are able to remain together. Sodium dodecyl sulfate detergent is an anionic detergent that inserts itself into the phospholipid bilayer, disrupting the hydrophobic interactions that make up the membrane. Sodium Hydroxide and SDS detergent lyse the cell and release its cellular contents including chromosomal and plasmid DNA, into the solution.

Step 4: Neutralization

A property of DNA is its ability to reanneal when pH conditions are neutralized. Under neutral conditions, hydrogen bonds reform between complementary base pairs, joining single strands to double-stranded DNA. Because the plasmid was so tightly coiled and small before the alkaline conditions were established, it can easily reanneal. The chromosomal DNA, however, because of its lengthy strands, does not anneal. In addition, the plasmid is circular-shaped, preventing complementary strands from separating as opposed to chromosomal DNA, which is linear.

Step 5: Centrifugation

Once the plasmid anneals, it dissolves into the solution. The potassium acetate reacts with the SDS detergent, Magnesium ions, and Calcium ions already present in the solution and forms potassium dodecyl sulfate (KDS), an insoluble white precipitate. The remaining chromosomal DNA strands, denatured proteins, and added chemicals stick together and precipitate out. The plasmid will remain in the supernatant. The solution can be centrifuged to isolate the plasmid from the remaining contents.

Steps of Alkaline lysis. Created in https://BioRender.com

Chemicals

• Sodium dodecyl sulfate detergent (SDS)
• Sodium Hydroxide
• Ethylenediaminetetraacetic acid (EDTA)
• Tris Hydrochloride (HCl) buffer
• Glucose
• Potassium acetate

Applications

Transformation involves the use of restriction enzymes to cut plasmids at specific locations to allow for insertion of a gene of interest. The recombinant plasmid is then introduced into a bacterial cell where it can replicate independently, allowing the bacterium to express the gene of interest. These plasmids can be used for cloning genes or altering gene expression.

Recombinant protein production is a process in which transformation is carried out with a recombinant DNA that codes for a specific protein. Once the recombinant plasmid has been introduced into the host cell, the protein is then produced by the host cell. Application of this process is common in the medical field with the production of insulin, hormones, and growth factors.

Gene therapy is the modification of an individual's genes to treat a disease or disorder. Gene therapy using plasmids is a potential treatment option for individuals who have a dysfunctional gene causing a disorder. Recombinant DNA of a functional copy of the defective gene can be incorporated into a plasmid host cell. This provides the host cell with a functional gene to treat the disorder.

DNA vaccines are a type of vaccine that uses plasmid DNA to trigger an immune response in the body. The plasmid DNA codes for an antigen which is detected by the immune system as foreign. The immune system carries out an immune response which builds immunity. If the actual virus the vaccine was created for enters the body, the immune system will be familiar with how to attack. The DNA vaccine can be personalized for the virus based on the plasmid recombinant DNA.