Steps in Recombinant DNA Technology or rDNA technology | Biotechnology

Summary

This video explains the steps involved in recombinant DNA technology, using human insulin production as a case study. It covers the process of identifying and isolating the gene of interest, inserting it into a vector, and introducing the recombinant DNA into a host organism, such as *E. coli*. The video then details how transformed cells are selected and how the introduced gene is expressed, resulting in the large-scale production of human insulin. Overall, it provides a clear, step-by-step guide to the foundational techniques of genetic engineering and gene cloning.

Takeaways

• 😀 Recombinant DNA technology involves identifying, isolating, and inserting a gene of interest into a suitable vector for replication.
• 😀 The first step in recombinant DNA technology is to isolate the gene of interest, which can be done using a genomic library or PCR amplification.
• 😀 A **vector** is a DNA molecule that carries the gene of interest into a host cell. Common vectors include plasmids and bacteriophages.
• 😀 Restriction enzymes are used to cut both the gene of interest and the vector DNA at specific sites, allowing the gene to be inserted.
• 😀 The gene is then inserted into the vector, and the ends are sealed using **ligase** to create a recombinant DNA molecule.
• 😀 The recombinant DNA is introduced into a host cell (commonly *E. coli*) through **transformation**, using techniques like electroporation or liposome-mediated transfer.
• 😀 Once the recombinant DNA is inside the host cell, researchers need to select the transformed cells, which can be done through antibiotic resistance or visual assays.
• 😀 In the case of insulin production, *E. coli* cells are genetically modified to produce human insulin, which is then harvested for medical use.
• 😀 After transformation, bacterial cells replicate and express the recombinant DNA, multiplying the gene of interest and producing the desired protein product.
• 😀 The final step involves large-scale culturing of the transformed cells to produce significant quantities of the gene product (e.g., insulin) for therapeutic use.
• 😀 Recombinant DNA technology is an essential tool in biotechnology and medicine, allowing for the production of proteins like insulin on a commercial scale.

Q & A

What is recombinant DNA technology?

Recombinant DNA technology is a technique used to identify, isolate, and insert a gene of interest into a vector (such as a plasmid) to form a recombinant DNA molecule. This process enables the production of large quantities of the gene or its product, like proteins, by transforming a suitable host organism.

What are the key steps involved in recombinant DNA technology?

The key steps in recombinant DNA technology include: 1) Identification and isolation of the gene of interest, 2) Insertion of the gene into a suitable vector, 3) Transformation (introducing the recombinant DNA into a host), 4) Selection of transformed cells, and 5) Expression or multiplication of the gene within the host.

How do you isolate the gene of interest?

The gene of interest can be isolated from various sources such as genomic libraries, cDNA libraries, or by chemical synthesis if the sequence is known. Additionally, if there are insufficient copies of the gene, it can be amplified using PCR (Polymerase Chain Reaction).

What is a vector in recombinant DNA technology?

A vector is a DNA molecule that can carry and replicate the gene of interest inside a host organism. Vectors such as plasmids are commonly used because they are capable of replicating independently within bacterial cells, allowing for the production of many copies of the gene or its product.

What role do restriction enzymes play in recombinant DNA technology?

Restriction enzymes are used to cut both the vector DNA and the gene of interest at specific sites. These cuts allow the gene of interest to be inserted into the vector in a controlled manner, forming a recombinant DNA molecule.

What is the purpose of ligase in the recombinant DNA process?

Ligase is used to seal the sticky ends of the recombinant DNA after the gene of interest has been inserted into the vector. It ensures that the gene remains attached to the vector, creating a stable recombinant DNA molecule.

What methods are used to introduce the recombinant DNA into a host cell?

Methods for introducing recombinant DNA into a host cell include electroporation, microinjection, liposome-mediated gene transfer, and chemical methods such as calcium chloride-mediated transformation. Bacteria can also naturally take up DNA from their environment in a process called transformation.

How do you select transformed cells from non-transformed ones?

Transformed cells can be selected using various techniques, such as growing cells on selective media containing antibiotics (if the recombinant DNA includes an antibiotic resistance gene). Other methods include visible markers or color changes in the case of enzyme activity, or hybridization techniques to identify cells with the correct DNA.

Why is *E. coli* commonly used as a host in recombinant DNA technology?

*E. coli* is commonly used because it is well understood, easy to manipulate, and capable of rapidly replicating the recombinant DNA. It is also a reliable and cost-effective host for gene expression and protein production.

What happens during the expression phase of recombinant DNA technology?

During the expression phase, the recombinant DNA inside the host cell is transcribed and translated to produce the desired protein product. For example, in the case of insulin production, the *E. coli* cells will produce human insulin as a protein.