DNA Cloning | Science & Technology for UPSC CSE PDF Download

Introduction

• DNA Cloning Process: DNA cloning involves generating multiple identical copies of a specific genetic component or DNA fragment. This process begins by inserting the target gene or DNA fragment into a circular DNA structure known as a plasmid. To accomplish this, specialized enzymes called restriction enzymes are employed to cut and join DNA segments, resulting in the creation of recombinant DNA.
• Introduction of Recombinant DNA: Subsequently, the recombinant plasmid is introduced into a bacterial cell. These bacteria then become carriers of the plasmid. Specific bacterial cells that have successfully taken up the plasmid are selected and cultivated.
• Amplification of Recombinant DNA: As these selected bacteria reproduce, they replicate the recombinant DNA carried by the plasmid. This DNA replication occurs during the bacterial cell's reproduction process and is passed on to their offspring.
• Purpose of DNA Cloning: DNA cloning is often necessary when a large quantity of DNA copies is required for research purposes or when creating new plasmids. In some cases, the cloned DNA fragment encodes a beneficial protein. The bacteria can be utilized as protein factories, and the colonies synthesized by these bacteria produce the protein.
• Example: Insulin Production: To illustrate this process, consider the production of insulin for diabetic patients. In this case, the human insulin gene is cloned into the DNA of Escherichia coli (E. coli) bacteria to enable the bacteria to produce insulin, which is then harvested for medical use.

DNA Cloning techniques

Isolation of Target DNA

• The target DNA can be synthetic, complementary, or genomic.
• If the genomic DNA fragment of interest can be recovered from gel electrophoresis, it's used directly.
• If not, mRNA templates are used to create complementary DNA (cDNA) fragments.
• Polyadenylated mRNAs are separated using affinity column chromatography.
• Reverse transcriptase is used to convert mRNAs into cDNAs.
• This cDNA must contain the unbroken coding sequence of the gene.

Insertion of Foreign DNA into a Vector

• The isolated cDNA is fragmented using a specific restriction enzyme.
• Cohesive ends are generated on both cDNA and the cloning vector using the same enzyme.
• Complementary single-stranded DNA sequences are added to the ends of linearized vectors.
• This allows double-stranded cDNA to be effectively inserted into the vector, creating sticky ends.

Transfer of rDNA into Bacterial Cell

• The rDNA must be introduced into a suitable bacterial host cell, which undergoes transformation.
• Transformation is the process of inserting a plasmid with foreign DNA into a cell.
• A gentle heat shock is applied to increase DNA uptake.
• Bacteria are cultured in an antibiotic selection medium to identify transformed cells.

Recombinant Clone Detection

• The final step is selecting or screening colonies with the recombinant plasmid.
• Antibiotic selection is an effective method.
• Altered bacterial cells are plated on a medium containing various antibiotics.
• Colonies that grow are those containing the plasmid, typically carrying antibiotic resistance genes.
• For example, the plasmid pBR 322 may carry tetracycline and ampicillin resistance genes, allowing identification based on antibiotic resistance.

DNA cloning procedures

Certainly, here's a breakdown of how DNA cloning can be employed to make bacteria produce a protein like human insulin:

Plasmid Modification

• Start by opening the circular DNA structure called a plasmid.
• Use restriction enzymes to cleave the plasmid DNA at specific sites.
• Simultaneously, the gene of interest (in this case, the human insulin gene) is obtained.
• Join the gene into the opened plasmid using DNA ligase. This creates a recombinant plasmid containing the insulin gene.

Introduction of Recombinant Plasmid into Bacteria

• The recombinant plasmid, now carrying the insulin gene, is introduced into a bacterial host cell.
• The host cell becomes a recipient of this plasmid.

Selection of Transformed Bacteria

• To identify which bacteria have successfully taken up the plasmid, antibiotic selection can be employed.
• The plasmid may contain an antibiotic resistance gene.
• Bacteria that have incorporated the plasmid will be able to grow in the presence of the antibiotic.
• Non-transformed bacteria will not survive in the antibiotic-containing environment, allowing the selection of the transformed cells.

Production of Protein

• Once the plasmid-carrying bacteria are identified, they can be cultured and multiplied.
• These bacteria act as "factories" for producing the protein of interest, in this case, human insulin.
• The bacteria follow their normal growth and reproduction cycles, generating insulin as they multiply.

Protein Purification

• After the bacteria have produced the protein, it needs to be purified.
• Various techniques, such as chromatography, can be used to extract and purify the protein from the bacterial cells.
• The purified protein can then be used for medical purposes or research, such as producing insulin for diabetic patients.

Cloning Techniques

Certainly, here's a breakdown of how DNA cloning is used to get bacteria to produce a protein, such as human insulin, with a focus on the key steps:

Cutting and Pasting DNA

• DNA is cut at or near a specific target sequence using restriction enzymes.
• These enzymes produce short, single-stranded overhangs at the cut ends.
• If the overhangs match, two DNA fragments can base pair with each other.
• DNA ligase, often referred to as molecular glue, is used to join the fragments together, filling gaps in the DNA backbone.
• The DNA of interest (the target gene) is digested with a specific restriction enzyme, and the plasmid is also digested to create compatible ends.
• DNA ligase is then used to combine these fragments, creating a recombinant plasmid containing the target gene.

Bacterial Transformation and Selection

• The recombinant plasmid, along with other DNA fragments, can be introduced into bacteria through a process called transformation.
• Bacterial cells are treated with a shock, often through heat or electrical methods, to encourage them to take up foreign DNA.
• Plasmids often carry antibiotic resistance genes, allowing bacteria that have taken up the plasmid to survive in the presence of a specific antibiotic.
• Bacteria lacking the plasmid will not survive in the presence of the antibiotic.
• Not all transformed bacteria will contain the correct plasmid due to variations in the ligation process, so multiple colonies are typically screened to identify the correct ones.
• Techniques like polymerase chain reactions (PCR) and restriction enzyme digestion can be used to verify the presence of the desired plasmid.

Manufacture of Protein

• Once a bacterial colony containing the correct plasmid is identified, it can be grown into a large culture of plasmid-carrying bacteria.
• These bacteria act as protein factories, producing the protein of interest (e.g., human insulin).
• If the plasmid contains the human insulin gene, the bacteria transcribe the gene into mRNA and then translate it to produce multiple molecules of human insulin protein.
• After production, the bacterial cells can be lysed to release the protein.
• However, the lysate contains various proteins and macromolecules besides the target protein.
• To purify the target protein from the cell components, several biochemical techniques are employed.

Applications of gene cloning

Here's some additional information and examples for each of these applications:

Biopharmaceuticals

• Insulin Production: Recombinant DNA technology has been pivotal in producing human insulin. Prior to this technology, insulin for diabetes treatment was extracted from the pancreases of animals. Today, human insulin is synthesized using E. coli or yeast cells that have been genetically engineered to produce the hormone.
• Tissue Plasminogen Activator (tPA): tPA is a protein used to dissolve blood clots and is vital in treating stroke patients. It can be produced through gene cloning techniques, ensuring a stable and abundant supply for medical use.
• Human Growth Hormones (HGH): Cloning genes for human growth hormone allows for the production of therapeutic HGH for individuals with growth disorders.

Gene Therapy

• Cystic Fibrosis Gene Therapy: As you mentioned, gene therapy can be used to introduce functional copies of genes into individuals with genetic diseases. In the case of cystic fibrosis, introducing a normal copy of the CFTR gene can improve lung function and overall health for patients with this condition. This is still an active area of research with various delivery methods being explored.

Gene Analysis

• Functional Genomics: Cloning allows scientists to isolate specific genes of interest and study their function. For example, researchers can clone genes responsible for drug resistance in bacteria to understand the mechanisms and potentially develop new antibiotics.
• Recombinant Proteins: Beyond biopharmaceuticals, gene cloning is used to produce recombinant proteins for research purposes. For instance, scientists might clone a gene encoding a specific enzyme to study its biochemical properties.