Gene Cloning Methods

• Gene cloning involves creating copies (clones) of a single gene, useful in biomedical and industrial research.
• Two primary methods for gene cloning exist: recombinant DNA technology and polymerase chain reaction (PCR).

Recombinant DNA Technology

• This technology joins DNA segments from different sources.
• It is an in vivo method, meaning it occurs in living cells.
• Well-suited for large-scale gene cloning in industrial settings.
• Produces the gene's product, in addition to copies of the gene itself.
• Involves selecting and isolating a gene of interest, inserting it into a vector, and transforming a host with the recombinant DNA.

Components of Recombinant DNA Technology

• Essential components include the gene of interest, molecular scissors, a vector (molecular carrier), molecular glue, and an expression system.

Gene of Interest

• The gene to be cloned, obtainable through:
  • Artificial gene synthesis: Creating a gene in vitro using a DNA synthesizer.
  • Synthesis from mRNA: Using reverse transcriptase (from retroviruses) to create complementary DNA (cDNA).
  • Cleavage from chromosomal DNA: Using restriction endonucleases.

Restriction Endonucleases

• Enzymes that cleave DNA at specific sequences by breaking phosphodiester bonds.
• Discovered in 1970, with many different types isolated since.
• Naturally found in bacteria, where they defend against viral infections by inactivating viral DNA.

Recognition Sites

• Restriction enzymes cleave DNA at specific sequences called recognition or restriction sites.
• These sites feature palindromic sequences.
• Palindromic sequence: Is a four to eight base pair DNA sequence with symmetrical arrangement in reverse order.
• Restriction enzymes create either:
  • Staggered cuts: Resulting in fragments with single-stranded "sticky ends."
  • Blunt cuts: Resulting in fragments without overhangs.

Molecular Carriers (Vectors)

• Vectors carry foreign DNA into a host cell for multiplication.
• Common vectors are small, circular DNA molecules of bacterial origin.
• Essential characteristics of a cloning vector:
  • Origin of replication site.
  • Antibiotic resistance genes.
  • Restriction sites for various enzymes.
• Examples of vectors: Plasmids, lambda phage DNA, Cosmids (plasmid/phage DNA combination), Yeast artificial chromosomes (YACs).

Molecular Glue (DNA Ligase)

• This enzyme forms phosphodiester bonds between adjacent nucleotides, joining DNA fragments.
• Used in rDNA experiments to connect different DNA fragments (vector and foreign DNA) annealed by sticky ends.

Expression System

• A suitable host organism for the recombinant vector to express (multiply).
• Selection depends on the vector used.
• Ideal expression systems have short generation times and simple genetic systems; bacterial cells are often preferred.

Mechanism of Recombinant DNA Technology

• Involves forming recombinant DNA (gene of interest + vector DNA), transforming a host, and identifying transformed clones.

Formation of Recombinant DNA

• Isolate and purify vectors and the gene of interest.
• Digest vector DNA (e.g., plasmid) with the same restriction enzyme used to cleave the gene of interest to create compatible sticky ends.
• Incubate vector and gene of interest together with DNA ligase, which connects them via phosphodiester bonds, forming the recombinant DNA molecule.

Transformation of Expression System

• Transformation is the insertion of recombinant DNA into the expression system.
• Introduce recombinant plasmids into bacterial cells (expression system).
• Bacterial cells uptake the plasmid, especially when treated with calcium chloride to increase permeability.
• As the cell reproduces, a bacterial clone forms, each new cell containing at least one plasmid with the gene of interest.

Identification of Transformed Clones

• Add an antibiotic (to which the plasmid has a resistance gene) to the medium.
• Transformed clones survive and grow due to antibiotic resistance.
• Untransformed clones are killed by the antibiotic.
• The cloned gene can be isolated from the transformed bacterial clone for further analysis, or its protein product can be separated and used.

Polymerase Chain Reaction (PCR)

• Technique to amplify a single gene or DNA piece into thousands to millions of copies via in vitro replication.
• DNA polymerase repeatedly polymerizes a specific DNA sequence.
• Taq polymerase: A heat-stable DNA polymerase from Thermus aquaticus (a bacterium found in hot springs) is used.

Components of PCR

• Template DNA (to be amplified), free nucleotides (dNTPs), primers, and Taq polymerase in a suitable buffer.
• The PCR mixture is placed in a thermocycler or PCR machine.
• Thermocycler: Regulates temperature during various PCR steps.

Mechanism of PCR Reaction

• A PCR cycle includes denaturation, primer annealing, and extension/polymerization, each requiring specific temperatures.

Denaturation

• The template DNA is heated to 94°C for one minute.
• dsDNA becomes ssDNA.

Primer Annealing

• Forward and backward primers anneal (hybridize) to complementary regions on the ssDNA template.
• Usually performed at a lower temperature, around 54°C for 2 minutes.

Extension or Polymerization

• Taq polymerase synthesizes new DNA strands to the 3' ends of primers, using dNTPs.
• The optimal temperature for this step is 72°C for one minute.
• One target DNA molecule is converted into two molecules.
• Second cycle: Starts with denaturation at 94°C, denaturing the newly synthesized DNA into single strands to act as templates again.