Recombinant DNA and Cloning Quiz

Recombinant DNA is a method used to make an identical copy of an animal, not to be confused with PCR or animal cloning.

Three methods to deliver Recombinant DNA to a host cell: Transformation, Phage Introduction, and Non-Bacterial Transformation.

Transformation: 1) Select a piece of DNA to be inserted into a vector, 2) Cut the DNA with a restriction enzyme and ligate it into the vector with DNA Ligase, 3) Insert the vector into a host cell, such as E. Coli, using selectable markers for identification.

Non-Bacterial Transformation: Similar to Transformation but does not use bacteria for the host, instead, DNA is injected directly into the nucleus or cells are bombarded with microprojectiles coated with DNA.

Phage Introduction: Transfection using a phage instead of bacteria to produce recombinants.

Recombinant DNA technology led to the development of various techniques such as Southern, Northern, and Western blot hybridization, and DNA sequencing.

Southern blot hybridization: DNA hybridization technique using restriction enzymes and DNA ligase to identify specific DNA fragments.

Northern blot hybridization: RNA hybridization technique using similar principles to Southern blot.

Western blot: Protein hybridization technique using antibodies to detect specific proteins.

Sequencing of cloned DNA: Initially based on Sanger method (enzymatic reaction) and Maxam-Gilbert method (chemical reaction) to determine the order of nucleotides in a DNA sequence.

Recombinant DNA technology has been effectively used in producing human proteins like insulin and growth hormone for medical treatments.

Insulin production: Isolate the insulin DNA, add it to a plasmid, cut the plasmid, add the insulin gene, join the plasmid and insulin DNA, insert the plasmid back into the bacterium, and extract the insulin from the bacterial culture.

Recombinant DNA technology involves constructing new DNA molecules by combining DNA fragments from different sources.

Four key developments led to the creation of the first recombinant DNA organism: isolation of DNA, cutting and joining DNA, and transferring DNA into host cells.

Obtaining rDNA (recombinant DNA):

• Isolate DNA: donor and vector DNA are obtained and purified.
• Cutting DNA: restriction enzymes are used to cut DNA at specific sequences.
• Joining DNA: DNA ligase is used to join cut DNA fragments.
• Placing gene in vector: the gene is inserted into a vector (plasmid or phage) using restriction enzymes and ligase.
• Transferring DNA: the vector carrying the gene is transferred into a host cell.
• Selection of host cells: cells carrying the gene are selected using a marker.
• Replication of cells: genetically identical cells (clones) are produced by replicating the cells carrying the gene.

Isolation of donor and vector DNA:

• Plasmid DNA is isolated using ultracentrifugation.
• Genomic DNA is denatured and precipitated during alkaline lysis.
• Phages can also be used as vectors for cloning.

Cutting DNA:

• Restriction enzymes EcoRI and HindIII cut DNA with sticky and blunt ends, respectively.
• Both strands of DNA are cut at specific points within the target sequence.
• Sticky ends help the cut DNA molecules join back together through complementary base-pairing.

Types of vectors:

• Plasmids: up to 15 thousand bases.
• Phages: up to 25 thousand bases.
• Cosmids: up to 45 thousand bases.
• Bacteriophage P1: 70 to 100 thousand bases.
• P1 artificial chromosomes (PACs): 130 to 150 thousand bases.
• Bacterial artificial chromosomes (BACs): 120 to 300 thousand bases.
• Yeast artificial chromosomes (YACs): 250 to 2000 thousand bases.

Cloning: creating multiple copies of the foreign DNA fragment in an organism.