L4: DNA Technology

• DNA Technology

  • Methods for studying and manipulating genetic material
  • Biotechnology: Manipulation of organisms in their genetic components to make useful products
  • Genetic Engineering: Direct manipulation of genes for practical purposes
• DNA Technology

  DNA Sequencing

  • Process of determining the nucleic acid sequence – the order of nucleotides in DNA.
  • ○ DNA fragments are amplified (copied) to yield an enormous number of identical fragments.
  • The first automated procedure of DNA Sequencing was called “Dideoxy Sequencing”.
  • ○ Developed in the 1970s by biochemist Frederick Sanger, who received a Nobel Prize in 1980 because of it.
  • ○ It is still used for routine small-scale sequencing jobs
• DNA Technology

  DNA Cloning

  • Methods to isolate a segment of DNA carrying that gene and make multiple identical copies of it.
  • ○ A clone is a group of identical cells descended from a single ancestral cell.
  • DNA Cloning is commonly approached with the use of bacteria, most often, Escherichia coli (E. coli).
  • ○ Foreign DNA is inserted into a plasmid, and the recombinant dna (plasmid) is inserted into a bacterial cell.
  • ○ Reproduction in bacterial cell results in cloning of plasmid including the foreign DNA
  • ■ Results in production of multiple copies of a single gene
• DNA Technology

  DNA Cloning

  • “Dolly” the sheep is a well-known successful experiment of DNA cloning.
• DNA Recombinant Technology

  • Combines two genetic materials from two different organisms resulting in amplification of target DNA.
  • ○ The Gene of Interest, is a fragment of DNA that carries the target gene.
  • ■ Like the bioluminescence of jellyfish, or fungus that can infect an insect, making a pesticide.
  • ○ The human genome is the map of the locations of all the genes.
• DNA Recombinant Technology

  • A Recombinant consists of two molecules that are to be re-combined
  • Recombinant DNA
  • ○ Nucleotide sequences from two different sources, that are combined into the same DNA molecule.
  • Also called gene cloning / dna cloning.
• DNA Recombinant Technology

  Cloned Insulin

  • Insulin is a protein that regulates blood sugar level, and can only be secreted by the pancreas through protein synthesis.
  • Back in 1922, insulin was extracted from the pancreas of animals, often pigs, and breeding one would normally take from months to years before their pancreas could be extracted
• DNA Recombinant Technology

  Cloned Insulin

  • Scientists in 1978 however found a way to produce more insulin without having to breed and slaughter animals– it is done with the cloning of human insulin DNA.
  • ○ This became one of the most important product of recombinant DNA in the 1980’s
  • The process of cloning human DNA starts by extracting the DNA responsible for the production of insulin from a human pancreatic cell and injecting it into the plasmid of an Escherichia coli bacteria.
• DNA Recombinant Technology

  Making a Recombinant DNA (1-2)

  • The target DNA (insulin-producing gene) and plasmid are extracted from their sources (human pancreatic cell and E. coli bacteria, respectively).
  • The plasmid is then cut at the restriction site with the use of a restriction enzyme.
  • ○ The enzyme that cuts the vector DNA (plasmid) is a restriction enzyme.
  • ○ The place where a restriction enzyme binds is called the restriction site, and it is a specific sequence (usually made of 4-8 base pairs) on a vector DNA.
• DNA Recombinant Technology

  Making a Recombinant DNA (3-4)

  • The gene of interest is then inserted to the vector DNA and is binded by a DNA Ligase.
  • ○ A DNA Ligase is an enzyme that will attach the gene of interest to the sticky / blunt ends of the plasmid.
  • After the gene of interest and vector is put in their proper places, the recombinant DNA is made.
• A Plasmid is a small, circular, double-stranded DNA molecule that carries

  accessory genes separate from those of a bacterial chromosome.

  ○ In DNA cloning, they are used as vectors carrying up to about 10,000

  base pairs (10 kb) of DNA.

  ○ Is just an accessory organ that can perform as a vector
• A Vector is a piece of DNA, usually a plasmid or a viral genome that is used to move genes from one cell to another.

  ○ Cloning Vector

  ■ DNA molecule that carry foreign DNA into a host cell

  ■ In gene cloning, the cloning vector is the original plasmid

  ○ Expression Vector

  ■ A cloning vector that contains a highly active bacterial promoter.

  ○ “Gene Carrier”
• The production of multiple copies of a single gene is a type of DNA cloning called gene cloning
• Bacterial Transformation is the process by which a bacterium (host cell) takes up a foreign DNA and expresses it

  ○ Can be done through heat shock, treatment of calcium chloride, or electroporation
• Gel Electrophoresis is a technique used to separate DNA fragments with different sizes (base pairs) through visualizing in an agarose gel
• Palindrome means they read the same way forward and backwards

  ○ i.e the opposite of racecar, is racecar.
• plasmid

  Are small circular DNA molecules that replicate separately from the much larger bacterial chromosome.
• restriction site

  A specific sequence on a DNA strand that is recognized and cut by a restriction enzyme.
• Biotechnology

  The direct manipulation of genes for practical purposes.
• Genetic Engineering

  The manipulation of living organisms or their components to produce useful products.
• Dolly

  The name of the first mammal being successfully cloned.
• False

  Palindrome means they read the same way upwards and downwards
• False

  Back in 1922, the pancreas of animals, often rats, were extracted to get insulin the organ produces from protein synthesis.
• True

  Dideoxy Sequencing was developed by Frederick Sanger, and won a Nobel Prize because of it.
• True

  A degradative enzyme that recognizes and cuts up DNA is called a restriction enzyme.
• False

  DNA Recombinant Technology can be used in the field of Medicine, Agriculture, and Metallurgy.
• Gel Electrophoresis

  Cutting DNA with a particular restriction enzyme produces DNA fragments that can be separated by?
• Recombinant DNA

  This is a DNA molecule that has been manipulated in the laboratory to carry

  nucleotide sequences derived from two sources often different species
• Size

  Gel electrophoresis separate DNA fragments on the basis of differences in their
• Recombinant Bacterium

  This will undergo bacterial transformation
• Clone

  A group of identical cells descended from a single ancestral cell.