Manipulating Genomes

Manipulating Genomes

1. PCR

   • It is a technique used for amplifying DNA allowing you to increase the amount of DNA from a small sample.
   • How does it work? - Artificial replication of DNA. There are 3 stages: Denaturing - Heat DNA sample to 94-96 to break hydrogen bonds. Annealing - Cool to 68 degrees. This allows the primer to hydrogen bond creating the first bit of the new double-stranded. Extending - Heat to 72, DNA polymerase binds to end and catalyses addition of nucleotides.
   • Uses of PCR: Tissue typing, detection of oncogenes, detecting mutations, identifying viral infections and monitoring spread of infectious diseases.

2. Electrophoresis

   • Separates different size fragments of DNA using an electric current.
   • Firstly DNA is cut into pieces. The gel is placed into the tank and the tank is filled with buffer. Loading dye is mixed with DNA and placed into the wells in the jelly. An electric current is passed through the gel. DNA carries a negative charged. Fragments migrate to the anode with the lightest moving the furthest.

3. DNA Profiling

   • A DNA profile is made up of the patterns of VNTR and STR.
   • Minisatellites are called VNTRS. These are 20-50 base pairs long. In every intron. They are repeated 50 to several 100 times in.
   • Microsatellites are called STRs. These are 2-4 base pairs long and are repeated 5-15 times.
   • Satellites appear in the same places on chromosomes. The number of repeats vary between individuals. Different lengths are inherited from both parents. An image of these patterns is known as a DNA profile.
   • How it works: DNA is cut with restriction enzymes. The fragments are separated by gel electrophoresis. Cut at defined points in introns so leave some repeats and satellites intact. Then the fragments are stained to reveal a pattern.
   • Used for forensics and paternity tests.

4. Genetic Engineering

   • 4 stages: obtaining the gene, gene placed in a vector, vector carries a gene into the cell and the cell is expressed in the novel gene.
   • Obtaining the gene: mRNA obtained from the cells where the gene is expressed. Reverse transcriptase forms a single strand of cDNA from mRNA.
   • Gene placed in a vector: Plasmids are mixed with restriction enzymes. The plasmids are cut at specific sites by the enzymes. This leaves exposed sticky ends. Free complementary nucleotides are added. DNA ligase then anneals the gene. Or the gene is sealed in a weakened virus to carry the DNA into the cell.
   • Getting the vector into the recipient cell: Heat shock, electrofusion, electroporation and T1 plasmids.
   • An example is golden rice. It is a precursor for vitamin A.

5. Gene Technology and Ethics

6. Genomes

   • It is an organisms complete set of DNA. This includes all of its genes.

7. DNA

   • Made up of two antiparallel chains.
   • A nucleotide is the monomer of a polynucleotide which is one of the chains. This consists of a pentose sugar, phosphate group and a nitrogenous base.
   • Two condensation reactions are required to produce a polynucleotide.