Technique, called the Polymerase Chain Reaction, (PCR) can also make large amounts of a desired gene or piece of DNA. PCR can be done without bacteria, inside test tubes, and can amplify billions of times samples with very little DNA (e.g. a single hair from a crime scene, or inside some fossils).
· PCR makes huge amounts of any gene, quickly.
1. heat the DNA to about 93ºC, which unwinds the DNA and separates the two strands.
2. Add some replication primers, and allow to cool.
3. Add heat resistant DNA polymerase (the replication enzyme) and free nucleotides. The DNA will copy itself.
4. Heat and repeat. The DNA will go on doubling itself each “generation”.
· After PCR has been performed, the sequence of bases on DNA can be determined (e.g. using the “Sanger Method”). Sequencing bases is useful for:
· study evolutionary relationships between organisms (e.g. humans and chimpanzees), and trace the origin of human races.
· map every single nucleotide on all human chromosomes (“the Human Genome Project”)
· PCR amplified DNA can be analyzed using a DNA probe (a specially synthesized single strand of radioactive DNA nucleotides) that will bind to a complementary DNA strand on the DNA being tested. This can be used to
detect viral infections, diagnose genetic disorders, and diagnose some cancers.
· A quick way of comparing the DNA from two different organisms is by using RFLP ANALYSIS. (Restriction Fragment Length Polymorphisms). This can provide a “DNA fingerprint” that is unique to each individual (except identical twins). RFLP analysis uses specific restriction enzymes that cut DNA at specific sequences. This produces fragments that, when separated using gel electrophoresis, produce patterns of bands that can be compared to another person’s pattern of bands.
· If the band pattern is identical, the DNA must have come from the same person.
· This can be used to identify a criminal from a blood or semen stain. It can also determine who the father of a child is, with a high degree of accuracy.
· RFLP analysis is also used to see whether a person carries a gene for a genetic disorder like cystic fibrosis or sickle-cell anemia, and can be used for prenatal diagnosis.
· RFLP analysis also contributes to our knowledge of evolution and evolutionary relationships by comparing human and animal DNA.