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PCR Applications

PCR Applications

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PCR Applications

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  1. PCR Applications

  2. The polymerase chain reaction (PCR) is a technique to amplify a piece of DNA very rapidly outside of a cell.

  3. Some applications of PCR. • Forensic medicine. • Preimplantation Genetic Diagnosis (PGD). • Archeology. • Paternity testing.

  4. The polymerase chain reaction (PCR).

  5. A cycle of PCR consists of three steps. • DNA denaturation at 95 degrees C. • Primer annealing at 50-60 degrees C. • DNA polymerization by a thermostable DNA polymerase at 72 degrees C.

  6. Starting with a single molecule of DNA, 25 rounds or cycles of PCR will produce about 10 million identical DNA molecules!!

  7. Forensic uses of PCR • PCR can be used to amplify DNA from a small amount of cells (about 1000 cells). • The amplified DNA from cells can be used in DNA fingerprinting analysis to determine who was at the crime scene.

  8. DNA fingerprinting using PCR in forensic investigations. • DNA is isolated from blood at a crime scene and amplified by PCR. • The amplified DNA is digested with restriction enzymes and resolved on an agarose gel. • Southern blot analysis is performed to give a DNA fingerprint.

  9. Restriction fragment analysis by Southern blotting.

  10. DNA fingerprints from a murder case.

  11. Individuals have unique DNA fingerprints because of restriction length polymorphisms (RFLPs).

  12. How reliable is DNA fingerprinting? • DNA regions chosen are ones known to be highly variable from one person to another. • In most forensic cases, the probability of two people having identical DNA fingerprints is between one chance in 100,000 and one in a billion. • The exact number depends on the number of probes used to different regions of human chromosomal DNA.

  13. Many argue that DNA evidence is more reliable than eyewitnesses in placing a suspect at the scene of a crime.

  14. Satellite DNA can be used as markers for DNA fingerprinting. • Satellite DNA consists of tandemly repeated base sequences within the human genome. • The most useful satellite DNA for forensic purposes are microsatellites having repeating units of only a few base pairs, and the number of repeats are highly variable from one person to another. • Microsatellite DNA is also called a simple tandem repeats (STRs).

  15. An example of simple tandem repeat (STR) alleles.

  16. STRs in DNA fingerprinting. • The greater the number of STRs analyzed in a DNA sample, the more likely the DNA fingerprint is unique to an individual. • PCR is used to selectively amplify particular STRs before electrophoresis. • PCR is especially valuable when DNA is in poor condition or available in minute quantities.

  17. PCR use in Pre-implantation Genetic Diagnosis (PGD). • PGD is a way to determine if human embryos from in vitro fertilization have genetic defects (for example, cystic fibrosis). • A cell is removed from an eight cell embryo and the DNA is analyzed by PCR for genetic defects. • Only healthy embryos are implanted into a mother’s uterus. • Should this technology be used for things like gender selection?