A cycle of PCR consists of three steps. <ul><li>DNA denaturation at 95 degrees C. </li></ul><ul><li>Primer annealing at 50-60 degrees C. </li></ul><ul><li>DNA polymerization by a thermostable DNA polymerase at 72 degrees C. </li></ul>
Starting with a single molecule of DNA, 25 rounds or cycles of PCR will produce about 10 million identical DNA molecules!!
Forensic uses of PCR <ul><li>PCR can be used to amplify DNA from a small amount of cells (about 1000 cells). </li></ul><ul><li>The amplified DNA from cells can be used in DNA fingerprinting analysis to determine who was at the crime scene. </li></ul>
DNA fingerprinting using PCR in forensic investigations. <ul><li>DNA is isolated from blood at a crime scene and amplified by PCR. </li></ul><ul><li>The amplified DNA is digested with restriction enzymes and resolved on an agarose gel. </li></ul><ul><li>Southern blot analysis is performed to give a DNA fingerprint. </li></ul>
Restriction fragment analysis by Southern blotting.
Individuals have unique DNA fingerprints because of restriction length polymorphisms (RFLPs).
How reliable is DNA fingerprinting? <ul><li>DNA regions chosen are ones known to be highly variable from one person to another. </li></ul><ul><li>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. </li></ul><ul><li>The exact number depends on the number of probes used to different regions of human chromosomal DNA. </li></ul>
Many argue that DNA evidence is more reliable than eyewitnesses in placing a suspect at the scene of a crime.
Satellite DNA can be used as markers for DNA fingerprinting. <ul><li>Satellite DNA consists of tandemly repeated base sequences within the human genome. </li></ul><ul><li>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. </li></ul><ul><li>Microsatellite DNA is also called a simple tandem repeats (STRs). </li></ul>
An example of simple tandem repeat (STR) alleles.
STRs in DNA fingerprinting. <ul><li>The greater the number of STRs analyzed in a DNA sample, the more likely the DNA fingerprint is unique to an individual. </li></ul><ul><li>PCR is used to selectively amplify particular STRs before electrophoresis. </li></ul><ul><li>PCR is especially valuable when DNA is in poor condition or available in minute quantities. </li></ul>
PCR use in Pre-implantation Genetic Diagnosis (PGD). <ul><li>PGD is a way to determine if human embryos from in vitro fertilization have genetic defects (for example, cystic fibrosis). </li></ul><ul><li>A cell is removed from an eight cell embryo and the DNA is analyzed by PCR for genetic defects. </li></ul><ul><li>Only healthy embryos are implanted into a mother’s uterus. </li></ul><ul><li>Should this technology be used for things like gender selection? </li></ul>
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