Polymerase Chain Reaction - PCR

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

Some applications of PCR.

Forensic medicine.
Preimplantation Genetic Diagnosis

(PGD). Archeology. Paternity testing.

The polymerase chain reaction (PCR).

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.

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

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.

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.

Restriction fragment analysis by Southern blotting.

DNA fingerprints from a murder case.

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

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.

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.

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).

An example of simple tandem repeat (STR) alleles.

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.

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 mothers uterus. Should this technology be used for things like gender selection?