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?