Agarose Gel Electrophoresis

1
Agarose Gel Electrophoresis

• Size Determination of Restriction Fragments

2
DNA Fingerprinting

• High-profile applications
• Low-profile applications
• Basic concepts of nucleic acid research

3
High-profile Applications

• Forensics (CSI) Can you find a match?
• Crime Scenes
• Paternity
• Human Remains
• Genome Projects
• Biological Weapons

4
Low-profile Applications

• Can you find similarities?
• Organ Transplants
• Anthropology and Human Relatedness
• Species Relatedness
• Food Identification

5
Important Characteristics of DNA Structure

• Nucleus command
  center
• Chromosomes
• Double-stranded
• DNA helix
• Encoded information in the form of bases
  genetic code

6
Important Characteristics of DNA Structure

• Genetic code
• Guanine
• Adenine
• Cytosine
• Thymine
• Phosphate-Sugar
• backbone
• NOTE
• Phosphate groups
• are negatively
• charged

7
Agarose (Horizontal) Gel Electrophoresis

• Malasian word
• for seaweed is
• agar-agar.
• Agarose is derived from red seaweed.
• Electrophoresis
• means carrying with electricity.

8
Agarose Gels

• When agarose is melted and then cooled it
  contains pores that can act like a sieve.
• Increasing agarose concentration decreases pore
  size.

• Increasing agarose concentration limits the size
  of molecules that can fit through the pores
  limits the size range of molecules that can be
  separated.

9
Agarose Electrophoresis is a DRAG!

• Negatively charged DNA is pulled through the
  agarose, from the cathode (-) to the anode ().
• The larger the DNA fragment, the slower it
  travels through the gel due to frictional drag.
  This relationship is size dependent (not sequence
  dependent).

gel electrophoresis buffer
10
Visualizing Genetic Differences Between
Individuals

• Position of bands on a DNA agarose gel is based
  on size (not DNA sequence contained within the
  fragment.)
• One band represents millions of DNA fragments !

(-)
()
11
Standards-Based Size Determination
SIZE 10,000 bp 8,000 bp 6,000 bp 4,000 bp 2,000
bp 1,000 bp 500 bp
DISTANCE ? mm ? ? ? ? ? ?
10,000 bp --
8,000 bp --
6,000 bp --
4,000 bp --
2,000 bp --
1,000 bp --
Produce a standard curve
500 bp --
12
Frequent Misconceptions

• Each band on the gel represents a single DNA
  strand...NOT TRUE.
• A single band/position in a lane contains only
  one type of DNA
• sequence...NOT TRUE.
• If one enzyme produces matching bands between a
  suspect and the
• crime scene, the suspect is guilty ! No
  doubt about it !
• .NOT TRUE.