Académique Documents
Professionnel Documents
Culture Documents
• Crime scene
• Human relatedness
• Paternity
• Animal relatedness
• Anthropology studies
• Disease-causing organisms
• Food identification
• Human remains
• Monitoring transplants
Stages of DNA Fingerprinting
• Stage 1:
Cells are broken down
to release DNA
• Evolved by bacteria
to protect against
viral DNA infection
• Endonucleases =
cleave within DNA
strands
• Over 3,000 known
enzymes
Restriction site
Enzyme Site
Palindrome
Recognition
Fragment 1 Fragment 2
The DNA Digestion Restriction Buffer provides
Reaction optimal conditions
Ethidium bromide
Ethidum bromide inhibits DNA polymerase and binds in vitro to both RNA and DNA. It
binds by a mechanism termed intercalation. Ethidium bromide intercalates into a
portion of a DNA double helix and fluoresens in UV light.
Visualization DNA Fragments by Thiazine dye
Thiazine Dye:
• Group of basic dyes whose molecules contain
the thiazine heterocycle
• Among the thiazine dyes, methylene blue is of
the greatest industrial significance.
• Examples: Azure A, Azzure B, methylene blue
Fast Blast:
- Contain cation compound
- Thiazine family of dyes
- The positively charged dye molecules are attracted to and bind to the
negatively charged phosphate groups on DNA molecules
DNA Fingerprinting Procedures
Analysis of Stained Gel
Determine
restriction fragment
sizes
• Create standard
curve using DNA
marker
• Measure distance
traveled by
restriction fragments
• Determine size of
DNA fragments
Electrophoresis of
restriction fragments
M: Marker
A-1: Allele 1 Fragments
A-2: Allele 2 Fragments
+
Fingerprinting Standard Curve: Semi-log
100,000
Molecular Weight
Determination
10,000
Size (bp) Distance (mm)
9,400 13.0
1,000
6,500 15.0
4,400 18.0
2,300 23.0
100
A
2,000 24.0 0 5 10 15 20 25 30
Distance, mm
Bands Distance
MARKER CS S1 S2 S3 S4 S5
Distan
Distanc Distanc Size Distanc Size Distanc Size Distanc Size Distanc Size Size
Size (bp) ce
e (mm) e (mm) (bp) e (mm) (bp) e (mm) (bp) e (mm) (bp) e (mm) (bp) (bp)
(mm)
11,0 23.130 19.0 …… 21.0 …… 21.0 ….. 19.0 …… 21.0 …... 21.0 ……
13,0 9.416 20.5 …… 23.5 …… 25.0 ….. 20.5 …… 29.5 …... 24.0 …..
18,0 4.361
23,0 2.322
24,0 2.027
Fast Blast Staining
Marker CS S1 S2 S3 S4 S5
Does not
need UV
light to
visualize
Ethidium Bromida Staining
Marker CS S1 S2 S3 S4 S5
Have to
use UV
light to
visualize