DNA
Fingerprint
DNA Fingerprint
• technique to identify people based on DNA
Gel Electrophoresis
• technique to separate fragments by size
+
sample well
gel
Tandem Repeats
• sequences of DNA that repeat
Short Tandem Repeats (STR) - 1,2,3 bases repeat
15-100 times
GACGACGACGACGAC
Variable Number of Tandem Repeats (VNTR) - 10-40
bases repeat 10’s to 1000’s
GACTTACAGCGACTTACAGCGACTTACAGC
e.g. DS180 (16 long, 16-40x)
Each Individual has 2 Alleles
Person A
Chromosome 1a GACGACGACGACGAC
Chromosome 1b
GACGACGAC
Person B
Chromosome 1a
GACGACGACGACGACGAC
Chromosome 1b
GACGACGACGAC
RFLP Analysis
Restriction Fragment Length Polymorphism
GACGACGACGACGAC
CTGCTGCTGCTGCTG
GACGACGAC
CTGCTGCTG
restriction
enzyme
RFLP Analysis
Restriction Fragment Length Polymorphism
Southern Blot transfer to nylon
sheet to make
single stranded
Add ProbeDNA complementary
to fragment
CTGCTG**
radioactive
Expose with X-ray film
PCR
• amplify TR region with PCR
• stain directly
GACGACGACGACGAC
CTGCTGCTGCTGCTG
GACGACGAC
CTGCTGCTG
Polymerase Chain Reaction (PCR)
• Heat DNA to open
• Primers attach and Taq polymerase extends
GGTTCCGACCGACCGACCGACCGACCGAGGTT
CCAA
CCAAGGCTGGCTGGCTGGCTGGCTGGCTCCAA
GGCTGGCTGGCTGGCTGGCTGGCTCCAA
GGTTCCGACCGACCGACCGACCGACCGAGGTT
CCAAGGCTGGCTGGCTGGCTGGCTGGCTCCAA
PCR Fingerprint
Gel
Electrophoresis
Stain
Ethidium
Bromide
PCR Fingerprint
Gel
Electrophoresis
Stain
Ethidium
Bromide
Lab 13
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Add loading dye and warm
Put in gel wells - 25 microliters
Add standard and control if available
Run gel for about 1 hour
Dry lab - Paternity suit
Dry lab - Criminal Case
Stain and photograph gel
Activity 21
• No class on Weds. or Fri.
• Obtain photographs of gels from website
• Complete activity 21 for Monday
Ideal Results
Ladder
proteins of known size
Molecular
Weight
Students' PCR products should show one or two bands with lengths be- tween 360 and 800 base pairs.
Activity 21
Ladder
proteins of known size
Molecular
Weight
Activity 21
Measure
Distances
for Ladder
e.g. 200 - 40 mm
400 - 35 mm
(do all!)
Activity 21
Ladder
proteins of known size
40
35
Molecular
Weight
Graph Ladder
50
45
Distance
Moved
(mm)
Distance Moved (mm)
40
35
30
25
20
15
10
5
0
0
120
240
360
480
600
720
840
Number of Base Pairs
Molecular Weight 1000’s
960
1080
1200
Draw Best Line
50
45
Distance
Moved
(mm)
Distance Moved (mm)
40
35
30
25
20
15
10
5
0
0
120
240
360
480
600
720
840
Number of Base Pairs
Molecular Weight 1000’s
960
1080
1200
Measure Sample Lines
e.g. 36 mm
25 mm
Activity 21
Me
3
Ladder
proteins of known size
40
35
36
25
Molecular
Weight
Determine Base Pairs
50
45
Distance
Moved
(mm)
Distance Moved (mm)
40
35
36
30
25
25
20
15
10
5
0
650
360
0
120
240
360
480
600
720
840
Number of Base Pairs
Molecular Weight 1000’s
960
1080
1200
Activity 21
Me
3
Ladder
proteins of known size
40
35
36
25
Molecular
Weight
360
650