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Forensic DNA Fingerprinting:

Using Restriction Enzymes


DNA Fingerprinting
• A technique used by scientists to distinguish
between individuals of the same species using
only samples of their DNA

• It is also termed DNA profiling


DNA Fingerprinting
Real World Applications

•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

If only a small amount of DNA


is available it can be
amplified using the
polymerase chain reaction
(PCR)
• In 1984, Sir Alec Jeffreys
(University of Leicester in England)
was able to distinguish differences
among individuals based solely on
their DNA composition.
• Highly polymorphic minisatelite
loci
• Then tremendous progress made
in the methodology of extracting
the DNA samples from such things
as blood, saliva, personal items
and in the identification of human
remains.
• In 1999, law enforcement agencies in both Great Britain and the
United States began switching to a new version of RFLP analysis
using shorter sequences called STRs ("Short Tandem Repeats").

• STRs are repeated sequences of a few (usually four) nucleotides,


e.g., TCATTCATTCATTCAT. They often occur in the untranslated parts
of known genes (whose sequence can be used for the PCR primers).

• The exact number of repeats (6, 7, 8, 9, etc.) varies in different


people (and, often, in the gene on each chromosome; that is,
people are often heterozygous for the marker).

• In the U.S., where 13 STRs — scattered over different chromosomes


— are examined, the chance that two people have the same
pattern is less than 1 in 1 trillion.
Stages of DNA Fingerprinting
• Step 2:
The DNA is cut into fragments using restriction enzymes.

Each restriction enzyme cuts DNA at a specific base sequence.


DNA restriction
enzyme

• 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

• Each enzyme digests


(cuts) DNA at a
specific sequence =
restriction site
• Enzymes recognize
4- or 6- base pair,
palindromic
sequences
(eg GAATTC)

Fragment 1 Fragment 2
The DNA Digestion Restriction Buffer provides
Reaction optimal conditions

• NaCI provides the correct ionic


strength

• Tris-HCI provides the proper pH

• Mg is an enzyme co-factor
2+
Stages of DNA Fingerprinting
Stage 3:
• Fragments are
separated on the basis
of size using a process
called gel
electrophoresis.
• DNA fragments are
injected into wells and
an electric current is
applied along the gel.
Stages of DNA Fingerprinting
DNA is negatively charged
so it is attracted to the
positive end of the gel.
The shorter DNA
fragments move faster
than the longer
fragments.
DNA is separated on basis
of size.
Stages of DNA Fingerprinting
Stage 4:
• The pattern of fragment distribution is then
analysed.
Visualization DNA Fragments by Ethidium bromide

C
ENI
NOG Ethidium bromide
RCI
CA

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 o
f 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

• Identify the related


samples
PstI EcoRI
CTGCAG GAATTC
Allele 1 GAGCTC GTTAAC
1 2 3
Restriction
CGGCAG GAATTC
Fragment Length Allele 2 GCGCTC GTTAAC
3
Polymorphism Different
Fragment 1+2
Base Pairs
(RFLP) No restriction site
M A-1 A-2

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)

Size, base pairs


23,000 11.0 B

9,400 13.0
1,000
6,500 15.0

4,400 18.0

2,300 23.0
100
A
0 5 10 15 20 25 30
2,000 24.0
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 …..

15,0 6.557 32.0 …… 30.5 …… 28.5 ….. 32.0 …… 29.5 ……

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
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