DNA

FINGERPRINTING
INTRODUCTION
DNA typing, DNA profiling
A process which uses DNA to show
relatedness or identity of individual
humans, plants, animals
Used in forensics, anthropology and
conservation biology
Teaching methods in molecular
biology, conservation biology and
biotechnology
Application of
DNA fingerprinting
Food identification
Identifying human remains
Determining relatedness of humans
Studying relatedness among ancient peoples
DNA testing of families
Identifying organisms that cause disease
Identifying birth parents (paternity testing)
Proving paternity
Determining effectiveness of bone marrow
transplants
Proving relatedness of immigrants
Confirming relatedness among animals
Method
May involve PCR analysis
RFLP (Restriction Fragment Length
Polymorphism) analysis
Compare band patterns produced by
cleavage of DNA samples when separated
on an agarose gel electrophoresis
Require: restriction enzymes and agarose
gel electrophoresis
1 sample from the crime scene and 5
samples from suspects in the case.


Restriction Enzymes
Molecular scissors: making cuts at specific
sequence of base pairs that is recognizes
Endonuclease restriction site
Bacterial enzymes: a natural defense
against bacteriophages a very primitive
immune system
Restriction site: specific palindromic
sequences of base pairs (e.g. GAATTC)
Palindrome
Restriction Enzymes
If a specific restriction site more than one
multiple fragments
Some restriction enzymes may leave a
short length of unpaired nucleotide bases,
called a sticky end at the DNA site where
they cut
Other restriction enzymes make a cut
across both strands creating double
stranded DNA fragments with blunt ends
The length of each fragment will depend
upon the location of restriction sites on
the DNA molecule
Restriction Enzymes
A sequence on one strand of DNA and its
complementary sequence on the other strand can
form a palindrom, i.e. GAATTC
CTTAAG
Palindromes can be detected by restriction enzymes
Restriction enzymes cut the palindromes at
restriction sites
Restriction enzymes recognize specific palindromes
Cutting DNA at restriction sites will produce DNA
fragments
Fragment size can be described by the number of
base pairs a fragment contains
Agarose Gel Electrophoresis
Separate DNA fragments based on their sizes.
DNA is an acid an has many negative electrical charges.
DNA fragments are loaded into an agarose gel slab, which
is placed into a chamber filled with a conductive buffer
solution.
A direct current is passed between wire electrodes at each
end of the chamber.
DNA fragments will be drawn toward the positive pole
(anode) when placed in an electric field.
Matrix of agarose gel acts as a molecular sieve through
which smaller DNA fragments can move more easily than
larger one.
The fragments will be visualized as bands in the gel after
the DNA is stained
Agarose Gel Electrophoresis
Loading Dye / Tracking Dye: BPB
(Brom Phenol Blue) and XC (Xylene
Cyanol)
TAE: Tris Acid EDTA buffer
DNA Staining: Ethidium Bromide or
Fast Blast DNA Stain
Quantitative Analysis of
DNA Fragment Sizes
Semilog Graph Paper linear Curve
Measure the distance (in mm) of each
DNA fragments / bands traveled from the
well: from the bottom of the well to the
center of each DNA band
Standard curve: DNA Marker (HindIII
lambda digest)
X axis: distance (mm)
Y axis: Size (base pairs)
Compare the fragment sizes of the
suspects and the crime scene