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