Methods of DNA analysis

Southern Blotting: Gel Transfer

 Gel-transfer hybridization—or Southern
blotting—is used to detect specific DNA
fragments. (A) The mixture of double-stranded
DNA fragments generated by restriction nuclease
treatment of DNA is separated according to length
by electrophoresis
 A sheet of either nitrocellulose paper or nylon
paper is laid over the gel, and the separated
DNA fragments are transferred to the sheet by
blotting. The gel is supported on a layer of
sponge in a bath of alkali solution, and the
buffer is sucked through the gel and the
nitrocellulose paper by paper towels stacked
on top of the nitrocellulose.
 As the buffer is sucked through, it denatures
the DNA and transfers the single-stranded
fragments from the gel to the surface of the
nitrocellulose sheet, where they adhere firmly.
 This transfer is necessary to keep the DNA
firmly in place while the hybridization
procedure (D) is carried out. (C) The
nitrocellulose sheet is carefully peeled off the
gel. (D)
 The sheet containing the bound single-
stranded DNA fragments is placed in a sealed
plastic bag together with buffer containing a
radioactively labeled DNA probe specific for
the required DNA sequence
 The nitrocellulose sheet is carefully peeled off
the gel. (D) The sheet containing the bound
single-stranded DNA fragments is placed in a
sealed plastic bag together with buffer
containing a radioactively labeled DNA probe
specific for the required DNA sequence.
 The sheet is exposed for a prolonged period to
the probe under conditions favoring
hybridization. (E) The sheet is removed from
the bag and washed thoroughly, so that only
probe molecules that have hybridized to the
DNA on the paper remain attached.
 After autoradiography, the DNA that has
hybridized to the labeled probe will show up
as bands on the autoradiograph. An adaptation
of this technique to detect specific sequences
in RNA is called Northern blotting.
 In this case mRNA molecules are
electrophoresed through the gel and the probe
is usually a single-stranded DNA molecule
DNA fingerprinting
 Like the fingerprints that came into use by detectives
and police labs during the 1930s, each person has a
unique DNA fingerprint. Unlike a conventional
fingerprint that occurs only on the fingertips and can
be altered by surgery, a DNA fingerprint is the same
for every cell, tissue, and organ of a person. It cannot
be altered by any known treatment. Consequently,
DNA fingerprinting is rapidly becoming the primary
method for identifying and distinguishing among
individual human beings.
 DNA fingerprinting is a laboratory procedure
that requires six steps
 Isolation of DNA.
 DNA must be recovered from the cells or
tissues of the body. Only a small amount of
tissue, like blood, hair, or skin, is needed. For
example, the amount of DNA found at the root
of one hair is usually sufficient.
 Cutting, sizing, and sorting. Special
enzymes called restriction enzymes are used
to cut the DNA at specific places. For
example, an enzyme called EcoR1, found in
bacteria, will cut DNA only when the
sequence GAATTC occurs.
 The DNA pieces are sorted according to size
by a sieving technique called electrophoresis.
The DNA pieces are passed through a gel
made from seaweed agarose (a jelly-like
product made from seaweed). This technique
is the DNA equivalent of screening sand
through progressively finer mesh screens to
determine particle sizes
3) Transfer of DNA to nylon. The
distribution of DNA pieces is transferred to a
nylon sheet by placing the sheet on the gel and
soaking them overnight.
 4-5) Probing. Adding radioactive or colored
probes to the nylon sheet produces a pattern
called the DNA fingerprint. Each probe
typically sticks in only one or two specific
places on the nylon sheet.
6) DNA fingerprint. The final DNA
fingerprint is built by using several probes (5-
10 or more) simultaneously. It resembles the
bar codes used by grocery store scanners.
 Uses of DNA Fingerprints
 DNA fingerprints are useful in several areas of
society. They are used by professionals in
human health and the justice system.
Diagnosis of inherited disorders
 DNA fingerprinting is used to diagnose
inherited disorders in both prenatal and
newborn babies in hospitals around the world.
These disorders may include cystic fibrosis,
hemophilia, Huntington's disease, familial
Alzheimer's, sickle cell anemia, thalassemia,
and many others.
 Early detection of such disorders enables the
medical staff to prepare themselves and the
parents for proper treatment of the child. In
some programs, genetic counselors use DNA
fingerprint information to help prospective
parents understand the risk of having an
affected child. In other programs, prospective
parents use DNA fingerprint information in
their decisions concerning affected
pregnancies
 Developing cures for inherited
disorders
 Research programs to locate inherited disorders on
the chromosomes depend on the information
contained in DNA fingerprints. By studying the DNA
fingerprints of relatives who have a history of some
particular disorder, or by comparing large groups of
people with and without the disorder, it is possible to
identify DNA patterns associated with the disease in
question. This work is a necessary first step in
designing an eventual genetic cure for these
disorders.
 Forensic or criminal
 FBI and police labs around the U.S. have
begun to use DNA fingerprints to link suspects
to biological evidence-blood or semen stains,
hair, or items of clothing-found at the scene of
a crime. Since 1987, more than 150 cases have
been decided with the assistance of DNA
fingerprint evidence.
 Another important use of DNA fingerprints in
the court system is to establish paternity in
custody and child support litigation. In these
applications, DNA fingerprints bring an
unprecedented, nearly perfect accuracy to the
determination
 Personal identification
 Because every organ or tissue of an individual
contains the same DNA fingerprint, the U.S. armed
services have just begun a program to collect DNA
fingerprints from all personnel for use later, in case
they are needed to identify casualties or persons
missing in action. The DNA method will be far
superior to the dogtags, dental records, and blood
typing strategies currently in use.
 Dermatoglyphics
 Dermatoglyphics (from ancient Greek derma
= "skin", glyph = "carving") is the scientific
study of fingerprints. The term was coined by
Dr. Harold Cummins, the father of American
fingerprint analysis, even though the process
of fingerprint identification had already been
used for several hundred years
 All primates have ridged skin, and it can also
be found on the paws of certain mammals and
on the tails of some monkey species. In
humans and animals, dermatoglyphs are
present on fingers, palms, toes, and soles, and
give insight into a critical period of
embryogenesis, between 4 weeks and 5
months, when the architecture of the major
organ systems is developing.
 Triradius: point of convergence of ridges from
3 different directions. Normally, there is:
 1 axial triradius: normally in t, close to the
wrist.
 4 subdigital triradii (a.b.c.d.).
 On the pad of the distal phalanx, sometimes on
thenar or hypothenar eminences, are triradii,
accompanied with the following patterns:
 worl: 2 triradii.
 loops and equivalents (ulnar or radial orientated): 1
triradius.
 arches: 0 triradius.
 From each palmar triradius a, b, c, d, and t, is
drawn the 3 lines separating the ridges at this
convergence point. The longest is the main
line (-- A B C D & T), ending at a side of the
palm numbered from 1 to 14
 T normally ends in 13.
 transversality index = A+B+C+D = 27 on the
Figure
 Genetic disorders
 Unusual dermatoglyphic patterns often relate
to genetic disorders
 One study of foetuses with chromosomal
abnormalities showed that the dermatoglyphic
patterns were delayed by more than two weeks
 Trisomy 21 (Down syndrome): People with Down
syndrome have mainly ulnar loops, and a
significantly different angle between the triradia a, t
and d (the 'adt angle').
 Other differences often include a
single transverse palmar crease ("Simian line")
(in 50%), and patterns in the hypothenar and
interdigital areas, lower ridge counts along
digital midlines, especially in little fingers,
which corresponds to finger shortening in those
with Down's syndrome
 There is less variation in dermatoglyphic
patterns between people with Down syndrome
than between controls, and dermatoglyphic
patterns can be used to determine correlations
with congenital heart defects in individuals
with Down syndrome by examining the left
hand digit ridge count minus the right hand
digit ridge count, and the number of ridges on
the fifth digit of the left hand
 Turner syndrome: Predominance of whorls,
although the pattern frequency depends on the
particular chromosomal abnormality
 47, XXY (Klinefelter's syndrome): Excess of
arches on digit 1, more frequent ulnar loops on
digit 2, overall fewer whorls, lower ridge
counts for loops and whorls as compared with
controls, and significant reduction of the total
finger ridge count
• Trisomy 13 (Patau syndrome): Excess of
arches on fingertips and
single transverse palmar creases in 60%.
• Trisomy 18 (Edward's syndrome) 6 - 10 arches
on fingertips and
single transverse palmar creases in 30%.
 Cri du chat (5p-): Excess of arches on
fingertips and single transverse palmar creases
in 90%.
 Inborn blindness : Dermatoglyphic analysis of
finger tip print patterns of blind children from
Bangalore