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Methods of DNA analysis Methods of DNA analysis
Methods of DNA analysis
Methods of DNA analysis
Southern Blotting: Gel Transfer Southern Blotting: Gel Transfer
Southern Blotting: Gel Transfer
Southern Blotting: Gel Transfer
Gel-transfer Gel-transfer hybridization—or hybridization—or Southern Southern blotting—is used to detect specific DNA blotting—is used to detect
Gel-transfer
Gel-transfer hybridization—or
hybridization—or Southern
Southern
blotting—is used to detect specific DNA
blotting—is used to detect specific DNA
fragments
fragments. (A) The mixture of double-stranded
. (A) The mixture of double-stranded
DNA fragments generated by restriction nuclease
DNA fragments generated by restriction nuclease
treatment of DNA is separated according to length
treatment of DNA is separated according to length
byby electrophoresis
electrophoresis
 AA sheet sheet of of either either nitrocellulose nitrocellulose paper paper or or nylon nylon
 AA sheet
sheet of
of either
either nitrocellulose
nitrocellulose paper
paper or
or nylon
nylon
paper is laid over the gel, and the separated
paper is laid over the gel, and the separated
DNA DNA fragments fragments are are transferred transferred toto the the sheet sheet byby
blotting. blotting. The The gel gel isis supported supported onon aa layer layer of of
sponge in a bath of alkali solution, and the
sponge in a bath of alkali solution, and the
buffer is sucked through the gel and the
buffer is sucked through the gel and the
nitrocellulose paper by paper towels stacked
nitrocellulose paper by paper towels stacked
onon top top of of the the nitrocellulose. nitrocellulose.
 As As the the buffer buffer isis sucked sucked through, through, itit denatures denatures the
 As
As the
the buffer
buffer isis sucked
sucked through,
through, itit denatures
denatures
the DNA and transfers the single-stranded
the DNA and transfers the single-stranded
fragments fragments from from the the gel gel toto the the surface surface of of the the
nitrocellulose sheet, where they adhere firmly.
nitrocellulose sheet, where they adhere firmly.
 This transfer is necessary to keep the DNA This transfer is necessary to keep the
This transfer is necessary to keep the DNA
This transfer is necessary to keep the DNA
firmly in place while the hybridization
firmly in place while the hybridization
procedure (D) is carried out. (C) The
procedure (D) is carried out. (C) The
nitrocellulose sheet is carefully peeled off the
nitrocellulose sheet is carefully peeled off the
gel. gel. (D) (D)
 The sheet containing the bound single- The sheet containing the bound single- stranded DNA fragments
The sheet containing the bound single-
The sheet containing the bound single-
stranded DNA fragments is placed in a sealed
stranded DNA fragments is placed in a sealed
plastic plastic bag bag together together with with buffer buffer containing containing aa
radioactively labeled DNA probe specific for
radioactively labeled DNA probe specific for
the the required required DNA DNA sequence sequence
 The The nitrocellulose nitrocellulose sheet sheet isis carefully carefully peeled peeled off off the the
 The
The nitrocellulose
nitrocellulose sheet
sheet isis carefully
carefully peeled
peeled off
off
the the gel. gel. (D) (D) The The sheet sheet containing containing the the bound bound
single-stranded DNA fragments is placed in a
single-stranded DNA fragments is placed in a
sealed plastic bag together with buffer
sealed plastic bag together with buffer
containing a radioactively labeled DNA probe
containing a radioactively labeled DNA probe
specific for the required DNA sequence.
specific for the required DNA sequence.
 The The sheet sheet isis exposed exposed for for aa prolonged prolonged period period toto
 The
The sheet
sheet isis exposed
exposed for
for aa prolonged
prolonged period
period toto
the probe under conditions favoring
the probe under conditions favoring
hybridization. (E) The sheet is removed from
hybridization. (E) The sheet is removed from
the the bag bag and and washed washed thoroughly, thoroughly, soso that that only only
probe molecules that have hybridized to the
probe molecules that have hybridized to the
DNA on the paper remain attached.
DNA on the paper remain attached.
 After autoradiography, the DNA that has After autoradiography, the DNA that has hybridized to the
After autoradiography, the DNA that has
After autoradiography, the DNA that has
hybridized to the labeled probe will show up
hybridized to the labeled probe will show up
asas bands bands onon the the autoradiograph. autoradiograph. An An adaptation adaptation
of this technique to detect specific sequences
of this technique to detect specific sequences
inin RNA RNA isis called called Northern Northern blotting blotting..
 In this case mRNA molecules are In this case mRNA molecules are electrophoresed through the
In this case mRNA molecules are
In this case mRNA molecules are
electrophoresed through the gel and the probe
electrophoresed through the gel and the probe
is usually a single-stranded DNA molecule
is usually a single-stranded DNA molecule
DNA DNA fingerprinting fingerprinting
DNA
DNA fingerprinting
fingerprinting
 Like Like the the fingerprints fingerprints that that came came into into use use byby
Like
Like the
the fingerprints
fingerprints that
that came
came into
into use
use byby detectives
detectives
and police labs during the 1930s, each person has a
and police labs during the 1930s, each person has a
unique DNA
unique DNA fingerprint
fingerprint..
Unlike
Unlike aa conventional
conventional
fingerprint that occurs only on the fingertips and can
fingerprint that occurs only on the fingertips and can
be altered by surgery, a
be altered by surgery, a DNA
DNA fingerprint
fingerprint is the same
is the same
for
for every cell, tissue, and organ of a person. It cannot
every cell, tissue, and organ of a person. It cannot
be altered by any known treatment. Consequently,
be altered by any known treatment. Consequently,
DNA fingerprinting is rapidly becoming the primary
DNA fingerprinting is rapidly becoming the primary
method for identifying and distinguishing among
method for identifying and distinguishing among
individual individual human human beings. beings.
 DNA fingerprinting is a laboratory procedure DNA fingerprinting is a laboratory procedure that requires six
DNA fingerprinting is a laboratory procedure
DNA fingerprinting is a laboratory procedure
that requires six steps
that requires six steps
 Isolation Isolation of of DNA. DNA.  DNA DNA must must bebe recovered recovered from
 Isolation
Isolation of
of DNA.
DNA.
 DNA
DNA must
must bebe recovered
recovered from
from the
the cells
cells or
or
tissues tissues of of the the body. body. Only Only aa small small amount amount ofof
tissue, like blood, hair, or skin, is needed. For
tissue, like blood, hair, or skin, is needed. For
example, the amount of DNA found at the root
example, the amount of DNA found at the root
of of one one hair hair isis usually usually sufficient. sufficient.
 Cutting, Cutting, sizing, sizing, and and sorting. sorting. Special Special enzymes enzymes called called restriction
 Cutting,
Cutting, sizing,
sizing, and
and sorting.
sorting. Special
Special
enzymes enzymes called called restriction restriction enzymes enzymes are are used used
toto cut cut the the DNA DNA at at specific specific places. places. For For
example, an enzyme called EcoR1, found in
example, an enzyme called EcoR1, found in
bacteria, will cut DNA only when the
bacteria, will cut DNA only when the
sequence sequence GAATTC GAATTC occurs. occurs.
 The The DNA DNA pieces pieces are are sorted sorted according according toto size size
 The
The DNA
DNA pieces
pieces are
are sorted
sorted according
according toto size
size
by a sieving technique called
by a sieving technique called electrophoresis
electrophoresis..
The The DNA DNA pieces pieces are are passed passed through through aa gel gel
made from seaweed agarose (a jelly-like
made from seaweed agarose (a jelly-like
product made from seaweed). This technique
product made from seaweed). This technique
is the DNA equivalent of screening sand
is the DNA equivalent of screening sand
through progressively finer mesh screens to
through progressively finer mesh screens to
determine determine particle particle sizes sizes
3) Transfer of DNA to nylon. 3) Transfer of DNA to nylon. The The distribution distribution
3) Transfer of DNA to nylon.
3) Transfer of DNA to nylon. The
The
distribution
distribution of
of DNA
DNA pieces
pieces isis transferred
transferred toto aa
nylon
nylon sheet
sheet byby placing
placing the
the sheet
sheet onon the
the gel
gel and
and
soaking
soaking them
them overnight.
overnight.
 4-5) 4-5) Probing. Probing. Adding Adding radioactive radioactive oror colored colored probes probes toto the
 4-5)
4-5) Probing.
Probing. Adding
Adding radioactive
radioactive oror colored
colored
probes probes toto the the nylon nylon sheet sheet produces produces aa pattern pattern
called the DNA fingerprint. Each probe
called the DNA fingerprint. Each probe
typically sticks in only one or two specific
typically sticks in only one or two specific
places on the nylon sheet.
places on the nylon sheet.
6) DNA fingerprint. The final DNA 6) DNA fingerprint. The final DNA fingerprint is built by
6) DNA fingerprint. The final DNA
6) DNA fingerprint. The final DNA
fingerprint is built by using several probes (5-
fingerprint is built by using several probes (5-
10 or more) simultaneously. It resembles the
10 or more) simultaneously. It resembles the
bar codes used by grocery store scanners.
bar codes used by grocery store scanners.
Uses Uses ofof DNA DNA Fingerprints Fingerprints  DNA fingerprints are useful in several areas of
Uses
Uses ofof DNA
DNA Fingerprints
Fingerprints
DNA fingerprints are useful in several areas of
DNA fingerprints are useful in several areas of
society. They are used by professionals in
society. They are used by professionals in
human health and the justice system.
human health and the justice system.
Diagnosis of inherited disorders Diagnosis of inherited disorders  DNA DNA fingerprinting is used to diagnose
Diagnosis of inherited disorders
Diagnosis of inherited disorders
 DNA
DNA fingerprinting is used to diagnose
fingerprinting is used to diagnose
inherited disorders in both prenatal and
inherited disorders in both prenatal and
newborn babies in hospitals around the world.
newborn babies in hospitals around the world.
These disorders may include cystic fibrosis,
These disorders may include cystic fibrosis,
hemophilia, Huntington's disease, familial
hemophilia, Huntington's disease, familial
Alzheimer's, sickle cell anemia, thalassemia,
Alzheimer's, sickle cell anemia, thalassemia,
and and many many others. others.
 Early detection of such disorders enables the Early detection of such disorders enables the medical
Early detection of such disorders enables the
Early detection of such disorders enables the
medical staff to prepare themselves and the
medical staff to prepare themselves and the
parents for proper treatment of the child. In
parents for proper treatment of the child. In
some programs, genetic counselors use DNA
some programs, genetic counselors use DNA
fingerprint information to help prospective
fingerprint information to help prospective
parents understand the risk of having an
parents understand the risk of having an
affected child. In other programs, prospective
affected child. In other programs, prospective
parents use DNA fingerprint information in
parents use DNA fingerprint information in
their decisions concerning affected
their decisions concerning affected
pregnancies pregnancies
Developing Developing cures cures for for inherited inherited disorders disorders  Research Research programs programs toto
Developing
Developing cures
cures for
for inherited
inherited
disorders
disorders
 Research
Research programs
programs toto locate
locate inherited
inherited disorders
disorders onon
the chromosomes depend on the information
the chromosomes depend on the information
contained in DNA fingerprints. By studying the DNA
contained in DNA fingerprints. By studying the DNA
fingerprints of relatives who have a history of some
fingerprints of relatives who have a history of some
particular disorder, or by comparing large groups of
particular disorder, or by comparing large groups of
people with and without the disorder, it is possible to
people with and without the disorder, it is possible to
identify DNA patterns associated with the disease in
identify DNA patterns associated with the disease in
question. This work is a necessary first step in
question. This work is a necessary first step in
designing an eventual genetic cure for these
designing an eventual genetic cure for these
disorders. disorders.
Forensic Forensic oror criminal criminal  FBI FBI and and police police labs labs around around
Forensic
Forensic oror criminal
criminal
 FBI FBI and and police police labs labs around around the the U.S. U.S. have have
begun to use DNA fingerprints to link suspects
begun to use DNA fingerprints to link suspects
toto biological biological evidence-blood evidence-blood or or semen semen stains, stains,
hair, or items of clothing-found at the scene of
hair, or items of clothing-found at the scene of
a crime. Since 1987, more than 150 cases have
a crime. Since 1987, more than 150 cases have
been decided with the assistance of DNA
been decided with the assistance of DNA
fingerprint fingerprint evidence. evidence.
 Another Another important important use use of of DNA DNA fingerprints fingerprints inin the the
 Another
Another important
important use
use of
of DNA
DNA fingerprints
fingerprints inin
the the court court system system isis toto establish establish paternity paternity inin
custody and child support litigation. In these
custody and child support litigation. In these
applications, DNA fingerprints bring an
applications, DNA fingerprints bring an
unprecedented, nearly perfect accuracy to the
unprecedented, nearly perfect accuracy to the
determination determination
Personal Personal identification identification  Because Because every every organ organ or or tissue tissue ofof
Personal
Personal identification
identification
 Because
Because every
every organ
organ or
or tissue
tissue ofof anan individual
individual
contains the same DNA fingerprint, the U.S. armed
contains the same DNA fingerprint, the U.S. armed
services have just begun a program to collect DNA
services have just begun a program to collect DNA
fingerprints from all personnel for use later, in case
fingerprints from all personnel for use later, in case
they are needed to identify casualties or persons
they are needed to identify casualties or persons
missing in action. The DNA method will be far
missing in action. The DNA method will be far
superior to the dogtags, dental records, and blood
superior to the dogtags, dental records, and blood
typing strategies currently in use.
typing strategies currently in use.
Dermatoglyphics Dermatoglyphics  Dermatoglyphics Dermatoglyphics (from (from ancient ancient Greek Greek derma derma == "skin", "skin",
Dermatoglyphics
Dermatoglyphics
 Dermatoglyphics
Dermatoglyphics (from
(from ancient
ancient Greek
Greek derma
derma
== "skin",
"skin", glyph
glyph = "carving") is the scientific
= "carving") is the scientific
study of
study of fingerprints
fingerprints. The term was coined by
. The term was coined by
Dr.
Dr. Harold
Harold Cummins
Cummins, the father of American
, the father of American
fingerprint analysis, even though the process
fingerprint analysis, even though the process
of fingerprint identification had already been
of fingerprint identification had already been
used for several hundred years
used for several hundred years
 All All primates have ridged skin, and it can also primates have ridged skin, and
 All
All primates have ridged skin, and it can also
primates have ridged skin, and it can also
be found on the paws of certain mammals and
be found on the paws of certain mammals and
onon the the tails tails of of some some monkey monkey species. species. InIn
humans humans and and animals, animals, dermatoglyphs dermatoglyphs are are
present on fingers, palms, toes, and soles, and
present on fingers, palms, toes, and soles, and
give insight into a critical period of
give insight into a critical period of
embryogenesis
embryogenesis, between 4 weeks and 5
, between 4 weeks and 5
months, when the architecture of the major
months, when the architecture of the major
organ organ systems systems isis developing. developing.
 Triradius: Triradius: point point of of convergence convergence of of ridges ridges from from 3
 Triradius:
Triradius: point
point of
of convergence
convergence of
of ridges
ridges from
from
3 different directions. Normally, there is:
3 different directions. Normally, there is:
1 axial triradius: normally in t, close to the
1 axial triradius: normally in t, close to the
wrist.
wrist.
4 subdigital triradii (a.b.c.d.).
4 subdigital triradii (a.b.c.d.).
 On On the the pad of the distal phalanx, sometimes on pad of the distal
 On
On the
the pad of the distal phalanx, sometimes on
pad of the distal phalanx, sometimes on
thenar or hypothenar eminences, are triradii,
thenar or hypothenar eminences, are triradii,
accompanied with the following patterns:
accompanied with the following patterns:
 worl:
worl: 22 triradii.
triradii.
loops and equivalents (ulnar or radial orientated): 1
loops and equivalents (ulnar or radial orientated): 1
triradius. triradius.
 arches:
arches: 00 triradius.
triradius.
 From each palmar triradius a, b, c, d, and t, is From each palmar triradius
From each palmar triradius a, b, c, d, and t, is
From each palmar triradius a, b, c, d, and t, is
drawn the 3 lines separating the ridges at this
drawn the 3 lines separating the ridges at this
convergence convergence point. point. The The longest longest isis the the main main
line (-- A B C D & T), ending at a side of the
line (-- A B C D & T), ending at a side of the
palm numbered from 1 to 14
palm numbered from 1 to 14
 TT normally normally ends ends inin 13. 13.  transversality transversality index index == A+B+C+D
 TT normally
normally ends
ends inin 13.
13.
 transversality
transversality index
index == A+B+C+D
A+B+C+D == 2727 onon the
the
Figure
Figure
Genetic Genetic disorders disorders  Unusual Unusual dermatoglyphic patterns often relate dermatoglyphic patterns often relate to
Genetic
Genetic disorders
disorders
 Unusual
Unusual dermatoglyphic patterns often relate
dermatoglyphic patterns often relate
to genetic disorders
to genetic disorders
One study of foetuses with chromosomal
One study of foetuses with chromosomal
abnormalities showed that the dermatoglyphic
abnormalities showed that the dermatoglyphic
patterns were delayed by more than two weeks
patterns were delayed by more than two weeks
 Trisomy Trisomy 2121 ((Down Down syndrome syndrome):): People People with with Down Down syndrome syndrome
 Trisomy
Trisomy 2121 ((Down
Down syndrome
syndrome):): People
People with
with Down
Down
syndrome syndrome have have mainly mainly ulnar ulnar loops loops,, and and aa
significantly different angle between the triradia a, t
significantly different angle between the triradia a, t
and d (the 'adt angle').
and d (the 'adt angle').
 Other Other differences differences often often include include aa single transverse palmar crease single transverse
 Other
Other differences
differences often
often include
include aa
single transverse palmar crease
single transverse palmar crease ("Simian
("Simian line")
line")
(in 50%), and patterns in the hypothenar and
(in 50%), and patterns in the hypothenar and
interdigital interdigital areas, areas, lower lower ridge ridge counts counts along along
digital midlines, especially in little fingers,
digital midlines, especially in little fingers,
which corresponds to finger shortening in those
which corresponds to finger shortening in those
with with Down's Down's syndrome syndrome
 There is less variation in dermatoglyphic There is less variation in dermatoglyphic patterns between people
There is less variation in dermatoglyphic
There is less variation in dermatoglyphic
patterns between people with Down syndrome
patterns between people with Down syndrome
than between controls, and dermatoglyphic
than between controls, and dermatoglyphic
patterns patterns can can bebe used used toto determine determine correlations correlations
with congenital heart defects in individuals
with congenital heart defects in individuals
with Down syndrome by examining the left
with Down syndrome by examining the left
hand digit ridge count minus the right hand
hand digit ridge count minus the right hand
digit ridge count, and the number of ridges on
digit ridge count, and the number of ridges on
the the fifth fifth digit digit of of the the left left hand hand
 Turner syndrome: Predominance of whorls, Turner syndrome : Predominance of whorls, although the pattern frequency
Turner syndrome: Predominance of whorls,
Turner syndrome : Predominance of whorls,
although the pattern frequency depends on the
although the pattern frequency depends on the
particular
particular chromosomal
chromosomal abnormality
abnormality
 47, XXY 47, XXY (Klinefelter's ( Klinefelter's syndrome syndrome):): Excess Excess of of arches arches
47, XXY
47, XXY (Klinefelter's
( Klinefelter's syndrome
syndrome):): Excess
Excess of
of
arches
arches onon digit
digit 1,
1, more
more frequent
frequent ulnar
ulnar loops
loops onon
digit digit 2, 2, overall overall fewer fewer whorls, whorls, lower lower ridge ridge
counts
counts for
for loops and whorls as compared with
loops and whorls as compared with
controls, and significant reduction of the total
controls, and significant reduction of the total
finger finger ridge ridge count count
∑ Trisomy Trisomy 1313 ((Patau Patau syndrome syndrome):): Excess Excess ofof arches on fingertips and arches
∑ Trisomy
Trisomy 1313 ((Patau
Patau syndrome
syndrome):): Excess
Excess ofof
arches on fingertips and
arches on fingertips and
single transverse palmar creases inin 60%.
single transverse palmar creases
60%.
∑ Trisomy Trisomy 1818 ((Edward's Edward's syndrome syndrome)) 66 -- 1010 arches arches onon fingertips fingertips
∑ Trisomy
Trisomy 1818 ((Edward's
Edward's syndrome
syndrome)) 66 -- 1010 arches
arches
onon fingertips fingertips and and
single transverse palmar creases inin 30%.
single transverse palmar creases
30%.
 Cri du chat (5p-): Excess of arches on Cri du chat (5p-): Excess of arches
Cri du chat (5p-): Excess of arches on
Cri du chat (5p-): Excess of arches on
fingertips
fingertips and
and single transverse palmar creases
single transverse palmar creases
inin 90%.
90%.
 Inborn blindness : Dermatoglyphic analysis of Inborn blindness : Dermatoglyphic analysis of finger finger tiptip
Inborn blindness : Dermatoglyphic analysis of
Inborn blindness : Dermatoglyphic analysis of
finger finger tiptip print print patterns patterns of of blind blind children children from from
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