SPDX WEEK 15: DNA ANALYSIS  The DNA polymer is made up of alternating
sugar molecules (deoxyribose) and phosphates.
APPLICATION OF DNA ANALYSIS IN FORENSIC
SCIENCE
 Makes it possible to associate a DNA sample to
a specific person with a high degree of certainty
 Thus, giving law enforcement and
forensic science in new and powerful
identification tool that complements
finger prints and other methods of
identification.
THE NATURE OF DNA
 Deoxyribonucleic acid (DNA) is a molecule that
is found in nearly all cells.
DNA TYPING
 Notable exemptions are RBCs which have no
nucleus
 DNA is a special type of molecule known as a
polymer.
 Which is a molecule made up of
repeating simple units called
monomers.
 DNA is located in two regions in a cell: the
nucleus and mitochondria.
 Both of it can be both in DNA typing
however, mitochondrial DNA is of
different length and shape.
 Unlike nuclear DNA, mitochondrial DNA
is inherited only from mother and is
used for typing under different
circumstances than nuclear DNA.
 It is used for typing under different
circumstances than nuclear DNA.
NUCLEAR DNA
RESTRICTION FRAGMENT LENGTH
POLYMORPHISM (RFLP)
 Nuclear DNA is found in a geometric shape
called a double helix.
 A helix is a spiral-shaped object.
 DNA looks like two helices that wrap
around each other.
 Consider a ladder, made up of two poles
held together by a series of rings.
 Dangling off each sugar molecule is one of four
bases or nucleotides: adenine (A), guanine (G),
cytosine (C), and thymine (T).
 To recall, when an adenine base and a thymine
base come into proximity, they form a bond to
each other.
 Likewise, when cytosine and guanine get near
each other they will bond.
 The order of these pairs of bases is the principle
of genetics and inheritance. The order of the
base pairs contains a sort of blueprint or genetic
code that determines many of the characteristics
of a person.
 The restriction fragment length
polymorphism (RFLP) was the first DNA typing
method to be widely adopted by forensic
biologists in the analysis of crime.
 Dr. Alec Jeffries applied the technique of
restriction fragment length
polymorphism (RFLP) to a real criminal
case and showed that this technique
could associate a person to biologic
evidence through DNA typing.
 But it is not used any longer in forensic
applications, having been surpassed by
methods with higher powers of
discrimination and whose results can be
obtained faster using much less biologic
material.
 RFLP was eventually supplanted by methods
derived from the polymerase chain reaction
(PCR), a technique that is used primarily to
increase the amount of DNA by amplification.
 For a while, many forensic science laboratories
used both RFLP and PCR methods in tandem.
Today, most laboratories use a typing method
known as short tandem repeats (STRs), which
combines some of the attributes of both PCR
and RFLP.
 DNA is extracted from biologic material and then
severed into small fragments called mini
satellites or variable number tandem repeats
(VNTRs) using restriction enzyme.
 The length polymorphism present in the variable
number tandem repeats (VNTRs) is used to
discriminate a population of people.
  In forensic analysis, four to six of these highly
polymorphic loci are analyzed, and the results
enable forensic scientists to generate DNA
profiles that approach individuality.
 The use of RFPL typing in forensic science has
decreased in recent years in favor of PCR-
based methods.
 But it is still used in some forensic laboratories
that performs paternity testing.
POLYMERASE CHAIN REACTION (PCR)
 Around the same time in the 1980s that RFLP
was being developed for forensic use, another
major advance was the development of forensic
applications of the polymerase chain reaction
(PCR).
 PCR had been used since the 1970s for making
copies of (amplifying) DNA using polymerase
enzymes.
 PCR overcame one of the major problems
facing forensic biologists, which is in RFLP
methods of DNA typing would require relatively
large quantities of DNA.
STEPS IN PCR
1. Denaturation
 The DNA is added to the PCR tube that
contains the reaction mixture and is then
heated to 95°C.
 Under these conditions, the double-
stranded DNA denatures.
 Each strand will be the template for the
formation of a new piece of double-
strained DNA.
 As the double-stranded DNA denatures,
the bonds between the base pairs that
holds the strands together would break
resulting in single-stranded DNA.
 As long as the temperature is
maintained which is 95°C, the strands
will remain apart.
2. Annealing
 This is the attachment of a short strand
of synthetic DNA to each of the
separated strands known as the
primers.
 This are called the primers
because they will mark the
starting points for the addition of
new bases to complete the
reproduction of each strands.
 The thermal cycler temperature drops
to 60°C for this step.
3. Extension
 Single bases (nucleotides) are added to
the primer.
 Under the influence of
polymerase single bases are
added to the primer one by one.
 Each base is complementary to
a single nucleotide present on
the strand being duplicated.
 The entire complementary strand is built
up, and a new piece of double-stranded
DNA is produced.
 This process occurs at each of
the complementary single
strands created by the
denaturation process. The end
result is that two identical pieces
of double-stranded DNA are
produced.
 The temperature is raised once again to
94°C and the process repeats.
 This produces about one billion copies
of the original DNA, enough for
additional typing.
 So the end result is that 2
identical pieces of double
stranded DNA are produced.
This complete its one PCR
cycle.
 The 2 strand of DNA denature
forming 4 single strand are in
turn subjected to annealing and
extension forming 4 new double
strand
 The process continuous until a
sufficient amount of DNAs
produced typically 25 to 40
cycles which takes about 3
hours.
  This would illustrate the PCR amplification
process. Each cycle double the amount of DNA
under ideal conditions.
 30 cycles would produce over 1 billion copies
and take about 3 hours.
DNA Typing of PCR Product
 After the completion of Amplification process,
the product must be detected. There a number
of ways to do this.
 The first DNA region widely subjected to
amplification and typing for forensic purposes by
PCR is the HLA (Human Leukocyte Antigen) DQ
alpha (now called DQA1) gene.
 This gene exhibit sequence
polymerphism. Neither DQ alpha nor
polymarker DNA typing is used in
forensic science anymore. This method
has largely surpass by STRs.
 DQ alpha and a number of other genes
collectively called polymarker are typed using a
method called reverse dot blot.
 This process involves identifying the particular
alleles present by reacting them with color-
forming reagents on specially treated nylon
strips.
 One reason for this is that the alleles in
these markers do not vary to a great
extent in the human population. As a
result, it is not possible to generate DNA
type that is rare enough to associate
with just one individual.
 A more important reason is that this
method of DNA typing is not capable of
resolving multiple DNA types that are
present in mixtures, such as vaginal
swabs obtain after rape cases.
SHORT TANDEM REPEATS (STR)
 The advantages of STR over the other methods
of DNA analysis.
 STR markers exhibit high variability in a
population, thus giving rise to high
degree of association of evidence with a
suspect.
 A small size of their repeats makes STR
much less sensitive to degradation of
DNA. Unlike in PCR, methods are very
sensitive to contamination by foreign
DNA. For this reason, DNA extraction
are always done in a location physically
isolated from the place where
subsequent applications will be
performed.
 Finally, there are many microsatellites to
choose from for forensic purposes.
Thousands of them have been identified
and many are used for commercial and
medical purposes.
GENDER IDENTIFICATION
 Amelogenin
 A locus on the sex determination
chromosome.
 One of the regions of this locus is six
base pairs longer in males than in
females.
 This locus is not an STR but can be
analyzed at the same time as STRs.
 There are 2 approaches in
gender identification using the
DNA typing.
 On the sex determination
chromosome there is a locus
called AMELOGENIN
 One of the regions of this locus
is 6 base pairs in male than in
females.
 Females have 2 x-
chromosomesannd will only
show 1 amelogenin.
 Males have 1 x and 1 y
chromosome and will show 2
bands (one 6 basepair longer
than the other)
 Y-STRs
 The Y chromosome, found only in
males, also contains STRs.
 They can be typed even on small or
degraded samples or mixtures with
large quantities of female DNA.
 The other approach to gender
identification utilizes Y-STRs
 Y chromosome found only in
males also contains STRs
 Y-STRs are useful when typing
mixed samples that contain
sperms and is guaranteed to
have a male fraction
 But it should be noted that y
chromosome analysis produces
only haplotype and is thus, not
as informative as common STR
analysis.
MITOCHONDRIAL DNA (mtDNA)

 Other than the nuclear, we also have the

mitochrondrial DNA and these are the
applications in forensic science
 Applications in Forensic Science:
1. mtDNA is very powerful for tracing
family lines back through the maternal
side.
 All male and female
mitochondrial DNA comes from
the mother there is no mtDNA
from the father.
 This means that except from the
mutation mentioned previously,
every descendant of a woman
should have the same mtDNA.
2. mtDNA often shows a high degree of
variation between unrelated people,
making it a powerful tool in forensic
typing.
 However because there are only
2 hypervariable regions in
mtDNA, the population statistics
are not near as discriminating
as with nuclear DNA.
3. mtDNA can be useful in typing samples
that have low quantities of cellular DNA,
or in exhibits that are degraded or very
old.
 Many forensic science
laboratories that perform
genomic DNA analysis do not
perform mtDNA analysis.
 Those that do mtDNA analysis,
generally use DNA sequencing.
They determine the entire base
pair sequence into two
hypervariable regions rather
than relying on length
polymorphism.