Before we start

discussing DNA profiling

techniques
Let’s spend a
couple of minutes
learning a few
additional,
important
concepts…
Do you remember the chain of
nucleotides (bases) building the DNA
double helix? You also should
remember there are four types:
Adenine, Thymine, Guanine, and
Cytosine (A, T, G, and C). We can write
out the DNA string of genetic
information in the form of a sequence of
letters such as GATCAGTACC… A GENE
is a complete sequence of DNA bases
that will cause a function  e.g. it may
define hair color or cause the
production of a specific protein. The
combination of the basic gene formats
causes different genetic functions and
this accounts for our differences from
each other and from other living things.
Gene are located in CHROMOSOMES
in the nucleus of the cell.

Each chromosome in every cell

contains two versions or strings of
 Chromosomes
Chromosomes are long, stringy aggregates
of genes that carry heredity information and
they are always coupled.

A chromosome
structure, with its
different regions
 Chromosomes
As an example, gender is determined by the presence or absence
of the Y chromosome, so we can have the combination of two X
chromosomes resulting in female gender XX or a combination of a
X and Y chromosome resulting in male XY

A electronic microscope
picture of the XY
chromosome. Notice the
curled aggregates of
genes made of DNA
strands
GENES are units of heredity information that
consist of DNA and are located on
CHROMOSOMES in a specific point called
LOCUS, plural LOCI. Genes can exist in
alternative forms called ALLELES.

(Exon and Intron are sequences of

DNA having specific function in
protein synthesis )

An ALLELE is an alternate
form of a GENE (one
member of a pair) that is
located at on a specific
chromosome . For example,
the gene for flower-color
exists in two forms, one
form or allele for purple
flowers shape and the other
GENES determine inherited features such as hair, skin and
eye color. For example the gene for eye color exist in
several forms, e.g. one allele for blue eyes and one for
brown eyes
 Oh I almost forgot…

Two more definitions:

Phenotype

Any feature or characteristic of an organism or any group of

characteristics phenotype refers to any of the detectable
attributes of a living thing. The phenotype is the result of the
interaction of the gene and environmental components. E.g.
“Mediterranean” phenotype with darker skin and dark hair,
“Nordic” phenotype with fair skin and lighter colored hair

Genotype
The sum of genes within a cell or within the cells of an organism.
Usually, the word genotype is used to describe only the one or the
few genes being studied at a time. For example, the gene
associated with eye color would have the genotypes BB and Bb,
which lead to brown eyes, or the genotype bb, which leads to blue
eyes.
 And now let’s move on…

DNA Profiling

“Ithas long been an axiom

of mine that the little
things are infinitely the
most important”

Sherlock Holmes
DNA profiling is based on the analysis
of non coding portions of DNA, that are
highly variable between individuals
(other portions –the coding portions-are
shared by all human beings). These
portions consist of repeats of specific
sequences of DNA .
We do remember that DNA is made
of 4 bases, Adenine, Thymine,
Guanine,, Cytosine, don’t we ?  ( A,
T, G, C) and how these bases form
sequences such as
ATGCGTAGGGACT...
These repeated sequences can be of
different length, and present in a
variable number of repeats ( even 10
000! )

In the first analysis techniques (still

in use today, the Variable Number of
Tandem Repeat (VNTR) were used.
So a TR (tandem repeat) is a short sequence of
DNA that is repeated in a head-to-tail fashion at
a specific chromosomal locus. Tandem repeats
are interspersed throughout the human genome.
Some sequences are only found at one site in the
the human genome-a single locus-for many
tandem
repeats, the number of repeated units vary
between
individuals. Such loci are termed VNTRs

One VNTR in humans is a 17 base pairs sequence

of DNA repeated between 70 and 450 times in the
genome. The total number of base pairs at this
locus could vary from 1190 to 7650.  
 When profiles from a single VNTR locus
from unrelated individuals are compared,
the profiles are normally different.
However two individuals may have the
same profile at one or two loci by chance.
However, the chance of more than one
person having the same DNA profile at
more than 3 or 4 different VNTR loci is
very small. When DNA profiles are used for
forensic purposes, 4-6 different VNTR loci
are analyzed.
 Short Tandem
Repeats
Although determination of VNTRs is still
used in
forensics, in Europe the standard is now
the determination of Short Tandem
Repeats (STR) much shorter but highly
STR are repeats
variable among individuals. They consist
of sequences of three, four or five bases
that can be repeated from 80 to 400
times. Also these sequences, as VNTRs
are located in the DNA non-coding
regions. They are the genetic
fingerprints of each individual
 Short Tandem Repeats
 In each individual genoma hundreds
of different STR can be present, each
of them under 5-10 different forms
(alleles) in the population, that
usually have different length
 Each individual can have only one
allele per each STR (homozygote) or
two different allele (heterozygote)
 Short Tandem Repeats
 Theadvantage of using STR is that
we need much smaller quantities of
DNA to perform the analysis, or the
equivalent of 8 cells (each cell
contains about 6 picograms of DNA,
and we need 50 picograms to
perform the analysis)
 PCR (Polymerase Chain
Reaction)
 The analysis of the DNA fingerprint is
performed by taking into consideration
several different STRs
 These are amplified by PCR, a reaction
that allows to analyze different STR at the
same time (STR multiplex system,to
quickly identify specific STR. Short
Tandem Repeats polymorphisms are
genetic that may be used to identify a
DNA sequence )
 We detect the DNA by measuring the
length of the fragment (by
electrophoresis)
 PCR: How is it performed
DNA strand
PCR is composed of cycles of heating
and cooling. A cycle begins with
denaturing the DNA by heating
(breaking apart the double strands of
the DNA molecule into single-
stranded DNAs). Then, primers (short
pieces of DNA complementary to a
specific sequence) bind to their
specific part of every single-stranded DNA strand
DNA during cooling. This step is
called annealing.
ANNEALING:
In the final step, extension, the
DNA STRAND after denaturation
temperature is raised again and a
and primers binding to specific
specific enzyme called Taq portions of single-stranded DNA
polymerase is used to add
complementary bases to the DNA
from the end of the primer along the
rest of the single-stranded DNA
templates, thereby making multiple
copies of the DNA segment between
 DNA Amplification
 The entire process is repeated a large number of times
(cycles) to yield a large amount of the desired DNA region.
After 25 PCR Cycles you can achieve 3,2 x 107 copies from
one target molecule.
cycle
Target gene
cycle

cycle

cycle cycle

DNA
Template

copie Billions copies

copies copies copie
s s
After the amplification cycles have
terminated, by means of agarose gel
electrophoresis we check whether
the PCR generated the anticipated
DNA fragment by size separation of
the PCR products. The size(s) of PCR
products is determined by
comparison with a molecular weight
marker, which contains DNA
fragments of known size, run on the
gel alongside the PCR amplification
product
 Example of STR: HUNTH01
901 aaatccatcc aaaaaatcca agatggccag aggtccccgg ctgctgcacc cagcccccac
961 cctactccca cctgcccctg cctccctctg ccccagctgc cctagtcagc accccaacca
1021 gcctgcctgc ttggggaggc agccccaagg cccttcccag gctctagcag cagctcatgg
1081 tggggggtcc tgggcaaata gggggcaaaa ttcaaagggt atctgggctc tggggtgatt

1141 cccattggcc tgttcctccc ttatttccct cattcattca ttcattcatt cattcattca

1201 ttcattcacc atggagtctg tgttccctgt gacctgcact cggaagccct gtgtacaggg
1261 gactgtgtgg gccaggctgg ataatcggga gcttttcagc ccacaggagg ggtcttcggt
1321 gcctccttgg gcactcagaa ccttgggctc cctggcacat ttaaaatggg tttttattta

1381 tggaccttga ttgaaatgtg gtgtgagttg tagcagtgtc atttccaggt accttctcag

Amplification of a STR
will yield two bands if the
individual is heterozygote
and one if homozygote

STR
Bands are strasformed in peaks
through machine reading
 Summary of PCR steps

 Extraction of DNA from sample

 Amplification of SRT

 Electrophoresis

 Statistic validation

STR
 By performing the analysis on 5
different STR the probability to find
the same DNA print in two different
people is equal to 1 out of 10 billions
(Birmingham Forensic Science
Service)
 Nevertheless, we must keep in
account that some alleles are more
frequent in certain populations.
 Data banks exist that help
researchers to evaluate the results,
by excluding those that could be
artifacts due to abnormal distribution
of specific STR in certain populations
 In
order to exclude doubts about
results, generally the analysis is
performed on 13 or 14 different
STRs, by repeating the PCR (if
sample is available)
 REAL TIME PCR
THE FUTURE
A newly developed technique, Real Time
PCR, allows the amplification process to
become quantitative. The reaction is
“monitored” throughout the amplification
process during the reaction at every cycle,
and not at the end like in PCR. There is no
need of post PCR manipulation and the
risk of environmental contamination is
reduced, thus making the quantitation of
DNA easier, faster and more precise
 References
1) L'analisi del DNA nelle
indagini forensi-Regione Piemonte,
provincia di Torino www.torinoscience.it
2) The Biology Project-University of Arizona-
www.biology.arizona.edu
3) Jeffreys AJ, Wilson V, Thein SL (1985),
Individuals specific fingerprints of human
DNA.
Nature 316;76-79.
4) Mullis KB (1990), La scoperta della
reazione a catena della polimerasi. Le
Scienze
262;XXIII:32-39.
5) Polizia Cantonale www.polizia.ti.ch
6) Molecular Biology, R.Weaver,WCB Mc
Graw-Hill (2001)
7) Improved DNA Analsysis Through Real
Time PCR Analysis Curtis Knox and Benjamin
Krenke, Forensics Magazine
Issue: April/May, 2007