Chapter 1

DNA Fingerprinting: Discovery,

Advancements, and Milestones

Jahangir Imam, Romana Reyaz, Ajay Kumar Rana,

and Vrijesh Kumar Yadav

Abstract  The discovery of DNA fingerprinting is one of the most fascinating sci-
entific discoveries till date. It is not only limited to the laboratory research but also
showed a huge potential in forensic science and criminal justice system. It was one
of the milestones in resolving crimes by exploring the polymorphism of human
DNA in noncoding regions. Since its inception, DNA fingerprinting has taken a
great leap in terms of advancements in technology, accuracy, and reliability of the
results as well as rapidity of the process for its more efficient application in justice
delivery systems. This has become the most valuable armory of the judiciary system
to aid in the conviction of guilty as well as exoneration of the innocent. Advancement
of DNA fingerprinting technique from RFLP to STR and now NGS has sped up the
process of DNA profiling with better discriminating power among individuals with
greater efficacy. In this prospect, the current chapter elaborately recapitulates the
process of advancement in DNA fingerprinting describing the use of different STR
kits, i.e., autosomal STRs, Y-STRs, X-STRs, miniSTRs, etc., for forensic applica-
tions. We have also highlighted the importance of SNPs and amalgamation of NGS
kits in forensic application. Notably, the importance of wildlife forensic has been
discussed for the identification of species as well as its geographic origin. Another
important budding aspect of RNA-based identification of forensically relevant bio-
logical fluids has also been discussed in much detail.

Keywords  DNA fingerprinting · Criminal justice system · STRs · Forensic

analysis

J. Imam (*) · R. Reyaz · A. K. Rana · V. K. Yadav

DNA Fingerprinting Unit, State Forensic Science Laboratory, Department of Home,
Jail and Disaster Management, Government of Jharkhand, Ranchi, India

© Springer Nature Singapore Pte Ltd. 2018 3

H. R. Dash et al. (eds.), DNA Fingerprinting: Advancements and Future
Endeavors, https://doi.org/10.1007/978-981-13-1583-1_1
4 J. Imam et al.

1.1  Introduction

DNA or deoxyribonucleic acid is the hereditary polymer molecule made up of four

different nucleotides, i.e., adenine (A), thymine (T), guanine (G), and cytosine (C).
These nucleotides arranged in a definite sequence represent the genetic makeup of
all known living organisms and some viruses. In humans, the DNA is present within
the nucleus of every cell (except mature red blood cells) and is organized into 46
different chromosomes (22 pairs of autosomes and one pair of sex chromosomes).
A haploid DNA is approximately three billion base pair in length. Additionally,
99.9% DNA sequences are similar between two individuals [70]. The difference of
0.1% of the DNA sequence between two individuals is usually present along the
noncoding regions of the DNA.  This difference of sequences is targeted by the
forensic scientists to discriminate between individuals by matching DNA isolated
from varied samples, i.e., blood, tissue, body fluid, or hair/nails, with high level of
accuracy. The difference in DNA sequence basically occurs in terms of around 2–9
base pair repetitions called as microsatellites or of 10–100 bps called as minisatel-
lites [39, 57]. In earlier times, Sir Alec Jeffreys analyzed minisatellites or variable
number tandem repeats (VNTR) using Southern blotting to visualize DNA markers
[73]. However, currently, microsatellites of around 4 bp repeats are being analyzed
along with fluorescent tags of respective markers using multiplex amplification of
at least 20 core loci [46, 71] as defined by the Federal Bureau of Investigation
(FBI), USA [12, 75]. These loci along with their respective repeat patterns are
inherited from parents to their offspring by a random process during meiosis, and
every individual can be discriminated from another (as calculated by FBI among
Caucasian population) by a probability factor of 1.683 × 10−15 [20], i.e., one indi-
vidual can have the same DNA pattern with another only in a population of 594
trillion. The exception is monozygotic twins who carry the same loci with the same
repeat pattern.

1.2  Discovery

DNA fingerprinting was initially used to find human genetic diseases by linking
particular DNA sequences with the help of segregating markers which were present
in close proximity within a chromosome [12, 15]. Eventually, it was also used for
criminal investigations and forensic science, when an undaunting Ph.D. scholar
Alec J.  Jeffreys from Leicester University, United Kingdom, took a scientific
responsibility to nab the culprit of famous twin girl’s rape and murder case from
Narborough using classical DNA fingerprinting assay using VNTR method [12, 15,
73]. The geneticist Alec J. Jeffreys discovered that short repetitive DNA sequences
are almost unique to every individual, and he called them “minisatellites.”
In 1984, at the Leicester University, UK, Dr. Jeffreys was studying hereditary
diseases in families and was also focusing on developing methods to resolve
1  DNA Fingerprinting: Discovery, Advancements, and Milestones 5

p­ aternity and immigration disputes by linking genetic elements between individu-

als, and he published his results in Nature journal [28, 29]. He discovered that vari-
able number tandem repeats (VNTR) is a part of junk DNA and these repeats vary
from one individual to another. He employed various restriction enzymes in his
technique to cut these variable DNA sequences and generate length polymorphism.
He then run these digested sequences in a very long agarose gel and demarcated the
variations from individual to individual. He named his technique “genetic finger-
printing” and demonstrated that this restriction fragment length polymorphism
(RFLP) of DNA was unique to each individual and cannot match on earth except
for identical twins. However, his technique was launched into the world of forensic
science when two murders were committed in a little village of Narborough in the
city of Leicestershire, east of Birmingham, UK. In 1983, 15-year-old lady Lynda
Mann was found raped and murdered, and in the same village after 3 years in 1986,
Dawn Ashworth, also 15, was also raped and murdered in similar manner. Dr.
Jeffreys was asked to do DNA analysis of the semen samples recovered from the
two victims with the blood sample of a suspect named Richard Buckland, aged 17.
Some of the detectives of this case were in suspicion as Buckland was below
14 years of age when the first crime occurred. From the DNA analysis of the sam-
ples, Dr. Jeffreys demonstrated and proved that the same killer’s semen is present
in both the crime scenes and this does not match with Buckland blood sample’s
DNA. After this, the law enforcement took an exhaustive task to match the blood
types and then DNA of total 4582 men from the three towns. Dr Jeffrey did genetic
fingerprinting of the 10% men who were tested same for blood group (Group A)
and isoenzyme, i.e., phosphoglycerate mutase (PGM). However the DNA could not
match with any one of them. After several months, a resident heard a conversation
in a pub, where a person named Ian Kelly confessed that he had taken bribe for
replacing his photo with a photo of a local baker named Colin Pitchfork in his pass-
port and had submitted his own blood instead of Pitchfork [32]. Twenty-seven-
year-old Colin Pitchfork was arrested on September 19, 1987, and when his DNA
was compared with the semen samples, it was indistinguishable, i.e., identical.
Pitchfork was found guilty in both rapes and murders, and in 1987, Pitchfork
became the first person in the world to be identified and convicted as a result of the
DNA fingerprinting. He was sentenced to life imprisonment on January 22, 1988
for both murders and is currently in jail. In 1994, Professor Jeffreys was knighted
by The Queen of England, and today he is still a professor in the Department of
Genetics at the University of Leicester in Great Britain.

1.3  Advancement and Milestones in DNA Fingerprinting

DNA fingerprinting, since its discovery around two and half decades back, has
taken a great leap in its advancement and made the justice delivery system more
efficient and accurate in the investigation of criminal and civil cases [28–30]. This
is much like a valuable armory in the hands of judiciary which aids in the conviction
6 J. Imam et al.

of the guilty as well as exoneration of the innocent [10]. It has also been proven
helpful in linking relationship of reference samples to dead remains of missing per-
son and in mass disasters like plane crash, vehicle collision, earthquakes, etc. [10,
51]. With the discovery and innovation of new techniques for DNA extraction and
genotyping, the generation of DNA profiles is becoming more and more accurate
and easy, even for challenged and trace DNA samples. The known fact about DNA
fingerprinting is its uniqueness among human populations, and this attribute makes
it the method of choice [19]. DNA profiling generally involves the five basic steps
from sample preparation, DNA extraction, DNA quantitation, DNA amplification to
capillary electrophoresis, and profile generation [60]. With the advancement in dif-
ferent fields of science, new technologies are regularly introduced and validated in
forensic laboratories to aid the process of DNA fingerprinting with improved sensi-
tivity and informativeness. Day by day, crimes are increasing which poses a para-
mount pressure on judiciary system. In this regard, the use of automation techniques
for sample preparation and data interpretation by the forensic laboratories will be
useful to meet this increasing throughput demands on the laboratories [10].
DNA fingerprinting requires good scientific skills and data interpretation ability
which is essential in result outcome. Earlier, the methodology used for DNA extrac-
tion to profile generation had lots of limitations for the type and quality of biological
samples available for forensic investigation. The advancements in genomic and
post-genomic era have put the forensic science one step ahead, and now it is much
faster, higher, and stronger in crime investigation and judiciary system [10], faster
in terms of rapid DNA instrumentation, recovering higher and good quality of data
from biological evidences and stronger conclusion on complex evidences [10]. The
development in forensic DNA analysis is possible because the pioneer work, pro-
gression, and innovation transpire over the past three decades (Table 1.1) [9, 60].

1.3.1  Current Status of DNA Fingerprinting Technique

The discovery of DNA fingerprinting is nothing less than bliss, not only to the sci-
entific world but also to the judiciary system. Alec Jeffreys laboratory was the only
one to work on DNA fingerprinting during 1985–1987, and his work and contribu-
tion in solving civil and criminal cases with the help of DNA fingerprinting is pio-
neer in establishment and adoption of this technique in judicial investigations
worldwide. The last three decades are the golden periods in the field of forensic
science with the advent and implementation of new techniques, the use of advanced
commercial DNA typing kits with various genetic marker systems, and NGS in
forensic science.
Since the discovery of DNA fingerprinting and its application in forensic judi-
ciary system, the procedure for biological sample collection from the crime scene
and DNA extraction methodology from different biological specimens are well
established [24, 25, 43, 58, 60, 65, 69, 74]. Here we will discuss the advancement
and emerging techniques and methodologies of DNA fingerprinting. Forensic sci-
1  DNA Fingerprinting: Discovery, Advancements, and Milestones 7

Table 1.1  Timelines and milestones for forensic DNA analysis

Timeline Major area Activities and milestones
1985–1995 Development and 1. Discovery of DNA fingerprinting and first publication by
exploration Alec Jeffreys
2. RFLP using VNTRs
3. Rapid and sensitive PCR assays
4. Formation of DNA profiling groups
1995–2005 Stabilization and 1. Improvement in PCR technology
standardization 2. Development of sensitive, fast, and genotypic precision
STR-based DNA analysis
3. National DNA database formation by the United Kingdom
with six STR markers
4. 13 core STR markers designated by the United States for
FBI CODIS software
5. Multiplexing and capillary electrophoresis
6. Autosomal and Y-STR kit released
7. Expansion of forensic research in different laboratories
8. Importance of Human Genome Project (HGP) in forensic
DNA technology development
2005–2015 Augmentation 1. Faster and efficient DNA extraction methods adopted
2. Automation
3. Accumulation of DNA database
4. Advancement in different STR kits
5. Development and validation of new STR kits
6. Encroachment of DNA forensic in wild life
7. Forensic DNA phenotyping (FDP)
8. Implementation of microbial DNA fingerprinting of human
fingerprints
9. Advancement in forensic RNA typing
10. Introduction and exploitation of NGS in forensic science
2015–2025 Sophistication 1. Development of tools for rapid DNA testing outside of
laboratories or at crime scene
2. Huge population database formation
3. Development of cost effective forensic DNA analysis
4. More better multiplexing system and DNA typing
5. More research in the field of wild life forensic, microbial
DNA fingerprinting and forensic RNA typing
6. Development of cheaper and more robust STR kits
7. Development of skills and ability to interpret forensic
evidence results
8. Higher sensitive methodologies applied to casework and
probabilistic software approached to complex evidence
8 J. Imam et al.

ence has gone through several stages of development since its discovery and
­application in the 1980s [19]. The first generation of DNA analysis RFLP-based
profiling is obsolete now from the forensic point of view, because it was not suitable
for degraded and challenging biological forensic samples as it was not able to ana-
lyze the samples with accuracy. The PCR-based second generation of DNA analysis
based on dot-blot methods was then developed but could not be fruitful enough as it
was not helpful with DNA fragments which were longer in length. The third genera-
tion is STR (short tandem repeats) based which is easy, suitable, and most widely
accepted for DNA analysis, but, sometimes, for highly degraded DNA samples,
getting DNA profile becomes difficult. The fourth generation of DNA analysis
which is introduced in forensic science is NGS (next-generation sequencing) which
has attracted the forensic community with its high-throughput capacity and low cost
[4]. There is a continuous effort to develop more effective, cheap, and fast DNA
profiling techniques with more discriminatory power to address the application of
forensic science in different fields (Table 1.2). Here, we have highlighted some of
the recent progresses made in the analysis of STRs, SNPs, low-template DNA,
mitochondrial DNA, DNA methylation, microbial forensic, and NGS in forensic
and illustrate how different technologies can be integrated for new-generation
forensic science.

1.3.1.1  E
 volution of Capillary Electrophoresis as a Tool for Forensic DNA
Analysis

Capillary electrophoresis (CE) is one of most important instrumental advancements

to be implemented for forensic DNA typing. After PCR invention, scientists con-
sider it as the second most needed development. Application of capillary electro-
phoresis in forensic DNA analysis not only makes the work easier but also makes it
more accurate and authentic, which is of paramount importance in forensic DNA
analysis [63]. The application of capillary electrophoresis is not only limited for
biological samples but also has a huge importance in the analysis of gunshot resi-
dues, explosive residues, and drugs. For forensic DNA analysis, STR profiling
(highly polymorphic markers) which is based on fragment analysis is of great value
for human identification (HID) due to the single-base resolution capability of CE
[63]. Introduction of capillary electrophoresis in STR typing circumvents the
tedious and expensive approach of DNA sequencing for STR typing. The approach
of CE like precise sizing, its sensitivity for the detection of fluorescence emitted by
different dyes, automatic electrophoresis, and data collection software are key fac-
tors in the worldwide adoption of CE as the preferred platform for forensic DNA
analysis. The most common CE systems used in forensic DNA analysis include the
ABI PRISM® 310, 3100, 3100 Avant, 3130, 3130xL, 3500, and 3500xL Genetic
Analyzers (GAs). The advanced CE automated machines are developed with
advanced features which are useful for forensic scientists. It has many advantages
like normalization of peak height, accurate sizing of fragments, sample injection,
single-base resolution, high run-to-run precision, good temperature control and
Table 1.2  Different commercial STR kits for forensic application produced after the year 2000
S.No. STR kit name No. of markers Application and advantages Make
1 Identifiler™, Amelogenin Plus 15 marker (13 Greater sensitivity and improved performance on inhibited Life Technologies
IdentifilerPlus™ and CODIS + D2S1338 and forensic samples. More robust master mix and modified
IdentifilerDirect™ D19S433) thermal cycling parameters
2 PowerPlex® 16 and Amelogenin Plus 13 CODIS Enhanced buffer system with improved robust performance Promega
PowerPlex® 16HS marker Corporation
3 PowerPlex® 18D Amelogenin Plus 15 marker (13 Improved allelic ladder featuring many additional alleles not Promega
CODIS + D2S1338 and found in original PowerPlex® 16 PowerPlex® 16HS kit. It is Corporation
D19S433 + Penta E and Penta D) also compatible with direct amplification from FTA card
punches as well as non-treated paper
4 NGM™ Amelogenin Plus 15 marker It enables simultaneous, multicolor fluorescence detection in a Life Technologies
(ENFSI loci -mini- and single PCR by using five-dye chemistry
midi-STRs)
5 NGM SElect™ and NGM Amelogenin Plus 16 marker It enables simultaneous, multicolor fluorescence detection in a Life Technologies
SElect™ express (ENFSI loci- mini- and single PCR by using five-dye chemistry
midi-STRs)
6 PowerPlex® ESX-16 and Amelogenin Plus 15 marker It enables simultaneous, multicolor fluorescence detection in a Promega
PowerPlex® ESI-16 (ENFSI loci – mini- and single PCR by using five-dye chemistry Corporation
midi-STRs)
7 PowerPlex® ESX-17 and Amelogenin Plus 16 marker It enables simultaneous, multicolor fluorescence detection in a Promega
1  DNA Fingerprinting: Discovery, Advancements, and Milestones

PowerPlex® ESI-17 (ENFSI loci – mini- and single PCR by using five-dye chemistry Corporation
midi-STRs)
8 ESSplex and ESSplex SE Amelogenin Plus 15 marker It enables simultaneous, multicolor fluorescence detection in a Qiagen, Hilden,
(ENFSI loci – mini- and single PCR by using five-dye chemistry Germany
midi-STRs)
9 PowerPlex® ES Amelogenin Plus 8 marker (SE33 Addition of highly polymorphic SE33 locus. It has limited use Promega
locus) in relationship testing due to highest mutation rate Corporation
(continued)
9
Table 1.2 (continued)
10

S.No. STR kit name No. of markers Application and advantages Make
10 SEfiler™ and SEfilerPlus™ Amelogenin Plus 10 marker Addition of highly polymorphic SE33 locus. It has limited use Qiagen, Hilden,
(SE33 locus) in relationship testing due to highest mutation rate. Improved Germany
synthesis and purification processes, enhanced sensitivity for
inhibited samples
11 PowerPlex® 21 Amelogenin Plus 20 marker (13 It can work with a variety of sample types, including casework Promega
CODIS + D2S1338 and samples. It is also compatible with direct amplification from Corporation
D19S433 + Penta E and Penta FTA card punches as well as non-treated paper
D + 3 marker)
12 GlobalFiler™ Express Amelogenin Plus 23 marker (13 Additional loci included (MiniFiler loci) which are designed Life Technologies
CODIS + D2S1338 and to meet the expanded US core loci requirements. It is
D19S433 + 7 marker + Y Indel optimized for efficient amplification of low level DNA and to
gender loci) overcome common inhibitors of the PCR. Optimized for the
amplification of single-source samples
13 PowerPlex® Fusion Amelogenin Plus 23 marker (13 Additional loci included (MiniFiler loci) which are designed Promega
CODIS + D2S1338 and to meet the expanded US core loci requirements. It is a Corporation
D19S433 + Penta E and Penta dual-purpose kit in that it can be used for common casework
D + 6 marker) samples as well as direct amplification of reference samples
stored on paper with only minor changes to the
PCR amplification conditions
14 PowerPlex® CS7 System Seven STR loci It is used as a confirmatory kit in paternity applications Promega
(nonstandard STR marker Corporation
system)
15 Investigator HDplex kit Amelogenin Plus 13 marker It is developed specifically to discriminate closely related Qiagen, Hilden,
(nonstandard STR marker (highly polymorphic markers) individuals. It is designed for difficult forensic and paternity Germany
system) cases
16 MiniFiler ™ Amelogenin Plus 8 CODIS For genotyping degraded DNA samples. First commercial kit Life Technologies
marker designed to amplify miniSTRs
17 PowerPlex Y 12 Y-STR loci First sex-chromosome STR kit developed to identify male Promega
lineages Corporation
J. Imam et al.
S.No. STR kit name No. of markers Application and advantages Make
18 Yfiler ™ 17 Y-STR loci Most commonly used sex-chromosome STR kit to identify Applied
male lineages as it works well in most outbred populations Biosystems
19 Argus Y-12 QS 12 Y-STR loci + internal control Sex-chromosome STR kit developed to identify male lineages. Qiagen, Hilden,
The internal control system provides helpful information Germany
about PCR efficiency and about the presence of inhibitors in
tested samples
20 PowerPlex® Y23 12 Y-STR loci of Yfiler Kit plus It allows Y-STR analysis of both human forensic samples and Promega
six additional new informative database samples. It features fast amplification time and better Corporation
loci for male-lineage tolerance of inhibitors of the PCR when compared to previous
differentiation generations of Y-STR multiplexes
21 Argus X-12 kit 12 X-STR loci Simultaneous amplification of 12 X-chromosomal markers for Qiagen, Hilden,
kinship and paternity testing, as well as population genetics Germany
and anthropological studies. Also suited for forensic stains,
such as female traces in male background
1  DNA Fingerprinting: Discovery, Advancements, and Milestones
11
12 J. Imam et al.

automation, better sensitivity, high throughput, user-friendly, and easy software fea-
tures to analyze the raw data to the level of precise accuracy [63]. Definitely the
incorporation of CE in forensic application must be considered as a milestone for
the service of the mankind.

1.3.1.2  S
 TR and Next-Generation STR Genotyping Kits for Forensic
Application

Forensic DNA typing has been constantly evolving driven by innovations from aca-
demic laboratories as well as kit manufacturers [47]. Much technological advance-
ment took place during the last 30  years, but the PCR-based STR genotyping is
central to all. STRs are now the markers of choice for various human identification
(HID) applications as the STR loci are considered polymorphic as they are unique
to each individual [8]. The basis of individual identification by STRs is the measure-
ment of length of different alleles which exhibit the highest variability among indi-
viduals [21]. Mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeats of STRs are
available, but tetranucleotides are commonly used and preferred in STR analysis
because the chance of stutter production is minimal and it can analyze the ampli-
cons that are one repeat less than the true allele [60]. Now, PCR-based multiplexing
of STRs and capillary electrophoresis enables the analysis of several different loci
at the same time with better accuracy and ready to available data for interpretation
[44]. In 1997, the United States established a core set of 13 STR loci known as the
Combined DNA Index System (CODIS) loci. The 13 STR loci set had strong distin-
guishing power with average random match probability of one in a quadrillion
(1 × 10−15). After the establishment of CODIS loci, Promega Corporation (Madison,
Wisconsin) and Life Technologies developed the commercial kits to meet the
demand of the forensic community [47].

1.3.1.3  Multiplex STR Kits

After 2000, Promega designed the PowerPlex 16 System, and Life Technologies
came up with AmpFLSTR Identifiler PCR Reaction kits which were capable of
amplifying all 13 CODIS markers in a single PCR reaction along with the amelo-
genin sex identification marker [14, 38]. The AmpFLSTR Identifiler PCR Reaction
Kit and the PowerPlex 16 System have been widely used for forensic database gen-
eration and casework analysis, both. This helps in the generation and addition of
millions of STR profiles to the DNA databases helping the criminal judiciary sys-
tem. With huge criminal cases happening frequently, forensic laboratories are con-
tinuously seeking and adopting enhanced technologies which help them to process
database and casework samples more efficiently and effectively. The AmpFLSTR
1  DNA Fingerprinting: Discovery, Advancements, and Milestones 13

Identifiler Plus PCR Amplification Kit and PowerPlex HS System were developed
for various challenging forensic samples for greater sensitivity and improved per-
formance. Various autosomal STR kits have been produced after the year 2000 for
challenging and inhibited biological samples which are listed in Table 1.2. Later on
in 2011, CODIS announced an increase in the number of CODIS loci to reduce the
likelihood of adventitious matches, to increase international compatibility, and to
increase the discrimination power (Mulero and Hennessy). Life Technologies devel-
oped GlobalFiler™ and GlobalFiler™ Express PCR Amplification kits, and
Promega Corporation developed PowerPlex Fusion System as a next-generation
STR kit to meet this challenge by incorporating extra loci in the kits, hence making
it more robust (Table 1.2). For genotyping degraded DNA samples, the first com-
mercial STR kit, “MiniFiler Kit,” was developed to amplify “miniSTRs.” The
improvement was done to amplify degraded samples by repositioning the primers as
close to STR repeat region, as possible (Table 1.2) [11, 13, 66]. New STR kits were
developed for human identification by incorporating highly polymorphic STR
locus, SE33 (a tetranucleotide STR). Inclusion of SE33 in multiplex has shown a
huge advantage due to its structural variations and polymorphism with differentia-
tion of around 1 bp [55, 59]. This marker also possesses the highest mutation rate,
thus limiting its application in forensic use such as relationship testing [16, 49].
PowerPlex® CS7 System (Promega Corporation) and Investigator HDplex Kit
(Qiagen), a nonstandard STR marker system, were also developed for very special
cases which involved in kinship analysis and testing with samples deficient of close
relatives. It has been developed specifically to discriminate closely related individu-
als for difficult forensic and paternity cases (Table  1.2). Many sex-chromosome
STR kits were also developed to identify male and female lineages (Table 1.2).
The next-generation commercial kits listed in the Table 1.2 have been developed
by improving the performance with inhibited samples, increased sensitivity, high
throughput with direct amplification, increased CODIS loci, and paternity- and
lineage-­specific STRs which provide a “driving force” for the progress of forensic
science.

1.3.1.4  ABO Typing with Multiplexes STRs

Few years back, Jiang et al. [31] developed a successful technique, in which both
ABO genotyping and STR analysis were combined in a single reaction, where a
forensic scientist could get both the information from a biological sample in a single
reaction. The developed technique has a combination of all the 15 autosomal STR
loci, gender-determining locus amelogenin, and markers for six ABO genotypes.
This was an important development as it is more accurate and has better confirma-
tory power than normal ABO blood group typing.
14 J. Imam et al.

1.3.1.5  Autosomal SNPs and Indels in Forensic Analysis

Many STRs have been developed (autosomal, MiniFiler, and sex chromosome
STRs) in lieu to overcome the challenges of generating DNA profiles from forensic
samples. This is only possible because of the greater variability of DNA polymor-
phisms. But forensic science always faces tough challenges in terms of low-level
DNA or degraded DNA, to obtain complete STR profiles. Degraded DNA or low
template (LT) causes problems in STR typing either with allele dropouts or allele
drop-ins which in many times are unable to get even with increased sensitivity and
miniSTR typing [1, 2]. The possible alternatives to increased PCR sensitivity of
STRs for degraded DNA or low template (LT) are to use SNPs and insertion/dele-
tions (indels). SNPs used in LT DNA can result in fewer allele drop-ins [5]. SNPs
(single-nucleotide polymorphisms) and indels (insertion/deletion polymorphisms)
are the most common short binary markers of the human genomic variations. SNPs
allow allele detection of comparatively small amplicon size ranges (about 41 bp)
and therefore can be a better option with highly degraded samples [6]. But still,
SNPs have not been whole heartedly adopted in forensic science as the marker of
choice, for highly degraded samples, as new and advanced STR technologies have
come up which can enhance the profiling performance even for highly degraded
DNA [7]. Therefore, SNPs are not very much suitable for normal forensic casework
and database entries when working with DNA mixtures. It is most suitable for iden-
tification of missing persons and relationship establishment. STRs present better
information than single SNPs, but as we increase the number of SNP markers, they
could provide better discriminating power. One of the valuable characteristics of
SNPs is variation at heterozygosity level of the genome. Other important advan-
tages of SNPs is that it does not require separations on the basis of size which makes
multiplexing and automation easier compared to the STR analysis and low mutabil-
ity rate making it more stable genetic marker [18].
Two SNP multiplexes have been developed for forensic identifications: a 52-SNP
assay developed by the SNPforID consortium, comprising a 52-plex PCR followed
by tandem 21- and 29-plex primer extension reactions [50, 61] and a 44-plex PCR
followed by tandem 18- and 26-plex extensions [41] based on the Kidd Lab forensic
identification marker panels, consisting of a list with almost twice as many ID-SNPs
than the 44 collated in this assay [53]. The 52-plex multiplex is best suited for
highly degraded DNA samples, i.e., for very old skeletal remains or body recovered
from the river or sea. This shows its importance in identification of missing person.
Here, it is also important to note that the number of SNPs required to match the
informativeness of STRs is higher in relationship testing than in identification appli-
cations. Table 1.3 listed a few techniques and variations of SNP analysis.
Indels (insertion/deletions) comprise about 5% of known polymorphisms in
human genome and are thus considered as potential markers in forensic identifica-
tion as they have combined the application of both SNPs and STRs [48]. The advan-
tages of indels over SNPs are as follows: first, ease of analyzing indels from very
short amplicon size as compared to SNPs, and, second, the ease of doing indel
analysis by combining the advantage of direct PCR-to-capillary electrophoresis
Table 1.3  List of few techniques and variants of SNP analysis
Sl. No. SNP techniques Details Application References
1 SNaPshot assay 1. Capable of multiplex SNP analysis in small amount of SNPs analysis for degraded DNA
template DNA samples samples
2. Highly sensitive, can analyze samples shorter than 70 bp
amplicons
2 TaqMan assay 1. Capable of multiplex SNP analysis in small amount of SNPs analysis for degraded DNA
template DNA samples samples
2. Highly sensitive, can analyze samples shorter than 70 bp
amplicons
3 Y-SNPs multiplex system 1. It is sensitive, human specific Applicable for Chinese Han
2. Informative in cases of degraded DNA and male-male mixture population
3. Resistant to high background of female DNA in male-female
mixture
4 Mitochondrial DNA 1. Mt DNA SNPs are lineage markers Lineage-specific SNP analysis for
SNPs (mtSNPs) 2. Not capable of genetic individualization degraded teeth and bone DNA
3. Can be employed for degraded bone and teeth samples samples
5 Nucleosome SNP assay 1. Based on the principle that histone-DNA complexes found in Best suited for highly degraded Freire-­Aradas
1  DNA Fingerprinting: Discovery, Advancements, and Milestones

(18-plex single-base nucleosomes offer protection from DNA degradation process DNA samples. It provides a new et al. [18]
extension assay) 2. It offers better results than other existing forensic SNP assays marker set that can be used to
supplement existing SNP assays
6 21 SNP multiplex system 1. Target amplicon length for 21 SNPs is from 63 bp to 192 bp This system provides a reliable
2. More efficient in the analysis of degraded DNA compared technique for individual
with standard STR typing identification of nondegraded and
degraded DNA samples
15
16 J. Imam et al.

t­yping, as this is not possible with SNP typing using SNaPshot assay. Different
indel typing kits were developed like Investigator DIPplex Kit (Qiagen, Hilden,
Germany) and indel-plex. Both have shown successful application for typing highly
degraded DNA samples in forensic science.

1.4  N
 ext-Generation Sequencing and Its Application
in Forensic Sciences

Next-generation sequencing (NGS), the technology which has overcome the limita-
tions of conventional Sanger sequencing, has grown rapidly in recent years in the
field of genomics research because of its high-throughput capacity and low cost and
ancient DNA analysis [54, 62]. Over the last 10 years, NGS methods and platforms
have evolved, and sequencing quality has now reached a level where NGS can be
launched in forensic science, and in the last 2 years, there has been an explosion in
scientific articles, with forensic applications of NGS. Since the number of casework
samples which require DNA processing is increasing day by day, the CE-based
methods which have fixed capabilities are sometimes unable to stand. Therefore,
NGS technology and platforms show promising results in DNA testing and identifi-
cation of missing persons, kinship testing, ancestry investigation, and other human
identification applications. In NGS technology, simultaneous amplification of mul-
tiple STR marker types and SNPs can be achieved in a single run for large number
of samples.

1.4.1  Next-Generation Sequencing Kits or Systems in Forensic

1.4.1.1  HID-Ion AmpliSeq Ancestry Panel

HID-Ion AmpliSeq ancestry panel (Life Technologies) enables simple and fast tar-
get selection of hundreds of SNPs using multiplex PCR. Thousands of primer pairs
can be used in a single tube for target amplification followed by next-generation
sequencing (NGS) on the Ion PGM™ System. This ready-to-use panel consists of
165 autosomal markers that provide biogeographic ancestry information. Fifty-five
of these markers were selected based on a poster by Dr. Kenneth Kidd [35, 36], and
123 markers were selected based on a publication by Dr. Michael Seldin [37]. Ion
AmpliSeq technology makes it possible to multiplex 165 PCR reactions in one tube
with only 1 ng of input DNA. With small amplicon sizes, the panel is optimized for
degraded DNA samples that provide the biogeographic ancestry information and
guide the investigation process.
1  DNA Fingerprinting: Discovery, Advancements, and Milestones 17

1.4.1.2  Precision ID NGS System for Human Identification

Precision ID NGS System for human identification (Applied Biosystem) for human
identification can help in solving tough cases by getting more information from the
challenging samples. It is the combination of Ion Chef System and Ion S5 or Ion S5
XL Systems with forensically relevant Precision ID panels that utilize Ion AmpliSeq
technology. It includes the same 21 autosomal STRs, along with Y indel and amelo-
genin sex marker found in the GlobalFiler DNA amplification kit. Instead of using
SE33, this panel includes nine additional multiallelic STR markers (for a total of 33
targets) to aid in mixture interpretation of complex casework samples.

1.4.1.3  ForenSeq™ DNA Signature Prep Kit

ForenSeq™ DNA Signature Prep Kit (Illumina) is developed by incorporating mul-

tiple STRs kits which include over 200 forensically relevant genetic markers in a
single, streamlined workflow, eliminating the need for multiple STR kits [64]. This
kit includes global autosomal STRs, Y-STRs, X-STRs, identity-informative SNPs
(iiSNPs), phenotypic-informative SNPs (piSNPs) (eye and hair color), and biogeo-
graphical ancestry SNPs (aiSNPs) in a single platform, which is not available with
traditional CE-based methods. This kit overcomes the limitations of other CE-based
kits for degraded, mixed, or PCR-inhibited samples.

1.4.1.4  PowerSeq™ Systems

PowerSeq™ systems (Promega) for forensic identification include three systems:

(a) PowerSeq™ Auto includes 23 STR loci and amelogenin, (b) the PowerSeq™
Mito system generates ten small amplicons (adapted from Eichmann and Parson)
covering the control region of the mitochondrial genome, and (c) PowerSeq™ Auto/
Mito/Y combines both sets of amplicons in one multiplex plus 23 Y-STR loci.
PowerSeq™ Auto system is a 24-plex kit for analyzing autosomal STRs, amelo-
genin, and DYS391. PowerSeq™ Mito system is based on sequencing of the
mtDNA control region (HV1 and HV2). PowerSeq™ Auto/Mito/Y system has been
configured for the simultaneous analysis of 22 autosomal STRs, amelogenin, 23 Y
STRs, and the control region of the mitochondrial genome [3, 17, 72].

1.4.2  Forensic Application Prospects of NGS Technology

Forensic science technology has embraced DNA technology as the main weapon to
address various crimes and help the judiciary. Today, PCR- and CE-based DNA typ-
ing is the backbone of forensic science and criminalistics. Various STRs
18 J. Imam et al.

Table 1.4  Forensic application prospects of NGS technology

Sl.
No. Application Advantages
1 STR typing High throughput, low cost, simultaneous detection of large numbers
of STR loci (autosomal and sex chromosome STRs), and the ability
to distinguish alleles with similar length facilitate the identification
of mixed DNA samples and analysis of complex paternity cases
2 Mitochondrial Important in maternal lineage identification; whole mitochondrial
genome analysis sequence for identification with high discrimination power
3 Y-chromosome Establishes paternal relationship between male individuals
analysis
4 Forensic NGS is suitable for whole-genome typing of microbial pathogens
microbiological during forensic and epidemiological investigations which can detect
analysis even the rare polymorphisms and thus give forensic data higher
resolution and greater accuracy for accurate identification of
criminals and biological terrorists
5 Animal and plant NGS technology has allowed the DNA typing in plant and animal
DNA analysis species identification
6 Ancestry studies and NGS technology for whole-genome sequencing will be helpful in
phenotypic determination of ancestry and personal characteristics like ethnicity,
inferences physical and physiological characteristics, and age
7 Epigenetic analysis Epigenetic changes like methylation pattern can be easily studied
and employed in forensic genetics with the aid of NGS technology
8 MicroRNA analysis NGS has proved to be very critical in analysis of millions of
miRNA sequences for rapid identification of organ and
developmental stage-specific expression and expression in different
diseases which is a powerful tool in forensic analysis

(autosomal, miniSTRs, sex chromosome, phenotypic STRs) are CE based, and CE

still is the method of choice for forensic analysis because of its accuracy, specificity,
discriminatory power, and easy handling. The use of NGS in forensic science as in
human identification (HID) and determination of phenotypic traits leads to its larger
application in forensic analysis. Definitely NGS technology has a lot of advantages
over tradition CE-based typing, and there is little doubt that NGS will be imple-
mented and used in forensic laboratories in the future. Table 1.4 presents the over-
view of NGS application prospects in forensic application.

1.5  RNA Profiling and Its Application in Forensic Science

The presence of biological evidences at the crime scene and its correct screening for
the possible source of DNA have always been the challenges for the forensic expert.
Many conventional biochemical and immunological assays are there for the screen-
ing of biological fluids, but they are time-consuming and laborious and even con-
sume the important evidentiary material. Because of this problem, many forensic
scientists bypass these preliminary screening processes and directly proceed for
1  DNA Fingerprinting: Discovery, Advancements, and Milestones 19

DNA analysis which many times lead to failure to provide the information regard-
ing the nature of crime [23]. In recent times, molecular approaches for the identifi-
cation of body fluids have been developed which have significantly improved the
sensitivity. The use of RNA profiling strategies is considered the better option for
the identification of forensically relevant biological fluids and tissues such as saliva,
vaginal secretions, menstrual blood, and skin [23].
The forensic identification of human body fluids and tissues by means of mes-
senger RNA (mRNA) profiling is a long-studied methodology that is being increas-
ingly applied to casework samples [68]. From a singleplex PCR technique to
multiplex RT-PCR platform, mRNA profiling has evolved in a big way in providing
expression of data on multiple genes simultaneously [40]. A single mRNA-based
system, 19-plex system, has been developed for the discrimination of common
forensic body fluids as well as skin cells [40]. This 19-plex system is able to estab-
lish both the donor and the cell type of the samples. This 19-plex mRNA assay
showed good results with body fluids, with high sensitivity and specificity. The
19-plex mRNA assay targets six different cellular origins to provide better assess-
ment, which is important in forensic casework.

1.6  Wildlife Forensics

Soon after the discovery, the forensic DNA profiling has revolutionized criminal
investigation process in humans. Today, forensic DNA analysis has become an
indispensable tool for different criminal cases and helps in the arrest of many per-
petrators as well as exonerations of many innocent individuals who were wrongly
convicted. As the forensic DNA analysis is growing day by day and its applica-
tions are also being introduced in other fields, its need is also felt in the investiga-
tion of wildlife-related crimes and wildlife conservation. Wildlife and their
products constitute the third most illegally traded commodity worldwide, after
arms and drugs [34, 42, 45]. An important difference between crimes against
humans and wildlife is that an animal becomes a “silent witness” to a human crime
scene and it’s like no “victim” to provide information regarding the investigation.
Another important issue with wildlife forensic science is that various species of
animals or plants have to be analyzed as against single species in human identifi-
cation cases [33]. Various sample types can be there in wildlife forensic science
like whole animals (dead or alive), skins or skeletal of animals, exoskeleton and
shells, and animal body parts such as the leg, wings, head, fur, scales, teeth, beak,
claws, skin, carcass, horns, organ parts, blood samples, and many more [33].
Therefore, good and improved preservation techniques are required to support
the prosecution in smuggling, poaching, and laundering of wildlife and their
­products [45]. In wildlife forensics, species identification is more important than
individual identification. In addition, geographical region of the samples can also
be analyzed.
20 J. Imam et al.

Table 1.5  List of currently available techniques in wildlife forensic analysis

Sl. No. Techniques used Application References
1 STR loci 1. Individual identification, i.e., Johnson et al. [33] and
investigation to determine if two samples van de Goor et al. [67]
are from the same individual
2. Pedigree analysis of a particular
individual
3. Dinucleotide repeat STRs are common
in identification of domesticated species
2 Mitochondrial 1. Most commonly used in forensic DNA Johnson et al. [33],
DNA (mtDNA) typing because: Ivanova et al. [27], Guha
typing  A. Mitochondrial loci are used for and Kashyap [22],
molecular taxonomy and phylogenetic Imaizumi et al. [26], Pun
 B. The presence of universal primers et al. [56] and Osborne
can be applied to unknown samples et al. [52]
 C. It works well with highly degraded
samples
2. Commonly used mitochondrial loci are
cytochrome b (cytb) and cytochrome
oxidase 1 (COI)
3. Other loci are ribosomal RNA genes,
D-loop region, and subunits of
mitochondrial encoded NADH
dehydrogenase
4. MtDNA sequencing for wildlife
forensic identification
3 Pyrosequencing 1. Direct sequencing of thousands of Karlsson and Holmlund
techniques small DNA fragments (degraded DNA) [34]
in a single run
2. Individual DNA typing of previously
unknown species
3. It is rapid, accurate, and flexible and
allows parallel processing
4 NGS or 1. Mass parallel sequencing for the Johnson et al. [33] and
high-­throughput identification of repetitive DNA Kidd et al. [35], [36]
sequencing sequences, for the identification of new
STRs and SNPs

DNA-based analysis is now frequently used and is most commonly applied in

species identification cases. Now it’s also being introduced for the analysis of geo-
graphic location of the species captured/claimed. The DNA markers applicable for
the wildlife forensic science are different from human identification (HID) markers.
As stated earlier, this is because of the complexities of species identification. STRs
and SNPs are used for individual identification, pedigree analysis, and assignment
of an unknown sample to a population [33]. Table 1.5 lists some of the currently
available techniques of wildlife forensic analysis.
1  DNA Fingerprinting: Discovery, Advancements, and Milestones 21

1.7  Conclusion

In this review, a brief overview of discovery, advancements, and milestones in the

field of forensic DNA analysis has been outlined. Many new discoveries took place
after the discovery of DNA fingerprinting, and these new approaches continue to be
explored for more effectiveness. DNA fingerprinting relies most basically on the
quality of DNA, DNA isolation from the biological samples has improved a lot, and
good-quality DNA can be extracted even from highly degraded samples. The dis-
covery of PCR and then advancements in real-time PCR have put the forensic sci-
ence on the road of successful DNA fingerprinting. The advancement in STR
technology and kits further improved the ability to decipher and interpret DNA
results from challenging samples which provided the opportunity for future advances
in forensic DNA analysis. The various forms of STR kits have revolutionized the
field of forensic DNA profiling. The evolution in capillary electrophoresis technol-
ogy and tough competition among the firms lead to cheaper and better kits, and this
is the reason why forensic DNA fingerprinting has advanced so much, and now, it is
helping the judiciary to solve the ever-increasing criminal and civilian cases. NGS
has also knocked the door of forensics, and the day is not very far when it will be
validated and incorporated as a conclusive technique to serve the judiciary system.
RNA profiling is also proving to be a helping hand of forensic science in many rel-
evant cases. The development in various molecular tools to investigate wildlife-­
related crimes has advanced the wildlife forensic analysis, and very soon, a
well-placed technique will be available for individual and species identifications.
Although various new techniques and scientific improvements are coming up, the
current methods of STR typing are reliable, valid, and widely applicable.

Acknowledgment  The authors are thankful to the Director of State Forensic Science Laboratory,
Ranchi, Jharkhand, for the support.

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