Enhancement STR profiling of teeth using magnetic

beads

Prepared By

Hind Hassan Al zahrani

Sarah Mosa Alatawi
Alanud Musaed Al-Sufyani

A research project plan submitted in partial fulfillment of the requirements for the
master's degree in (Genetic Fingerprinting and Forensic Diagnostics)

Under Supervision of

Dr. Amal Ahmed Al Yamani

Assistant Professor
of Genetic Engineering and Biotechnology Applications, Department of Biotechnology
College of Science - Taif University

1443-2022
 Dedication

To all those who are enlightened by the knowledge of the mind of others or

guided by the correct answer puzzled by his clients and showed with his grace the

humility of the scholars And his blessing is the mercy of those who know. To those who

taught me success and patience I dedicate this humble work to my father, who

provided me with support and to my mother, who gave me affection and love. I say to

them: You gave me life and hope and the emergence of a passion for knowledge .To my

brothers and all my family. And then to everyone who taught me a character who has

become a lightning rod that lights the road in front of me.

To candles that burn to light up for others .To everyone who taught me characters. I

dedicate this humble research to the lord almighty to find acceptance and success.

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 Acknowledgements

As we take our final steps in the master's thesis, we must return to the years we

spent in the university with our distinguished professors who have provided us with

great efforts to build the future generation of the nation. Before we proceed, we offer

our highest thanks, gratitude, appreciation and love to those who have received the

most sacred message in life.To those who paved the way for us the path of science and

knowledge .To all our distinguished professors .

"Be a scientist .. If you can't be educated, if you can't love scientists, if you

can't hate them"

I especially appreciate and thank: Dr . Amal Ahmed Al Yamani (university of Taif) Who

planted optimism in our path and provided us with assistance, facilities, ideas and

information, perhaps without feeling their role so we have all the thanks.

We also thank all those who helped to complete this

research and gave us help and provided us with a helping

hand and provided us with the necessary information to

complete this research, especially: colonel Khalid

Ghurmallah Alghamdi Chief of forensic Science training

center (criminal Evidences Administration, Jeddah Police).

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Abstract
Forensic dentistry, also known as forensic odontology, is primarily concerned with the
use of teeth and oral structures for identification in a legal context. Various forensic
odontology techniques help in the identification of the human remains in incidents such
as terrorist attacks, aeroplanes, train and road accidents, fires, mass murders, and
natural disasters such as tsunamis, earthquakes and floods, etc. (Disaster Victim
Identification-DVI).

Genetic markers can be generated from the teeth which can provide a DNA profile to
determine the age, sex, race/ethnicity, etc. which can give further clues regarding the
identity of the individuals. Especially, when dental remains are the only available
biological evidence for identification. Despite this, the main key challenge in DNA
analysis from teeth remains is the limited yield of DNA recovered from these samples
and the quality of DNA isolated to create a DNA profile. Therefore, enhanced DNA
recovery by extraction techniques would allow further advancements.

Here we present a genetic analysis and examination of DNA content and quality in the
different teeth samples of 30 volunteers from Taif ………. DNA was extracted from
coronal dentine, root dentine, cementum and pulp of 30 volenteers via a ……………
method. Then, real time quantification assay was used to establish Short Tandem
Repeat-based forensic DNA markers. We confirm that targeted sampling of teeth can
provide a reliable source of nuclear DNA for STR-based genotyping using ………….
extraction methods.

Keywords: Sex determination, Forensic odontology, Amelogenin,…………….

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‫الملخص العربي‬

‫‪5‬‬
Contents Page
Dedication ……………………………….…………………………………….....…… iv
Acknowledgments ...………………………………………………………….…….... v
Abstract ……………………………………………………………………………... vi
Arabic abstract ……...………………………………………….…………….……... 86
Table of contents………………………………………………….……………….…. xi
Chapter I: Introduction
Introduction ……………………………………………………………………...…. 15

Chapter II: Literature review

Literature review ………………………………………………………………..……..20
Structure of dental hard tissue …………………………………………………….…. 21
Dentin …………………………………………………………………………….…. .22

Enamel …………………………………………………………………………….…. 23
Cementum ………………………………………………...………………………….23
Dental pulp ………………………………………………………..………….……….. 24

DNA Evidence and Forensic Science …………………………………..…………… . 25

The role of AMEL gene for sex determination in forensic cases……….………. … 25

Use of Technology in Forensic Dentistry ……………………………………….……27

Chapter III: Materials and Methods

Sample Collection …………………………………………………………..…………30

Storage ……..…………………………………………………………………………..32

Reference samples and ethics approval ........................................................................33

Instrumentation ………………………………………………………………………..34
Method ……………………………………………………….………………………...35
Standard Curve …………………………………………………………….………..…40
Chapter IV: Results and Discussion
Conclusion ……………………………………………………….…………………….51
Appendix I ………………………………………………………………….…….……56
References……………………………………………………….………..…….………72

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List of Figures :
Figure 1Sexing of embryos using Amelogenin gene. Amplified amelogenin gene (AMELX, 262 bp;
AMELY, 202 bp). DNA ladder 100 bp (M). Male embryos lane (1), (3) & (5). Female embryos lane
(2) & (4). GAPDH (496 bp) lane (6) 15
Figure 2Normal tooth anatomy 20
Figure 3The AMELY and AMELX genes are located on the genome. A red line represents the
common location of Amelogenin on the Y and X chromosomes Elmrghni, S., et.al. (2012).(12) 26

Figure (1): Sexing of embryos using amelogenin gene………………………..……………………………………20

Figure (2): Normal tooth anatomy ……………………………………………….………………………………………….23

Figure (3): The AMELY and AMELX genes are located on the genome……………………………….…….29

Figure (4): Samples Preparation……………………………………………………………………………………….………31

Figure (5): Work flow over view…………………………………………………………………………..………….

……...35 

Figure (6): using Magnetic Beads for extraction bind to DNA On the right wall of tube …….…..37

Figure (7): Exclusion Based Sample Prep With Extraction ……………………..…………………….………….39

Figure (8): Washing and Elution…………………………………………………..……………………..…………….……..40

Figure (9): strategies used in real time PCR……………………….……………………………….…………………….43

Figure (10): Serial dilution of STD………………………………………………………………….………..….……………45

Figure (11): Principle of RT-PCR…………………………………………………………………...………………..………..45

Figure (12): shows how the ABI prism™ 7500 device ……………………………………………………………..46

Figure (13): shows the binding method of the messenger dye and the absorbent dye………….46

Figure (14) shows the presence of the unknown dye in the reaction mixture ……..47

Figure (15): Threshold Cycle ……………………………………………………………47

Figure (16): Amplification plot …………………………………………………………………………….…………….…...48

Figure (17): Analysis of Standard Curve………………………………………...…………………………………..……48

Figure (18): Schematic photograph showing replication of DNA by PCR………………..…………………49

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Figure (19): samples loaded on Genetic analyzer 3500 to detects the loci profiles…………………..53

Figure (24): show the Profiler Plus STR profiles corresponding to different teeth…….………..…….66

Figure (25): Profiler Plus STR from Male…………………………………………………………………………….…….67

Figure (26): Profiler Plus STR from Female……………………………………………………………………………….68

List of Tables

Table (1): …………………………………………………………….………….33

Table (2) :…………………………………………….…………………………50

Table (3): ………………………………………………….……………………60

Table (4):……………………….………………………………………………..72

8
Introduction:
Over the past 20 years, forensic genetic analysis has become the main technique in criminal
investigations for determination and human identification when working with human
biological traces and unknown corpses. The biological evidence used for DNA profiling
include hair, skin, semen, urine, blood, saliva and even body remains in burn cases.

The use of forensic genetics for identification purposes largely results from two rules. First, a
single cell with a cell nucleus contains the complete genetic information about an organism.
The second rule, which forms the basis of all branches of criminalistics, is Locard’s exchange
principle, formulated in 1928.4 According to this principle, contact between two objects
leads to an exchange of substances between them, so that the perpetrator both takes
material away from the scene of the incident and leaves traces of his or her presence. In this
manner, the biological matter left behind, containing complete information on the organism
of the perpetrator, enables individual identification (Kowalczyk, M et al.,2018) [38].

Forensic identification is on individualization through nuclear short tandem repeats (STRs),

which require relatively large quantities of DNA. Teeth are generally used for forensic
identifications and ancient DNA analyses because of it is a good source of DNA within the
mash, dentine, cementum and periodontal tendon filaments (Sweet et al., 1999). DNA could
be preserved for a long time, even after rotting of remains (Garish et al., 2010).

The anatomical location (within bony sockets) and morphological structure (particularly
covering of impenetrable enamel over the crown) provides special protection to
endogenous DNA especially from post-mortem degradation [18,31–33]. Furthermore, the
contrasting cellularity and mineral content of the four tooth tissues (enamel, dentine, pulp
and cementum) [34] creates a unique biochemical and anatomical setting to examine the
content.

In the forensic genetics, there are several molecular markers which are used for individual
identification by using various strategies have been adopted to utilize the minimum quantity
of sample to detect maximum variability in a single PCR reaction. It is known there are two
types of molecular markers can be divided one of them as known autosomal STRs and
another is DNA sequences that are found on the sex chromosomes such as Y-STRs, X-STRs

1
and AMEL gene. Apart from identifying age and ethnic origin, determining sex is an
important element of identifying human remains (Maulani et al., 2020) [39].

Short tandem repeats (STRs), which produce many possible genotype combinations [1] to
provide gene diversities for diverse human populations or species. Their analysis is based on
DNA typing of specific microsatellite loci with 2 to 7 base pairs length of core units repeat
both in autosomal and sex chromosomes. REF

The expanded Combined DNA Index System (CODIS) core STR loci, consisting of original
thirteen CODIS core loci (CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539,
D18S51, D21S11, FGA, TH01, TPOX, and vWA) and seven new additional CODIS core loci
(D1S1656, D2S441, D2S1338, D10S1248, D12S391, D19S433 and D22S1045), and 32 non-
CODIS STR loci (D1GATA113, D1S1677, D3S3045, D3S4529, D4S2366, D4S2408, D5S2500,
D6S1017, D6S1043, D6S474, D6S477, D7S1517, D7S3048, D8S1132, D9S1122, D9S925,
D10S1435, D11S2368, D11S4463, D12ATA63, D13S325, D14S1434, D15S659, Penta E,
D17S1290, D17S1301, D18S535, D19S253, D20S470, D20S482, Penta D, and
D22GATA198B05) were completely selected to Forensic Analysis System in ……………………..

Multiplecues SetB Kit for their forensic informativeness and rigorous genomic audit [24, 64].

Since the 1990s, the PCR-CE (capillary electrophoresis) profiling technique, which measures
length polymorphisms in STRs, has gradually dominated the forensic DNA practice [2, 3].

Due to the high variability in alleles and genotype distribution among unrelated individuals,
autosomal STRs DNA analysis serves as an effective human identification tool. The more STR
markers are investigated, the more informative individual profiles can be obtained, and the
less likelihood coincidence that can be observed. What is more, constant location at specific
loci in the genome promotes their stability for a long time even under unfavorable DNA
preservation conditions. REF

On the other hand, Y-chromosomal STRs (Y-STR) are used to determine the presence of
male DNA in mixed biological traces. However, the degree of Y-STR preservation is much
lower than for autosomal STR and this fact is especially noticeable in the analysis of highly
degraded DNA samples. However, Y-STR can be very useful in the determination of paternal

2
lineage relationships and these data can be used as supplementary information to
autosomal profiles. REF

Currently, Amelogenin is used as a sexing marker in most commercial short tandem repeat
(STR)-based tests. The Amelogenin gene, which is found on both the X and Y chromosomes
with a base pair size difference. However, large variations in Amelogenin-based sex
determination have been discovered, owing to population X and Y deletion. REF

The X-chromosome Amelogenin gene is 106 base pairs long, while the Y-chromosome
Amelogenin gene is 112 base pairs long. This information can help us distinguish between
male and female Amelogenins and emphasizes the fact that females have two identical
Amelogenin genes on the X-chromosome, whereas males have two different genes on both
sex chromosomes (Takayama, T, 2022).(6)

In the current study, human teeth samples have been selected to evaluate the most
effective method of nucleic acid extraction and purification for downstream quantification
and genetic profiling, also sex determination by qRT-PCR.

For that, this study has led to testing, and validation of advanced extraction along with new
instrumentation platforms providing a system that is a sensitive, rapid, and robust
extraction and genotyping of short tandem repeats (STR), by using magnetic beads to isolate
high quality of DNA from …. Tissues such as teeth for forensic DNA profiling which will have
led to a high and increasing demand for forensic DNA testing.

The aims and objectives

Aims:

•The aim of this study is to evaluate the DNA samples that will be extracted from the
different dental tissues by using a specific method namely magnetic beads that meet the
purpose of high-quality extracting DNA from dental tissues.
•Quality assessment of forensic DNA profiles.
•Investigation the role of AMEL gene as molecular marker can be used to detect the sex in
different forensic cases.
Objectives:

3
•It was considered a high level of sample homogeneity and minimal variation in the
environment or other factors was achieved to gain better results with this method.
•Isolate DNA from dental hard tissue (enamel and dentin)
•Genomic DNA isolation by using magnetic beads to separate DNA from proteins and RNA
from other tissue for purification DNA.
•Analysis the quality and purity of the DNA and use STR to determine the gender.

Problem Statement:

DNA extraction was becoming a complex technique to produce enough good quality
samples of DNA that can be used in the PCR (Liu et al., 2018) [21]. The accurate and actual
quality of the DNA was tough to extract, and, in some cases, it has been observed that the
quality of the DNA becomes deteriorated with the application of using chemical materials.
Therefore, the utilization of magnetic beads helps extract the DNA accurately.

Problem and the key issue:

The main issue that DNA extraction requires removal of impurities, and it requires high
proficiency, different strategies is followed to meet that demand which is aim to find
alternative techniques, or to improve the exciting methods. The main advantages of these
are related to the elimination of potential PCR inhibitors, and the feasibility of performing
automated, and more objective, laboratory procedures10,18-20

The common method is “phenol-chloroform”, “isoamyl alcohol” is used for extraction of

DNA, the main element that is used for extraction is “Lysis buffer” which contains “lysis” of
the cell membrane and the nuclear envelop, protein of the cell membrane are denatured by
using the “chloroform” and “phenol” which are known organic in nature (Sneha Singh et al.
2019) [22].

In Forensic genetics, DNA isolation is widely increasing in the forensic laboratory, which are
group in three different categories such as “Organic extraction”, “Solid phase DNA
extraction” and “use of resins” to find the accurate result quality standard forensic
laboratory is required (Austin et al. 2019) [23]. The validation of real time extraction is not

4
the qualification of the DNA and extraction of other useful DNA methods. These are the key
factors that have to take care in the DNA extraction, which is use for PCR diagnosis.

It is a worth having investigation because DNA identification is used to extract human tissue
and forensic stain and part has to be preserved over time. Therefore, to extraction of DNA
from teeth is highly extensive, here we divide the structure of DNA to yield and divide into
two quality and quantity for sample preparation. The use of this technique in the forensic
lab helps to identify the problem that is relating to amelogenin.

Nature and purpose of the research:

The main purpose of this research is to identify the role of the magnetic beads for extracting
the DNA from the teeth, to demonstrate the role of Short tandem repeat (STR) multiplexes
with the amelogenin (AMEL) gene as a gender marker have been used as a routine tool of
forensic DNA analysis. It has been reported that AMEL-based gender detection could
misidentify a known male as a female due to the dropout of amelogenin Y (AMELY) allele.

Achievement of this research:

The main achievement of this research is to illustrate the role of magnetic beads in the
extraction of DNA from teeth.

5
Literature review

The term "forensic" originated from the Latin word forensic, which means "before the
forum". It comes from Roman times; an adjective meaning association or uses within a
court (The Lawyer and Jurists, 2017) [25].

It refers to the application of scientific knowledge to determine legal disputes, both criminal
and civil (Morgan RM., 2019) [26]. It includes many disciplines such as criminology, digital
evidence analysis, fingerprint expertise, dentistry, nursing, pathology, toxicology, and
questionable documentation (Chisum WJ, Brent ET.2011) [27].

The analysis of human genetic variation has been a driving force behind advances in forensic
genetics. Until the 1980s, the identification (determination of sample properties) and
individualization (uniqueness) of biological evidence relied on the application of histology,
microscopy, immunology, biochemistry, and serology. Traditional genetic markers such as
blood group antigens, erythrocyte isoenzymes, serum/plasma proteins, hemoglobin
variants, and the human leukocyte antigen (HLA) system are used to personalize blood and
body fluid evidence (Gaensslen RE,2020) [28].

Another significant aspect of forensic science is forensic odontology that depends on the
skill of the dentist in personal identification during mass calamities, sexual assault and child
abuse to name a few.

This branch has been growing rapidly in its potential and its ability to identify the person by dental
remains, which is the only available evidence.

Teeth are the strongest part of the human body, and they can withstand big eruptions
without getting hurt. As a result, when other forms of identification, such as fingerprints and
facial features, are lost, the potential for DNA recovery from teeth increases.

6
Therefore, teeth are commonly used for forensic identifications and ancient DNA studies, because it
is considered a source of DNA, and offer great potential for examine short-term DNA degradation.

Their anatomical location (within bony sockets) and morphological structure (particularly covering of
impenetrable enamel over the crown) provides unique protection to endogenous DNA from post-
mortem degradation [18,31–33]. Furthermore, the contrasting cellularity and mineral content of the
four tooth tissues (enamel, dentine, pulp and cementum) [34] creates a unique biochemical and
anatomical setting to examine the content and postmortem degradation of DNA.

Structure of dental hard tissues:

There are 32 teeth in the permanent adult human dentition, 16 in the mandible and 16 in
the maxilla. There are four incisors, two canines, four premolars, and six molars in each
dentition. Food is sliced with the incisors, torn with the canines, grasped with the premolars,
and ground with the molars (i.e., masticating). There is a relatively little organic matrix in the
prisms, which are made up of hydroxyapatite crystallites. Dental enamel is an excellent
material for cutting and masticating food because of these qualities (i.e., processes that
involve friction and wear) Dentin, on the other hand, is harder than enamel but not as hard
as enamel. (Laurance-Young et al., 2011) [30].
Dentin

It is a heterogeneous substance with four major components: dentin matrix, dentinal

tubules, mineral (i.e. carbonate containing hydroxyapatite), and dentinal fluid. Dentinal
tubules (about 45 000 per mm2) Intertubular dentin is less mineralized than peritubular
dentin, according to histological investigation. Furthermore, root dentin has a different
matrix and mineral composition than coronal dentin. Waters provides an excellent overview
of tooth structure.

7
The dental crown is covered in enamel, which is a stiff, inert, and acellular substance. Dentin
makes up the majority of the tooth and serves as a harder foundation for the more stiff and
brittle enamel. Cementum covers the dentin in the root area, anchoring periodontal
ligament fibers that support a variety of dental movements during mastication and function
(Wang, X et al., 1999) [31].

Figure 2Normal tooth anatomy

The hard tissue of the tooth consists of enamel, dentine and cementum. Enamel is a hard
material composed almost exclusively of mineral — which is mainly composed of
hydroxyapatite (Ca10(PO4) 6(OH)2) — and covers the dentine on the crown of the tooth.
Cementum is a bone-matrix-like substance, composed of mineral and collagen; it covers the
root of the tooth (Pitts et al., 2017) [32].

Enamel
Dental enamel is composed of 96 percent inorganic elements, mostly in the form of
carbonated hydroxyapatite crystals, and is the most mineralized tissue in the human body.
Remains of the organic matrix and loosely attached water molecules are also found in
enamel. Enamel has an avascular and acellular structure that cannot renew or repair itself.
These characteristics represent the unique mechanisms that occur during enamel
development. The three basic steps of enamel creation (amelogenesis) are cyto-
differentiation, matrix secretion, and maturation. Secretion of the organic matrix from
ameloblasts (enamel producing cells) into the extracellular space proximal to the dentino-
enamel junction initiates amelogenesis (DEJ). Enamel matrix proteins, such as ameloblastin,
enamelin, and tuftelin, are non-collagenous proteins made up of hydrophobic amelogenins
and non-amelogenin proteins. Enamel proteinases, enamelysin (MMP 20), and Kal- likrein4

8
actively contribute in the selective breakdown and removal of the protein rich matrix during
mineral deposition.

The maturation stage of amelogenesis produces a very hard, highly miner- alized tissue
made up of exceptionally long, high aspect ratio hydroxyapatite crystals with a limited
number of organic components and water. Enamel crystallites are mostly calcium and
phosphorous in the form of hydroxyapatite (HAp), Ca10 (PO4)6(OH) 2, with little amounts of
sodium, magnesium, chlorine, carbonate, potassium, and fluoride. The organic matrix of
mature enamel makes up 1-2 percent of total enamel and serves as the apatite crystallites'
adhesive. (Goldberg, M et al., 2011) [33].

Cementum
The cementum seals the tubules of root dentin and serves as a support for periodontal
fibers that hold the tooth in its socket. Through the process of deposition, cementum
reverses root resorption by forming a smooth patch on thecemental surface.

Hard tissue covering tooth roots is composed of two types. The first type is called
intermediate cementum, which consists of cells originating from the inner epithelial layer of
the root sheath. A second type of cementum is cellular-acellular cementum, which is a
thicker deposit of a bone-like substance caused by cementoblasts that differentiate from
periodontal ligament fibroblasts.

By displaying cells in lacunae and cell processes within canaliculi, cementum simulates bone.
The cementum also appears to have incremental lines, but it does not have vascular and
neural supply that are typical of bone. With age, the cementum exhibits rough and irregular
surfaces due to an increase in resorption of the cemental surface as well as free, attached,
or embedded cementicles. These stones resemble the denticles in pulp. They are calcified
bodies that may be embedded, attached to cementum, or free in the periodontal ligament.
(Schroeder HE., 1991) [34].

Dental pulp
The dental pulp is a connective tissue that contains vascular, lymphatic, and nervous
elements that originate from neural crest cells and resides inside a cavity with rigid walls.
The pulp contains odontoblasts, highly specialized cells with a secretory function. In addition
to forming dentin, these cells also initiate the formation of enamel by interacting with the

9
dental epithelium early in tooth development. Also found in the pulp are fibroblasts,
undifferentiated mesenchymal cells, collagens I and II, proteoglycans, and glycoproteins and
water1.

A pulp's histologic structure provides insight into its unique architecture, which is suitable
for forming dentine and defending against pathogens.

Dentin tubules extend about one-third of the length of the odontoblasts; they form a
palisading layer along the walls of the pulp space. When the peritubular dentin gets thicker,
the tubules become more sclerotic. Most mineralized dentin is produced by odontoblasts.
As odontoblasts form semipermeable membranes, they play an important role in defense
since they express Toll-like receptors (see below), cytokines, and defensins, among other
immunologic mediators. Pulp is innervated by two types of sensory fibers: A*-fibers in the
peripheral area and C-fibers in the central area. The A*-fibers are responsible for the sharp
response to temperature changes. They extend between the odontoblasts, lose their myelin
sheath, and extend to a distance of 100 to 200 μm into the dentinal tubules. Patients with
symptomatic irreversible pulpitis may experience a dull ache coming from the unmyelinated
C-fibers. The pulp can also have AB-fibers and sympathetic fibers. The pulp vasculature plays
a critical role in its response to irritation (Yu, C., 2007) [35].

DNA Evidence and Forensic Science

DNA analysis of human skeletal remains are providing high-resolution insights into the
origin, migrations, health, biogeographic ancestry, phenotype and identification of deceased
individuals and populations for evolutionary, archaeological, medical, and forensic studies
(Higgins, 2015) [36]. Regardless of whether the remains were discovered at a crime scene,
after a mass disaster, or in other circumstances, timely and unambiguous identification is
critical. In some criminal incidents, such as mafia-related killings, offenders may employ
corrosive reagents to disintegrate the body to prevent victim identification. Forensic experts
are fascinated by the idea of destroying the body by soaking it in acid or other caustic
liquids.

In both natural and man-made disasters, dental recognition has long been used, particularly
in mass casualties during airline accidents. Teeth contain a large amount of DNA, which is
found in dentin, cementum, and periodontal ligament. The DNA extracted from the teeth

10
may be retained for a long time even after the remains have deteriorated. Human teeth may
be exposed to corrosive substances in forensic situations, industrial chemical spills at work,
and dental applications. For example, dental remains from the infamous Romanov case
provided conclusive evidence (Al-Owaidi., 2020) [37].

The role of AMEL gene for sex determination in forensic cases.

Both X and Y chromosomes are useful in determining the sex of the individual who was the
source of the material. Human sex identification is mostly based on the amelogenin gene
(AMEL) because of the differences in amplicons sizes observed between the X- and Y-specific
gene.9

The Amelogenin gene present on both the X chromosome, is known (AMEL X) and the Y
(AMEL Y) chromosomes of humans (Bailey et al. 1992) [40]. The (AMG) and its counterpart
(AMGL) have been pinpointed in the p22 region of the X chromosome and the Y
chromosome, respectively. On chromosome maps, one associated location is in the distal
short arm of the X chromosome in the p.22.1–p.22.3 region, and the other one is located
near the centromere of the Y chromosome (Sasaki and Shimokawa 1995).

However, it showed size differences between these two chromosomes and therefore, this
gene has been used to differentiate males from females (Mannucci et al. 1994; Haas-
Rochholz and Weiler 1997) [41, 42].

Therefore, the gender of an offender is frequently crucial preliminary information for an

inquiry. Many genetic profiling multiplex systems include Amelogenin testing to detect
whether the sample being analyzed is male or female. In addition, various companies
manufacture multiplex STR kits containing the Amelogenin system for individual and gender
identification, respectively.

Generally, several studies have confirmed AMEL gene accuracy in sex determination for
samples from the Tikrit population. Our experiments confirmed those genes were highly
reliable for human sex identification.

11
Figure 3The AMELY and AMELX genes are located on the genome. A red line represents the common location of Amelogenin
on the Y and X chromosomes Elmrghni, S., et.al. (2012).(12)

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

Are the sex chromosomes (for humans X and Y ) expressed in all somatic cells? Or just those
cells of organs that are related somehow to sexual phenotype (primary and secondary
sexual characteristics)?

● The X chromosome has hundreds of genes on it and many of these are controlling
characters related to organs other than the reproductive system.
● For example, gene for colourblindness present on X, is responsible for maintenance of
photoreceptors in healthy cone cells of retina.
● Then there are genes on X chromosome which allow hepatic cells to produce clotting
factors; hence mutation in such X linked genes cause hemophilia.
● So, it cannot be said that genes on X chromosome are expressed only in cells associated
with reproductive system; some of these genes are actually expressed in various somatic
tissues of both sexes through entire life.

12
 ● That is not entirely true for genes present on Y chromosome, because Y chromosome is
present only in males: i.e. Y linked genes are absent in all female cells.
● There is one AMELY gene on Y, that codes for extracellular enamel protein
amelogenin which expresses during development of tooth enamel, a nonreproductive
tissue but this is rather an exception.
● Most of the Y-linked genes are expressed in organs of male reproductive system such as

testes, prostate gland, etc.

● Expression of SRY gene of Y chromosome initiates sex differentiation very early in embryonic
life.
● So, expression of genes on Y chromosome are restricted in male reproductive organs only,
and some of these genes express for only a brief period of embryonic life.

Figure 1Sexing of embryos using Amelogenin gene. Amplified amelogenin gene (AMELX, 262 bp; AMELY, 202 bp). DNA
ladder 100 bp (M). Male embryos lane (1), (3) & (5). Female embryos lane (2) & (4). GAPDH (496 bp) lane (6)

.  REF

13
Chapter III: Methods and tools

14
Materials and reagents:

Sample Collection
The examined material consists of 30 samples of healthy human teeth (premolars and
premolars) obtained from adult and old patients (female and male) aged between 25-72 in
dental surgery clinics in Taif. All patients provided their consent to participate in this study.
All studied teeth were extracted for valid clinical reasons (periodontal disease or
orthodontic treatment).

Figure (4) : Samples Preparation

Table(1): Types of teeth samples were collected from 30 volunteers from Taif Society.

(their age and gender).

No. Of Age Sex The type of tooth and location

Sample

1 45 M Upper left third molar

15
2 33 M Upper right first premolar

3 52 M Lower right third molar

4 40 M Upper left first molar

5 55 M Upper right first premolar

6 36 M Lower right second molar

7 72 M Upper left second molar

8 65 M Lower right third molar

9 44 M Upper left second molar

10 25 M Lower right second molar

11 43 M Lower right first molar

12 60 M Upper left third molar

13 51 M Upper right third molar

14 29 M Lower right second molar

15 66 M Lower lift second molar

16 28 M Lower right first molar

17 44 M Lower lift third molar

18 54 M Upper right third molar

19 50 M Lower right third molar

20 49 M Lower lift second molar

21 30 M Upper left first premolar

22 61 F Lower right third molar

23 70 F Lower lift third molar

24 69 F Upper left third molar

25 56 M Lower right third molar

26 43 F Lower lift second molar

27 38 M Upper left third molar

28 49 M Upper left first molar

16
 29 46 M Upper left third molar

30 37 F Upper right third molar

Storage:

Immediately after extraction, each tooth was rinsed profusely with sterile saline. These
solutions were rubbed onto the root surfaces using a sterile cotton applicator cleaned with
sterile water only (we did not use abrasives, bleach, sandpaper, or any deep cleaning
mechanisms), and stored for at least 24h at −80 °C.

Kits:

● PrepFiler BTA Lysis Buffer (Applide biosystem, LOT 1904108, UK)

● Quantifiler Human DNA Quntification Kit (Applide biosystem, LOT 2109230, UK)
● AmpF STR Identifiler Plus PCR Amplification Kit ( Applide biosystem, LOT 201162,
UK)
● AmpF STR Identifiler Plus Kit PCR Reagents ( Applide biosystem, LOT 2109160, UK)

Reference samples and ethics approval:

DNA profiles obtained from the teeth of the patients. This project was approved by the
Research Ethics Committee of the Taif University. This study was carried out at Forensic
science Training Center Laboratory in Jeddah.

17
 Instrumentation:

Tooth
cutting ,Grinding

Spex certiprep
the teeth after cutting by saw

DNA Extraction Daigger vortex genie ThermoscientificPrepSEQ™ with Proteinase K

DNA
Quantification

Real time pcr system7500 Applied Biosystems™ Quantifiler™ DNA kit

DNA
Amplification

AmpFLSTR™ Identifiler™ Plus PCR

pcr system 9700 Amplification

Profiling

Applied Biosystems™ GeneScan™

Genetic Analyzer3500
600 LIZ™ dye Size Std

Figure(5): Work flow over view.

1
 Method:

Cryogenic Grinding
Each root was ground using a 6750/MILL freezer (SPEX CertiPrep), which was run with liquid
nitrogen to cryo-grind all dental root fragments and transferred to individual cylindrical
polycarbonate milling flasks. Frozen samples were ground with magnetically fixed stainless
steel end stoppers to seal the flask (volume = 25 ml). The flask was immersed in liquid
nitrogen during operation.

The program used for cryogenic grinding included the following parameters:

1. Precooling for 15 minutes (before grinding, when the vial was immersed in liquid
nitrogen for freezing samples).
2. Grinding for 1 minute (resulting from impact bar motion in each working cycle).

and the resulting powder was weighed in a precision balance Around 0.6 g (max. = 1.02 g;
min. = 0.34 g) of root powder was used for DNA Extraction. (Alves, et al. 2009) [47].

DNA Extraction
DNA was extracted from root powder samples by using the PrepFiler DNA Kit (Applied
biosystems) REF 444139 following the protocol recommended by the manufacturer to
maximize DNA extraction from teeth according to the manufacturer’s instructions (Zoranjic,
et al 2021) [48], following steps:

▪ 250 ml of BTA lysis buffer (Applied Biosystem) Lot No. 2008058.

▪ 7 ml of proteinase K (Applied Biosystem) Lot No. 4441400.

▪ 5 ml of DTT (DL-Dithiothreitol) Promega Lot No. 286351.

- They were added on the samples, followed by vigorous vortexing (DAIGGER Vortex Genie 2)

for 15 seconds.

- Then incubated at 56˚C at 1100 rpm (thermos scientific) at overnight.

2
- PrepFiler Express™ Forensic DNA Extraction System (Applied Biosystems) uses Patented

Magtration (magnetic filtration). The elution volume in all cases was 25µl. And finally, the

extracted DNA were stored at 4 ˚C.

Figure (6): using Magnetic Beads for extraction bind to DNA On the right wall of tube.

1. Magnetic Beads are Added to the Samples

When the magnetic beads are added to the samples, the DNA will bind to them. The beads

have several layers, with the core wrapped by a layer of magnetite. The design of the

surface of the magnetic beads is crucial to how the binding will occur. The greater the

surface area, the higher the chance that the target material will bind to the beads.

2. An External Magnetic Field is Introduced

A permanent magnet is used to attract the magnetic beads to the side of the containing

tube. With the nucleic acid material bound to them, the beads are practically immobile on

the edge of the containing tube.

3. Washing and Elution

3
The sample is washed while the beads remain immobilized. An elution buffer is then

introduced to wash away any unbound proteins. Finally, the magnetic field is removed, and

the DNA is released as a purified sample ready for measurement and analysis.

Figure (8): Washing and Elution

Below are some quick reminders about the use of magnetic beads:

4
● Handling magnetic beads: Follow the manufacturer's recommendations, such as the need to

wash beads before use and resuspend them to remain homogenous. Pipetting using

proper technique is also necessary to avoid bubbles.

● Capture: Allow a sufficient amount of time to pull out the beads from the suspension. When

pipetting, make sure that the tube is on the magnet, or the beads may drop into the

solution

● Washing and Elution: To lower non-specific binding, you can increase the number or

duration of washes. Ensure that you use enough volume of buffer to elute your desired

product.

Optimizing Pipetting Procedures Using Magnetic Beads for DNA and RNA Extraction

Proper liquid handling is crucial for manual or automated DNA or RNA extraction using

magnetic beads. This is where proper pipetting techniques and having the right

equipment count the most.

Important:

In the work steps, the steps and method of work are written only, without a description
of the principle or other reasons.

Please review the steps, are they correct and complete?

5
Quantification strategies in real-time RT-PCR

Accurate quantification of DNA samples is an important step in obtaining accurate and

reproducible short tandem repeat (STR) profiles. Quantitative real-time-PCR has improved
the speed and accuracy of DNA quantification over earlier methods, albeit at a significantly
greater cost per reaction (Zoranjic, et al 2021) [49].

Real-time PCR assays are commonly used to detect small changes in gene copy-number and
expression, and the reference values used in those applications are often in the femtogram
and picograms ranges and use standard curves based on a log scale. Adding the correct
amount of DNA to the PCR will result in high-quality results in the shortest time (Haque, K et
al 2003) [50].

All human genomic DNA content of all samples was quantified using the Quantifiler DNA
Quantification Kit REF 4343895 on an Applied Biosystems 7500 RealTime PCR System as

● 15 µl quantitative mix was added for each sample (Applied Biosystem) Lot No.
2109230

● 10 µl per sample Quantifiler Human DNA Quantification Kit

● 10 µl in the stander (16 standers: they are known samples of the primer of different
concentrations)

● The Plate was put in a device Real-Time PCR System 7500.

Examine the standard curve results to evaluate the quality of the results from the
quantification standard reactions.

Quantification standards were prepared by serial dilution of the Quantifiler DNA standard
Kit (Applied biosystems) Lot 2010061, UK. Included ranging from 50 ng/µL (Std. 1) to 0.021
ng/µL, or 21 pg/µL (Std. 6

Preparation of dilutions for DNA standard curves

1. Equilibrate rehydrated PCR Quantification Standard and Tris Buffer at room temperature.

6
2. Label 1.5 ml reaction tubes consecutively. Pipette 90 µl Tris Buffer to each reaction tube

3. Vortex PCR Quantification Standard briefly and spin down.

4. 1st dilution: pipet 10 µl of the undiluted PCR Quantification Standard to the first reaction
tube, close the tube and vortex briefly. Spin down briefly.

5. 2nd dilution: pipet 10 µl from the first dilution to the second reaction tube, close the tube
and vortex briefly. Spin down briefly.

6. 3rd dilution: pipet 10 µl from the second dilution to the third reaction tube, close the tube
and vortex briefly. Spin down briefly.

Figure(10) :Serial dilution of STD

7
Figure (11): Principle of RT-PCR.
(A) TaqMan probe. The principle of TaqMan probe is illustrated. Note that fluorescence
emitted from Fluorophore is quenched before it is removed by Taq polymerase. (B) A
diagram showing the relationship between the amount of DNA measured by copy number
and the amplification cycle.

8
Figure (12): shows how the ABI prism™ 7500 device

Diploid nucleotide reagents:

Double Dye nucleotide reagents are among the most popular selective reagents ever.

Reagents used a radioactive dye linked to a reagent with a sequence complementary to one

of the resulting cassettes. The messenger dye is a color-creating dye such as FAM, Thus, the

fluorescent emitted by the FAM reporter dye is inversely increased and the fluorescent

emitted by the TAMRA dye is correspondingly decreased.

9
Figure (13): shows the binding method of the messenger and the absorbent dye in the
diploid nucleotide

DNA Amplification Process:

▪ Prepare reagents. Thaw the AmpFlSTR® Identifiler® Plus Kit Master Mix and the
AmpFlSTR® Identifiler® Plus Kit Primer Set, then vortex 3 seconds and centrifuge
briefly before opening the tubes.
▪ Vortex the reaction mix for 3 seconds, then centrifuge briefly.
▪ Dispense 15 μL of the reaction mix into each reaction well of a MicroAmp® Optical
96-Well Reaction Plate or each MicroAmp® tube.
▪ Prepare the DNA samples:
▪ Dilute a portion of the test DNA sample with free water nuclease so hat 1.0 ng of
total DNA is in a final volume of 10 μL. Add 10 μL of the diluted sample to the
reaction mix. Plate or each MicroAmp® tube.
The final reaction volume (sample or control plus reaction mix) is 25 μL.

Passive Reference:

The unknown reference is the ROX (9-2) dye present in the 10X TaqMan Buffer buffer
solution and is not affected by the activity of the polymerase enzyme removal unit during
the polymerase chain reaction. The goal of developing this dye is to normalize the Nomalize
signal of the messenger dye during the data analysis process. This normalization process is
necessary to correct the fluctuation and difference in the intensity of the fluorescent
emitted by the FAM dye due to the change in the concentration or size of the sample.

10
Figure (14) shows the presence of the unknown dye in the reaction mixture.

Threshold Cycle CT:

The CT value is the period (of cycles of the thermal circulator) during which the first
significant determination of the difference in the fluorescent intensity ∆Rn occurs. The
minimum cycle CT is defined as the standard deviation rate of the correspondent finger
normalization value Rn multiplied by the form adjustment factor

Figure(15): Threshold Cycle

Figure(16) :Amplification Plot The x-axis represents the number of thermal cycles and the y-
axis represents the normalization values of the correspondent dye Rn. We note that the CT
minimum cycle that the device starts to identify a signal is associated with an increase in the
cumulative magnification product.

11
DNA Amplification

Samples were amplified on a GeneAmprep PCR System 9700 with an input of 0.5 ng or a
maximum DNA volume of 15 µL for low quantity samples. Reactions were cycled with the
following cycle conditions: 50°C for 2', 95°C for 10', followed by 40 cycles of 95°C for 30" and
60°C for 1'. QG reactions were cycled using the "Absolute Quantification" assay setting on
the instrument. Data were analyzed using Sequence Detection Software 2.0. Using
AmpFiSTR Identifiler Plus PCR Amplification Kit (Applied Biosystems) REF 4427368.

The AmpFℓSTR® Identifiler® kit (Identifiler, Applied Biosystems, and Foster City, CA, USA)
primer set was chosen for the development of a fast PCR protocol. The loci amplified by this
kit include the 13 Combined DNA Index System (CODIS) core STRs loci, widely used in the
sex-marker Amelogenin (Foster, A., & Laurin 2012) [51].

Figure( 18 ):Schematic photograph showing replication of DNA by PCR

12
The following table shows the loci amplified, their chromosomal locations, and the

corresponding fluorescent marker dyes. The AmpFlSTRTM IdentifilerTM Plus Allelic Ladder is

used to genotype the analyzed samples. The alleles contained in the allelic ladder, and the

genotype of the AmpFlSTRTM IdentifilerTM Plus Control DNA 9947A are also listed in the

table.

Table (2 ): AmpFlSTRTM IdentifilerTM Plus Kit loci and alleles.

Locus Alleles included in Identifiler™ Control

Chromosome Dye DNA
designation location Plus Allelic Ladder label 9947A
6-
D8S1179 8 8, 9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 FAMT 13
M
D21S11 21q11.2-q21 24, 24.2, 25, 26, 27, 28, 28.2, 29, 29.2, 30, 30
30.2, 31, 31.2, 32, 32.2, 33, 33.2, 34, 34.2,

35, 35.2, 36, 37, 38

D7S820 7q11.21-22 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 10, 11

CSF1PO 5q33.3-34 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 10, 12

D3S1358 3p 12, 13, 14, 15, 16, 17, 18, 19 VICTM 14, 15
TH01 11p15.5 4, 5, 6, 7, 8, 9, 9.3, 10, 11, 13.3 8, 9.3
D13S317 13q22-31 8, 9, 10, 11, 12, 13, 14, 15 11

D16S539 16q24-qter 5, 8, 9, 10, 11, 12,13, 14, 15 11, 12

D2S1338 2q35-37.1 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 19, 23

26, 27, 28
D19S433 19q12-13.1 9, 10, 11, 12, 12.2, 13, 13.2, 14, 14.2, 15, 14, 15
NEDTM
15.2, 16, 16.2, 17, 17.2

vWA 12p12-pter 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 17, 18
22,

23, 24

TPOX 2p23-2per 6, 7, 8, 9, 10, 11, 12, 13 8

13
 7, 9, 10, 10.2, 11, 12, 13, 13.2, 14, 14.2,
D18S51 18q21.3 15, 15, 19

16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27
AmelogeninX: p22.1-22.3 X, Y PETTM X
Y: p11.2

D5S818 5q21-31 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 11§§

FGA 4q28 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 26.2, 23, 24
27, 28, 29, 30, 30.2, 31.2, 32.2, 33.2, 42.2,

43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 50.2,

51.2

▪ Seal the MicroAmp® Optical 96-Well Reaction Plate with MicroAmp® Clear Adhesive
Film or MicroAmp® Optical Adhesive Film or cap the tubes.
▪ Vortex the reaction mix for 3 seconds, then centrifuge the tubes at 3000 rpm for
about 20 seconds.
▪ Amplify the samples in a GeneAmp® PCR System 9700 with the silver 96-well block.
Scientific, T. F. (2012) [52].

DNA Separations:

Following PCR amplification, the overall length of the STR amplicon is measured to

determine the number of repeats present in each allele found in the DNA profile. This length

measurement is made via a sized-based separation involving gel or capillary electrophoresis

(CE). Each STR amplicon has been fluorescently labeled during PCR, since either the

forward or reverse locus-specific primer contains a fluorescent dye. Thus, by recording the

dye color and migration time of each DNA fragment relative to an internal size standard, the

size for each STR allele may be determined following its separation from other STR alleles.

14
Commonly used instruments for STR allele separation and sizing include the ABI PRISM

3500 genetic analyzers (Applied Biosystems) .

There are a number of both biological and instrumental artifacts that often must be sorted

through in order to generate a complete and accurate STR profile. Biological artifacts include

stutter products, split peaks from incomplete adenylation, triallelic patterns, and variant

alleles containing mutations in the repeat or flanking regions that cause an allele to be off-

ladder. Instrumental artifacts arise from voltage spikes, dye blobs, and bleed-through between

dye colors.While multicolor fluorescence detection CE instrumentation, such as the ABI

PRISM 3500 genetic analyzer, presently dominate the field, efforts are ongoing to develop

microchip CE platforms to perform high-resolution DNA separations with eventual

integration of the PCR amplification and CE separations.A total of 10 μl of sample injection

volume was used with 1 μl of PCR product, 10 μl of the Hi-Di formamide (lot no. 1411363)

and 0.2 μl of the GeneScan 600 Size standard (lot no. 00924009).. Data analysis was carried

out using GeneMapper ID v3.2 software. Alleles were designated on the basis of the number

of allele repeats with the help of an allelic ladder provided by the manufacturers of the

multiplex systems used for the study. Peak detection threshold was set to 50 RFUs for allelic

designation.

Prepare the sample for Electrophoresis:

▪ Calculate the volume of Hi-Di™ Formamide

▪ Pipette the required volumes of components into an appropriately sized
polypropylene tube.
▪ Vortex the tube, then centrifuge briefly.
▪ Into each well of a MicroAmp® Optical 96-Well Reaction Plate,
add:
● 9 μL of the formamide: size standard mixture
● 1 μL of PCR product or allelic ladder

15
▪ Seal the reaction plate with appropriate septa, then briefly centrifuge the plate to
ensure that the contents of each well are collected at the bottom
▪ Heat the reaction plate in a thermal cycler for 3 minutes at 95 °C.
▪ Immediately place the plate on ice for 3 minutes.
▪ Prepare the plate assembly on the autosampler.

Figure (19 ): samples loaded on Genetic analyzer 3500 to detects the loci profiles.

16
 Chapter IV
Results and Discussions

17
Samples Qty Human Vol. of DNA Vol. of TE
Buffer

TAIF 01 0.2169 10 0
TAIF 02 0.0252 10 0
TAIF 03 0.0014 10 0
TAIF 04 41.428 0.07 9.93
TAIF 05 0.0938 10 0
TAIF 06 3.9312 0.76 9.24
TAIF 07 0.0682 10 0
TAIF 08 1.5826 1.9 8.1
TAIF 09 3.2651 0.92 9.08
TAIF 10 9.9627 0.3 9.7
TAIF 11 0.1935 10 0
TAIF 12 2.1042 1.43 8.57
TAIF 13 4.5372 0.66 9.34
TAIF 14 0.8542 3.51 6.49
TAIF 15 0.8152 3.68 6.32
TAIF 16 1.1988 2.5 7.5
TAIF 17 0.421 7.13 2.87
TAIF 18 0.0786 10 0
TAIF 19 2.3884 1.26 8.74
TAIF 20 0.0184 10 0
TAIF 21 0.1603 10 0
TAIF 22 0.0103 10 0
TAIF 23 0.4493 6.68 3.32
TAIF 24 0.3584 8.37 1.63
TAIF 25 0.8414 3.57 6.43
TAIF 26 1.8069 1.66 8.34
TAIF 27 1.2297 2.44 7.56
TAIF 28 3.436 0.87 9.13
TAIF 29 1.9325 1.55 8.45
TAIF 30 3.1412 0.96 9.04

18
 Results

This study shows that the extraction yield of the pulverized tooth, and the extraction

time was shortened and considerably reduced. On the other hand, we successfully get full

profiles for all teeth we used in our study by using magnetic beads which enhance the

quality of the extraction yields and enable us to get the profile for Representative Short

Tandem Repeats (loci D8S1179, D21S11, D7S820 , CSF1PO , D3S1358, TH01, D13S317,

D16S539, D2S1338, D19S433, vWA , TPOX , D18S51, Amelogenin , D5S818 and FGA) profiles

of DNA extracted from dental remain. This new method could be useful to improve DNA

extraction in special cases such as ancient or badly preserved human remains, which in

many situations.

There was no evidence of false positive or false negative results, and no substantial evidence
of preferential amplification within a locus, which indicates to the results obtained were
reliable.

The criteria entail the following:

(a) The number of peaks at a locus

(b) The relative height of stutter products

(c) Peak height ratios

Stochastic threshold levels and the efficiency of non-templated nucleotide addition also aid
in profile evaluation. While these guides should be applicable to the majority of samples for
determining the presence or absence of multiple contributors, hereafter, and these criteria
can be used when assessing the relevance of the data at each locus. This study reinforces
previous findings that multiplex STR typing is sufficiently robust for implementation into
forensic laboratories and will be effective for characterizing most human biological teeth
encountered at crime scenes.

19
The resulting STR profiles were characterized by strong allelic peak heights that were well
balanced at all 16 of the Profiler Plus loci with a slight but expected trend toward lower peak
heights for alleles at the larger loci.

We can find out homozygous and heterozygosis by looking at the peaks under each specific
locus. If a locus have a single peak then it is homozygous while two peaks under one locus
means that it is heterozygous. (Butler, J. M. 2007) [55].

However, there were no allele drop out, significant peak imbalance, split peaks, enhanced
stutter effects or other stochastic effects associated with having significantly over or under
estimated the amount of DNA needed to generate a high quality STR profile.

The Profiler Plus STR profiles corresponding to different teeth samples that were obtained
from both genders in different ages.

Sex chromosomal STR

The allele profile for male samples at Amelogenin locus was X, Y. The Genotyper™ profiles at
the Samples (1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,1,17,18,19,20,21,25,27,28,29) which
exhibited amplification of both X and Y alleles. The results are presented in Figure
The sample was taken from a Male. The number of homozygous loci: - 5 ; the number of
heterozygous loci: - 11;

Explanation: The AML loci have 2 peaks and underneath the peaks we can see that the
alphabets X and Y are written which means that the samples belong to males because a
male person has the karyotype of X, and Y chromosome, and the AML is used as a marker to
determine the sex of the people from which the sample has come from.

A simplified schematic of how an electropherogram for a single STR is generated. In the

individual has one allele with 11 repeats and another with 12, 21.2, 24 repeats. During PCR,
the DNA fragments are amplified, and a fluorescent tag is applied. After PCR, running
fragments of known DNA sizes (known as a DNA ladder or DNA standards) through the
system allows scientists to determine the sizes of alleles in the DNA samples. The alleles are
shown in the electropherogram as labeled peaks. This technique also allows measured as
number of base pairs versus time. Computer software uses data from the detector to
determine the sizes of alleles in the DNA samples, in this result 150 bp (allele 11,12) and 249

20
 bp (allele 21.2,24). The X chromosome-specific allele was amplified in all samples (Samples
22,23,24,26 and 30), which is known for female.

is known for female.

F05 TAIF-22
… D5S818 FGA

10 150
0 200 250 300

2600

10 11
23
X 24
4

The AMAL loci have One peak and underneath the peaks we can see that the alphabet X are
written which means that the samples are taken from a female because females have the
karyotype of X chromosome, and the AMAL is used as a marker to determine the sex of the
people from which the sample has come from. We can find out homozygous and
heterozygosity by looking at the peaks under each specific locus. If a locus has a single peak,
then it is homozygous while two peaks under one locus means that it is heterozygous.

Ageing
A comparative study of the ageing process on all samples demonstrated that all different
samples were fairly resistant to the effects of age. Especially, all samples were prepared and
examined under identical conditions showed similar patterns of amplification. Gender

21
determination in human societies has been important in evolutionary processes, prenatal
sex determination, Sex-Linked (X and Y chromosome), and forensic sex determination. There
are several advanced molecular biology approaches for sex identification that produce more
reliable and repeatable results. Our findings confirmed the utility of Amplification PCR of the
X and Y chromosomes in determining the sex of individuals utilizing the AMEL gene.

Discussion

In the present study has shown that the separation of nucleic acid is a highly

dynamic field of research and development. An increasing number of commercial vendors

offer magnetic particles, also in the form of a kit that is optimally suited for the application

desired. The increasing number of publications shows that magnetic particles of higher

potential are currently under research. Materials with more specific-binding properties and

a better separability are promising approaches. A higher degree of automation leads to

systems analysing a larger number of samples and higher sample volumes at the same time.

The complete process for STR typing includes sample collection, DNA extraction, DNA

quantitation, PCR amplification of multiple STR loci, STR allele separation and sizing, STR

typing and profile interpretation, and a report of the statistical significance of a match (if

observed). Magnetic Beads enhance the detection of STR alleles in general and particularly

Amelogenin (AMEL).

CONCLUSION

The present study was designed to determine the SRY and AMEL gene accuracy in sex determination

for samples from the Tikrit population. Our experiments confirmed those genes were highly reliable

for human sex identification with no positive or negative false results. Nevertheless, AMEL gene Y

22
was absent from the two male samples which makes these findings less acceptable. Therefore,

further research is required to establish the efficiency of the AMEL gene as a forensic genetic marker

for sex determination. Finally, the findings make an important contribution to the field of DNA

database in the Tikrit population, with relevant results from other previous studies [42,43].

23
Main Recommendations:

1. There is a need for more studies that must be conducted on a larger number of samples

to obtain a sufficient statistical analysis of the individual pattern in the distribution of

Saudi society.

2. Paying attention to the STR allele frequency of the Saudi people due to its stability

characteristic, and therefore it will be of great benefit to explore genetic diversity or

homogeneity between different tribal groups.

3. Forensic DNA profiling utilizes autosomal short tandem repeat (STR) markers

to establish identity of missing persons, confirm familial relations, and link persons of

interest to crime scenes

4. Maintain a sterile environment when handling DNA to avoid any contamination from

DNases

5. Ensure that no DNase is introduced into the solutions supplied with the kit

6. Make sure that all equipment coming in contact with DNA is sterile, including pipette

tips and tubes

7. Perform the recommended wash steps during the purification to obtain the best results

24
Appendix I: Raw Data

25
 Result :

Sample file Sample Name SOS SQ SSPK MIX OMR CGQ

A01
… D5S818 FGA

100 150 200 250 300 350 400

8000

X 10 21 24
4
Y 12

B01
… D5S818 FGA

100 150 200 250 300 350 400

2000

XX 10 23
Y 24
11 4

26
 D01 TAIF-0
… D5S818 FGA

100 150 200 250 300 350 400

6200
3100

21 25
X 12 4
Y
X

F01 TAIF-0
… D5S818 FGA

100 150 200 250 300 350 400

4600

26
X 12 4
Y 25
X

H01 TAIF-0
… D5S818 FGA

27
 100 150 200 250 300 350 400

1400

20 25
X 12 4
Y
X 13

B02 TAIF-0
… D5S818 FGA

100 150 200 250 300 350 400

4700

9 21 24
X 13 4
Y
X

D02
… D5S818 FGA

100 150 200 250 300 350 400

4400

28
 0

XX 9 22
Y
11

H02 TAIF-0
… D5S818 FGA

100 150 200 250 300 350 400

2800

21 23
X 12 4
Y
X

G03 TAIF-0
… D5S818 FGA

100 150 200 250 300 350

9900

22 24
X 13 4
Y 12
X

29
 H03 TAIF-1
… D5S818 FGA

100 150 200 250 300 350

8000

23
X 11
Y
X 12

A04 TAIF-1
D19S433 vWA 200 TPOX250 300
D18S51

100 150 350

9000

15 8 11 15
13 4 18
14 4
X

B04 TAIF-1
… D5S818 FGA

30
 100 150 200 250 300 350

2400

11 21.2
X
24
Y 12 4
X

31
 C04 TAIF-1
… D5S818 FGA

100 150 200 250 300 350

11000

22
X Y 12 23
X 4

D04 TAIF-1
… D5S818 FGA

100 150 200 250 300 350

7000

24
X Y 11 26
X 4

32
 E04 TAIF-1
… D5S818 FGA

100 150 200 250 300 350 400

6000

20
X Y 11 23
X 4

F04 TAIF-1

… D5S818 FGA

100 150 200 250 300 350 400

2400

20
X Y 11 12 23
X 4

G04 TAIF-1

33
 … D5S818 FGA

100 150 200 250 300 350

4400

23.5
X Y 12 25
X 4

H04 TAIF-1

… D5S818 FGA

150 350
100 200 250 300 400

4400

11 12 22
X Y 23
X 4

C05 TAIF-1

… D5S818 FGA

150 350
100 200 250 300 400

34
2800

10 11 23
X Y 24
X 4

D05 TAIF-2

… D5S818 FGA

150 350
100 200 250 300 400

2800

11 12
22
X Y 24
X 4

35
 E05 TAIF-2

… D5S818 FGA

150 350
100 200 250 300 400

2400

11 12
23
X Y 24
X 4

F05 TAIF-2

… D5S818 FGA

150 350
100 200 250 300 400

2600

10 11
23
X 24
4

G05 TAIF-23

36
 … D5S818 FGA

150 350
100 200 250 300 400

4050

1350

10 12
23
X 24
4

H05 TAIF-2

… D5S818 FGA

150 350
100 200 250 300 400

3900

0
23
10 12
22 4
X

37
 C06 TAIF-2

… D5S818 FGA

150 350
100 200 250 300 400

8000

12 21
X Y

E06 TAIF-2

… D5S818 FGA

150 350
100 200 250 300 400

10000

10 12 21 25
X

38
 G06 TAIF-2

… D5S818 FGA

150 350
100 200 250 300 400

3500

8 12 19 23
X

H06 TAIF-2

… D5S818 FGA

150 350
100 200 250 300 400

2100

10 12 20 23
X

39
 D07 TAIF-2

… D5S818 FGA

150 350
100 200 250 300 400

1100

11 13 21
X

F05 TAIF-3

… D5S818 FGA

150 350
100 200 250 300 400
23
Y
2600

10 11 23
X

References:

40
24
1) P. Shah, P. R. Velani, L. Lakade, and S. Dukle, “Teeth in forensics: a
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an Automated DNA Extraction Method for Bone and Teeth Samples and
Applicability to Two Forensic Cases. Forensic Sciences, 1(3), 194-201.

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45
 ‫‪53) Juroske, D. M. (2006). Quantitation of male and female DNA in mixed biological‬‬
‫‪samples using quantitative amplification of the human amelogenin locus.‬‬
‫‪Oklahoma State University.‬‬

‫‪54) Diegoli, T. M., & Coble, M. D. (2015). Characterization of X chromosomal short‬‬

‫‪tandem repeat markers for forensic use‬‬

‫‪55) Butler, J. M. (2007). Short tandem repeat typing technologies used in human‬‬
‫‪identity testing. Biotechniques, 43(4), Sii-Sv‬‬

‫‪:‬أمهية الدراسة‬
‫سيتم إجراء هذه الدراسة للكشف عن الوسائل املتاحة ليت تستخدم لزيادة كفاءة احلمض ‪ ‬‬
‫الن ووي ال وراثي املس تخلص من عين ات األس نان البش رية وحتدي د األمناط الوراثي ة مبا فيه ا‬
‫‪ .‬حتديد مورث األميلوجينني اخلاص بتحديد هوية وجنس العينات املفحوصة‬

‫‪:‬منهج البحث واإلجراءات‬

‫مت اس تخدام النهج الع ام يف التحلي ل يف ه ذه الدراس ة‪ .‬مت إج راء اس تخالص العين ات‬
‫باس تخدام تقني ة احلبيب ات املغناطيس ية‪ .‬بع د ذل ك مت عم ل قي اس لكمي ة احلمض الن ووي ال وراثي‬
‫ومن مث إظهار النتائج ‪ PCR‬باستخدام تقنية ال زمن احلقيقي ومن مث عمل تفاعل التسلسل املبلمر‬
‫مت ض بط األس لوب واألدوات التجريبي ة ‪ Genetic Analyzer 3500 .‬على أجه ز التحلي ل اجليين‬
‫‪.‬واالختبار واملراقبة وفقاً ملعايري املختربات الدولية‬
‫وسيتم إجراء الدراسة على ‪ 30‬عينة من األسنان املرفوعة من مدينة الطائف و الباحة خالل الفرتة‬
‫‪ 17/2/2022‬إىل ‪3/2022//7‬‬

‫‪46‬‬
47
‫‪:‬أهداف البحث وأمهيته‬
‫‪:‬أهداف البحث‬
‫اهلدف من ه ذه الدراس ة ه و تق ييم عين ات احلمض الن ووي ال يت س يتم استخالص ها من ‪1.‬‬
‫أنسجة األسنان املختلفة باستخدام طريقة حمددة وهي احلبيبات املغناطيسية اليت تليب الغرض‬
‫‪.‬من استخراج احلمض النووي عايل اجلودة من أنسجة األسنان‬
‫كواس م جزي ئي للكش ف عن اجلنس يف ‪ AMEL‬ميكن اس تخدام التحقي ق يف دور جني ‪2.‬‬
‫‪.‬حاالت الطب الشرعي املختلفة‬
‫‪:‬أهداف‬
‫مت اعتبار مستوى ٍ‬
‫عال من جتانس العينة وأدىن ح د من التب اين يف البيئة أو مت حتقيق عوامل ‪1.‬‬
‫‪.‬أخرى للحصول على نتائج أفضل باستخدام هذه الطريقة‬
‫عزل احلمض النووي من أنسجة األسنان الصلبة (املينا وعاج األسنان) ‪2.‬‬
‫باستخدام حبات مغناطيسية لفصل احلمض النووي عن الربوتينات و ‪ DNA‬عزل اجلينوم ‪3.‬‬
‫‪.‬نقي ‪ DNA‬من عينة األسنان للحصول على ‪RNA‬‬
‫‪.‬حتليل جودة ونقاء احلمض النووي واستخدام املعامالت املشبوهة لتحديد اجلنس ‪4.‬‬
‫‪:‬عرض املشكلة‬
‫أص بح اس تخراج احلمض الن ووي نش اطًا معق ًدا ومل ينتج عن ه عين ات جي دة النوعي ة كافي ة ميكن‬
‫كان من ‪ (Liu et al. ، 2018). (15).‬استخدامها يف تشخيص تفاعل البوليمريز املتسلسل‬
‫الص عب اس تخراج اجلودة الص حيحة والفعلي ة للحمض الن ووي ‪ ،‬ويف بعض احلاالت ‪ ،‬لوح ظ أن‬
‫ج ودة احلمض الن ووي تت دهور م ع اس تخدام املواد الكيميائي ة‪ .‬ل ذلك ‪ ،‬ف إن اس تخدام احلبيب ات‬
‫‪.‬املغناطيسية يساعد يف استخراج احلمض النووي بدقة‬

‫امللخص‬

‫‪48‬‬
‫مفيدا يف قضايا الطب الشرعي ‪ ،‬كما هو احلال يف الكوارث‬ ‫اخللفية‪ :‬ميكن أن يكون حتديد اجلنس ً‬
‫اجلماعية وحوادث النقل وحاالت الشخص املفقود أو االعتداء اجلنسي‪ .‬ميكن تتبع بقايا اجلسم‬
‫عن طريق احلمض النووي للضحية ‪ ،‬باستخدام عينات من مصادر خمتلفة مثل األسنان واألنسجة‬
‫‪.‬الطالئية الفموية واللعاب‬
‫اجلس م الرئيس ي‪ :‬الوامسات اجليني ة ال يت نش أت من الع اج ولب األس نان…‪ .‬ك انت ط رق حتلي ل‬
‫أداة رائع ة لتحدي د ‪ STR‬ح دد الطريق ة األنس ب تعترب ‪ PCR ،‬احلمض الن ووي املطبق ة هي‬
‫الف رد ألن ه ذه الطريق ة ت وفر نت ائج ممت ازة يف حتدي د اجلنس‪ .‬يكتش ف موق ع اجلين ات وأع داد‬
‫الكروموسومات اجلنسية واملواقف والشكل ‪ ،‬وهو أمر مفيد يف حتديد جنس األفراد‪ .‬حتديد كل‬
‫حيث يك ون ل دى ال ذكر ‪ X‬ش خص لتحدي د جنس ه ك أنثى ل ديها اث نني من الكروموس ومات‬
‫لتحديد ‪ STR‬كروموسوم‪ .‬هناك أنواع خمتلفة من عالمات ‪ Y‬واحد ويفتح ‪ X‬كروموسوم‬
‫اهلوي ة ومن بينه ا ‪ ،‬تع د تك رارات رب اعي النيوكليوتي د أح د أك ثر األش كال فعالية‬
‫ه ذا يس اعد على حتدي د اجلنس يف العملي ة اجلزيئي ة ‪(strbase.nist.gov ، 2022). (31).‬‬
‫احليوي ة بش كل فع ال ودقي ق‪ .‬خط وات عملي ة التحدي د ه ذه هي التض خيم ‪ ،‬وال رحالن الكه ريب ‪،‬‬
‫يف ‪ RNA‬والتفسري‪ .‬كل هذه اخلطوات مطلوبة لتحديد عزل احلمض النووي من بروتني اجلينوم‬
‫‪.‬لربط اخلرز املغناطيسي يف األسنان ‪ AMEL‬حتليل جني‬
‫االس تنتاجات‪ :‬أظه رت الطريق ة املس تخدمة جناح اس تخراج احلمض الن ووي جبودة عالي ة وحتدي د‬
‫اجلنس‪ .‬ميكن تأكيد أميلوجينني أنه كان مؤشرات موثوقة لتحديد اجلنس‪ .‬أثبت التحليل اجلزيئي‬
‫‪ ،‬فعاليته ودقته‬
‫الكلم ات املفتاحي ة‪ :‬حتدي د اجلنس ‪ ،‬طب األس نان الش رعي ‪ ،‬األميلوجي نني ‪ ،‬عين ات األس نان‬
‫‪.‬البشرية‬

‫تحديد األنماط الوراثية في عينات األسنان باستخدام مورث الجنس‬

‫‪49‬‬
 ‫‪:‬إعداد‬

‫هند حسن الزهراين‬

‫ساره موسى العطوي‬

‫العنود مساعد السفياين‬

‫أطروحة (ماجستير البصمة الوراثية واألدلة الجنائية)‬

‫‪.‬جامعة الطائف كلية العلوم ‪ ،‬قسم التقنية الحيوية ‪2022 ،‬‬

‫‪:‬ملخص‬

‫مفيدا يف قضايا الطب الشرعي ‪ ،‬كما هو احلال يف الكوارث‬

‫اخللفية‪ :‬ميكن أن يكون حتديد اجلنس ً‬
‫اجلماعية وحوادث النقل وحاالت الشخص املفقود أو االعتداء اجلنسي‪ .‬ميكن تتبع بقايا اجلسم‬
‫عن طريق احلمض النووي للضحية ‪ ،‬باستخدام عينات من مصادر خمتلفة مثل األسنان واألنسجة‬
‫‪.‬الطالئية الفموية واللعاب‬

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 ‫تحديد األنماط الوراثية في عينات األسنان باستخدام مورث الجنس‬
‫‪:‬إعداد‬

‫هند حسن الزهراين‬

‫ساره موسى العطوي‬

‫العنود مساعد السفياين‬

‫أطروحة (ماجستير البصمة الوراثية واالدلة الجنائية)‬

‫‪.‬جامعة الطائف كلية العلوم ‪ ،‬قسم التقنية الحيوية ‪2022 ،‬‬

‫تحت إشراف‬
‫د ‪ .‬أمل أحمد اليماني‬
‫أستاذ مساعد‬
‫الهندسة الوراثية و‬
‫تطبيقات التقنية‬
‫الحيوية‬

‫كلية العلوم‬
‫جامعة الطائف‬
‫هـ‪14432022-‬‬

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