“EXTRACTION OF DNA FROM SALIVA

SAMPLES”

A
Project Submitted to
THE DEPARTEMNT OF FORENSIC SCIENCE

In partial fulfillment of the requirement for the Degree of

MASTER OF SCIENCE
Session – 2023-24

INTERNAL GUIDE EXTERNAL GUIDE SUBMITTED BY

Miss. Isha Sahu Ms. Aparna Sharma Miss. Janvi Patidar

Assistant Professor Director of DNA Division M.Sc. Final Year

Department of Forensic Sci- DNA Division at SFSL Department of Forensic Science

ence

Govt. Holkar (Model, Auton- Directorate of Forensic Services, Govt. Holkar (Model, Autonomous)
omous) Science College, In- Junga, Himachal Pradesh Science College, Indore (M.P)
dore (M.P)

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 GOVT. HOLKAR (MODEL, AUTONOMOUS) SCIENCE COLLEGE
INDORE- 452017 (M.P.) INDIA
(NAAC Accredited "A++ " Grade)
DEPARTMENT OF FORENSIC SCIENCE

DR. GEETHA SARASAN

www.collegeholkar.org (HOD)
Contact No.-0731-2464047 email – [email protected]

CERTIFICATE

This is to certify that Miss. Janvi Patidar M.Sc. Final Year, has car-
ried out his project work entitled "Extraction of DNA from saliva sample " at workplace
Directorate of Forensic Services, Junga, Himachal Pradesh submitted to Department of
Forensic Science, Govt. Holkar (Model, Autonomous) Science College, Indore in the partial
fulfillment of the requirement for the Degree of Masters of Science. The work was carried out
by the student under the supervision of an External Guide Dr. Aparna Sharma and an Internal
Guide Prof. Isha Sahu.

HEAD OF THE DEPARTMENT EXTERNAL INTERNAL

Dr. Geetha Sarasan Dr. Aparna Sharma Miss. Isha Sahu
Department of Forensic Science Director of DNA Division (Assistant Professor)
Govt. Holkar (Model, Autono- Armour Department of Forensic Science
mous) Science College, Indore Directorate of Forensic Ser- Govt. Holkar (Model, Autono-
vices, Junga, Himachal Pra- mous) Science College, Indore
desh

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 DECLARATION

I hereby declare that the work entitled " Extraction of DNA from
Saliva Samples" presented in this project is an original piece of work carried out under the
guidance of internal guide Prof. Isha Sahu and external guide Dr. Aparna Sharma in the
name of external institute Directorate of Forensic Services, Junga which is being submitted
to the Department of Forensic Science, Govt. Holkar (Model, Autonomous) Science Col-
lege, Indore for partial fulfillment of the requirement for the award of Degree of Masters in
Science. This is further certified that this project work has not been submitted in part or full for
any other degree or diploma of this or any other institute.

Date-09/04/2024 Janvi Patidar

Place-Indore M.Sc. Final Year

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 ACKNOWLEDGEMENT

I would like to express my deepest appreciation to

my workplace guide Dr. Aparna Sharma for leading and supporting me through-
out my project with his/her gentle behavior and patience and knowledge.

I extend my sincere gratitude to Dr. Geetha Sarasan

Head of the Department, Department of Forensic Science, Govt. Holkar (Model,
Autonomous) Science College, Indore for invigorating me.

I record my humble thanks to my internal guide

Prof. Isha Sahu for encouraging me during my entire study.

I would like to record many thanks in the name of

all the members of Directorate of Forensic Services, Junga for helping, support-
ing and providing me resources and knowledge throughout my work, as they are
Mr. Anil Kumar and Dr. Ajay rana. I would like to take this opportunity to
thank laboratory staff of department who never let me run out for the chemicals
and supported me whole heartedly. Besides, this project made me realize the value
of working together as a team and I found a new experience in working in differ-
ent environment with different people, yet importantly, I would like to express
my heartful thanks to my beloved parents for their blessings and my friends
Hirdesh Chaurel, Sneha Vashinde and Harshraj Nishod for being with me and
supported me for the successful completion of this project.

Janvi Patidar
M.Sc. Final Year

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 TABLE OF CONTENTS

CHAPTER CONTENTS PAGE No.

No.
1. INTRODUCTION 9
2. THEORY 10-14
3. REVIEW OF LITERATURE 15-18
4. MATERIAL & METHODOLOGY 19-31
5. RESULT & DISCUSSION 32-35
6. CONCLUSION 36
7. REFERENCES 37

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 1.INTRODUCTION

Saliva starts the digestive process in the mouth.

Saliva is a food fluid that mixes with food in the mouth during chewing by teeth. It acts as a
digestive juice and softens the food, allowing for an easier digestion process. Salivary glands
produce this substance. Moreover, Saliva is a dark, colourless, opalescent fluid found in the
mouths of humans and other vertebrates at all times. Air, mucus, proteins, mineral salts, and
amylase make up this fluid. Saliva gathers Sthe mouth cavity. The human mouth excretes one
to two Liters of fluid every day.

Saliva (commonly referred to as spit) is an extracellular fluid produced and secreted by sali-
vary glands in the mouth. In humans, saliva is around 99% water, plus electrolytes, mu-
cus, white blood cells, epithelial cells (from which DNA can be extracted), enzymes (such
as lipase and amylase), and antimicrobial agents (such as secretory IgA, and lysozymes).

The enzymes found in saliva are essential in beginning the process of digestion of dietary
starches and fats. These enzymes also play a role in breaking down food particles entrapped
within dental crevices, thus protecting teeth from bacterial decay. Saliva also performs a lu-
bricating function, wetting food and permitting the initiation of swallowing, and protecting
the oral mucosa from drying out.

✓ Normally the daily production of whole saliva ranges from 0.5 to 1.0 litter.

✓ PH of saliva in between 5-8.

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 2. THEORY

2.1 Formation of saliva -

Formation of saliva occur in 2 stages:

STAGE 1: Production of primary saliva from the cells of secretory end pieces and interca-
lated ducts which is an isotonic fluid.

STAGE 2: The primary saliva is modified as it passes through the striated and excretory ducts
mainly by reabsorption and secretion of electrolytes the final saliva that reach the oral cavity
of hypotonic.

2.2 Function of saliva -

• Protective properties

• Lubrication

• Maintenance of mucous membrane integrity.

• Soft tissue repair

• Dilution and clearance

• Antifungal and antiviral activity

• Maintenance of PH

• Maintenance of tooth integrity

• Digestion

• Taste

• Excretion

• Water balance

• Oral hygiene

• Salivary diagnosis

• Diagnosis

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2.3 COMPOSITION OF SALIVA:

• Dilute aqueous solution present in oral environment.

11
 • 99% of this hypotonic fluid in water
• Remaining 1% consists dissolve organic and inorganic Constituents.

ORGANIC CONSTITUENTS.

Enzymes

A. Amylase
B. Maltase
C. Lingual lipase
D. Lysozyme
E. Carbonic anhydrase
F. Kallikrein

Other organic substance

1. Protein
2. Blood group antigen
3. Free amino acid
4. Non protein nitrogenous substance urea, uric acid creatinine

INORGANIC CONSTITUENTS

A) Sodium (0-80mg/100ml)
B) Potassium (60-100mg/100ml)
C) Calcium (2-11mg/100ml)
D) Phosphate (6-71mg/100ml)
E) Chloride (50- 100mg/100ml)
F) Fluoride (.01-.04mg/100ml)
G) Bicarbonate (0-40mg/100ml)
H) Thiocyanate (2mg)
I) Hydrogen ion (PH range is 5.0-8.0)

Gases dissolved in saliva

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 • Oxygen (0.18-25vol%)
• Nitrogen (0.9vol%)

Carbon dioxide (10-20 vol % unstimulated saliva)

2.4 SECRETION:

General Secretion

Saliva is always a hypotonic solution but it needs to be produced from concentrated extra-
cellular fluid and modified.

The fluid secreted from the acini is overall isotonic with the extracellular fluid:

• Sodium and potassium ions are equivalent

• Iodide ions are present at an increased concentration
• Chloride ions are present at a decreased concentration
• Bicarbonate is present at the same concentration

During ductal modification, there is little change in volume however concentrations of some
of the ions change:

• Sodium concentration decreases

• Potassium concentration increases
• Bicarbonate concentration decreases at rest and increases when stimulated

The ductal cells have a maximum rate of modification and therefore the more rapidly saliva is
produced, the less it is modified (excluding bicarbonate).

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 Fig – Diagram showing the modification of saliva.

Resting Saliva

At rest, the acinar secretion is highly modified and has the following characteristics:

• Low volume
• Very hypotonic
• Neutral or slightly acidic
• Few enzymes

Stimulated Saliva

When the production of saliva is stimulated, flow exceeds the ductal cells’ maximum rate of
modification and so the acinar secretion is modified less:

• High volume
• Less hypotonic than resting saliva
• Alkaline

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 3. REVIEW OF LITERATURE

3.1 Improved performance for forensic casework: Extraction and isolation

updates for the Maxwell® 16 instrument

Authors: M. Lindner, P.V. Mandrekar, J. Bessetti, C. Newton, B. Mankani, S. Krue-

ger, J. Krueger et.al., The DNA IQ™ System is an established chemistry for DNA recovery from
casework samples. Successful DNA recovery from most casework samples depends on the efficiency
of extraction, which refers to removal of DNA from a solid support such as a swab or fabric cutting,
and isolation, which refers to the recovery of DNA once it is extracted from the solid support.

We have recently improved the performance of the DNA IQ™ System on the Maxwell® 16
Instrument by increasing extraction and isolation efficiencies. First, we designed a new LEV
plunger using a proprietary material that increases isolation efficiency. Second, we improved
extraction efficiency by introducing an optimized DNA extraction buffer. In this article, we
demonstrate the resulting increase in DNA yield across a variety of samples, and compare
these results to data generated by organic extraction.

3.2 Evaluation of Maximum DNA Yield from a New Non -invasive Buccal
Collection Device Following Various Extraction Protocols

Authors: Mona Pißarreck , Fernando Moreira, and Megan Foley

Background: As the demand in genetic testing increases, various fields look toward collection
methods that are non-invasive and efficient in recovering deoxyribonucleic acid (DNA) for
testing that will allow for high first-pass success rates.

Objective: Two extraction methods (Prep Filer™ Express Forensic Extraction and the Max-
well® RSC Buccal Swab DNA Kits) were optimized to increase DNA yield from a buccal cell
collection device (Gentueri's Collect Eject™ Swab).

Materials and Methods: Buccal swabs were processed under varying incubation parameters
using a forensic workflow. The Prep Filer method was adjusted to test longer incubation
times and more aggressive agitation. The Maxwell method was adjusted to test incubation
temperatures and duration.

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Results: Quantitative results showed that increased agitation can yield more DNA through
the Prep Filer extraction, but longer incubation times did not increase DNA recovery. The re-
sults from the Maxwell study showed no significant difference between incubation tempera-
tures or times.
Conclusions: The results indicate that various applied genetic fields can utilize a non-inva-
sive, simple collection method using the Collect Eject device in conjunction with extraction
methods already implemented in laboratories to collect 5000 ng of DNA or greater from a
buccal cell collection.
3.3 Increasing DNA extraction yield from saliva stains with a modified
Chelex method

Authors:David Sweet a, Miguel Lorente b, Aurora Valenzuela b, JoséA. Lorente b, J. Car-

los Alvarez bet.al., Recovery, preservation and analysis of body fluid stains is an important
aspect of forensic science. PCR-based typing of DNA extracted from recovered stains is often
a crucial method to identify a perpetrator or exclude an innocent suspect. This paper reports an
improved method of extracting genomic DNA from saliva stains deposited on human skin in
simulated bite mark situations. Results of organic (phenol-chloroform) extraction and Chelex
extraction were compared to a modified Chelex method developed by the authors. Modifica-
tions include pre-extraction preparation with proteinase K and incubations at 56 °C and 100 °C
plus micro concentration of the solution. Quantification results using the classical Chelex ex-
traction method showed that 31.9 ± 4.22% of the deposited DNA was recovered, but using the
modified Chelex extraction method DNA recovery was increased to 47.7 ± 6.90%. The quantity
and quality of extracted DNA was shown to be adequate for PCR-based typing at two STR loci.

3.4 Collection and Extraction of Saliva DNA for Next Generation Sequencing

Authors: Michael R. Goode1, Soo Yeon Cheong1, Ning Li1, William C. Ray1,2, Christopher
W. Bartlett1,3 et.al., The preferred source of DNA in human genetics research is blood, or cell
lines derived from blood, as these sources yield large quantities of high-quality DNA. However,
DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmen-
tation that is suitable for a variety of DNA assays without the expense of a phlebotomist and
can even be acquired through the mail. However, at present, no saliva DNA collection/extrac-
tion protocols for next generation sequencing have been presented in the literature. This proto-
col optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient

16
quality and quantity for DNA assays with the highest standards, including microarray genotyp-
ing and next generation sequencing.

3.5 Isolation of Deoxyribonucleic Acid (DNA) from Saliva and Forensic Sci-
ence Samples Containing Saliva

DJ Walsh, M.S., AC Corey, B.S., RW Cotton, Ph.D., L Forman, Ph.D., GL Herrin, Jr,
Ph.D., CJ Word, Ph.D., DD Garner, Ph.D.et,al.,Saliva and saliva-stained materials were
examined as potential sources of deoxyribonucleic acid (DNA) for DNA analysis and identity
testing. In this paper, the authors demonstrate that DNA was isolated and DNA banding pat-
terns suitable for DNA typing were obtained from fresh saliva and various saliva-stained ma-
terials, such as envelopes, buccal swabs, gags, and cigarettes. Furthermore, DNA and DNA
banding patterns were obtained from actual forensic evidentiary samples containing mixed
saliva/semen stains. The DNA banding patterns obtained from saliva or saliva-stained mate-
rial were indistinguishable from the patterns obtained from blood or hair from the same indi-
vidual. Intact DNA was readily isolated and DNA banding patterns were obtained from saliva
stored at −20°C and dried saliva stains stored under varying conditions. We conclude that sa-
liva and saliva-stained material can be good sources of DNA for analysis and for DNA typing
in certain forensic settings.

3.6 Pre-Analytical Factors Affecting Extracellular DNA in Saliva

Ľubica Janovičov 1, Dominika Holániová 1, Barbora Vlková1 and Peter Celec 1, et.al.,
Salivary DNA is widely used for genetic analyses because of its easy collection. However, its
extracellular fraction in particular, similar to the extracellular DNA (ecDNA) in plasma,
could be a promising biomarker for oral or systemic diseases. In contrast to genetics, the
quantity of salivary ecDNA is of importance and can be affected by the pre-analytical pro-
cessing of samples, but the details are not known. The aim of our study was to analyse the
effects of centrifugation and freezing of saliva on the concentration of ecDNA in saliva. Fif-
teen healthy volunteers, free of any known systemic or oral diseases, were asked to collect
unstimulated saliva samples. Aliquots were centrifuged at 1600× g and frozen or directly
processed. The fresh or thawed cell-free saliva samples underwent subsequent centrifugation
at 16,000× g. The supernatants were used for DNA isolation and quantification using fluo-
rometry and real-time PCR. While freezing had minimal effects on the salivary ecDNA con-
centration, another centrifugation step decreased ecDNA considerably in both fresh and

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frozen samples (by 97.8% and 98.4%, respectively). This was mirrored in the quantitative
PCR targeting a nuclear (decrease by 93.5%) and mitochondrial (decrease by 97.7%) ecDNA
sequence. In conclusion, in this first study focusing on the technical aspects of salivary ec-
DNA quantitation, we show that, regardless of its subcellular origin, the concentration of ec-
DNA in saliva is mainly affected by additional centrifugation and not by the freezing of cen-
trifuged cell-free saliva samples. This suggests that most salivary ecDNA likely is associated
with cell debris and apoptotic bodies. Which fraction is affected by a particular disease
should be the focus of further targeted studies.

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 4. MATERIAL & METHODOLOGY

4.1 COLLECTION OF SAMPLES:

Saliva found at crime scenes is one of the most common types of evidence used to support
the involvement of a victim or suspect in a specific crime, such as sexual contact. Forensic
saliva samples are often mixed with other body fluids, such as sweat or vaginal fluid from the
victim in a sexual assault case. This mixture can cause difficulties when using DNA typing to
perform personal identification because there is currently no technique that is practically used
in most forensic laboratories for selectively isolating saliva cells or DNA from mixed sam-
ples. Moreover, because α-amylase activity, an indicator of most saliva identification meth-
ods, can be slightly detected in other body fluids, careful attention should be paid to saliva
identification tests of other body fluid-containing samples to determine whether the detected
activity is saliva- or non-saliva-derived.

Bitemarks are most frequently found in violent crimes, especially in those that involve sexual
assaults. Saliva samples can provide information on the assailant's blood type, secretor status,
and on the presence or absence of salivary amylase and other proteins. A saliva collection kit
should include a control and a sample tube, each containing eight 12-15 mm cotton threads
which have been obtained from rinsed sheeting. The vials should have a 2-3 mm hole melted
in their plastic stoppers to allow the threads to air dry after they have been moistened in the
swabbing process.

SCENE EXAMINATION: Forensic investigators thoroughly examine the crime scene for
any potential saliva stains or traces, such as on clothing, objects or surface.

Visible stains or suspected saliva spots are documented through photography and sketches.

Personal protective equipment (PPE): investigator should wear appropriate PPE, including
gloves, face masks, and protective clothing to prevent contamination of the sample and pro-
tect themselves from potential hazards.

Saliva sample collection usually involves spitting into a collection tube or using a swab to
collect saliva from inside of the mouth. It’s a sample and non-invasive process commonly
used in various medical tests and research studies.

At crime scene saliva should be found on toothbrush, washbasin, etc

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Collection tools: - sterile cotton swabs or specialized collection devices are used for sample
collection.

Collection tubes or envelops are labelled with unique identifiers, including the date, time, lo-
cation, and case number, to maintain chain of custody.

Swabbing technique: -

Swabs are moistened with a sterile buffer solution to enhance sample collection.

Investigators gently swab the saliva stain or suspected sources of saliva,

Ensuring to cover the entire area of interest.

✓ Before Saliva Collection

Good saliva collection requires documenting items which may affect results, as well as fol-
lowing procedures which avoid the possibility of contaminating saliva with substances that
could interfere with the immunoassay. Before starting saliva collection from a research par-
ticipant, we recommend the following precautions: Avoid foods with high sugar or acidity, or
high caffeine content, immediately before sample collection, since they may compromise the
assay by lowering saliva pH and increasing bacterial growth. Document consumption of alco-
hol, caffeine, nicotine, and prescription/over-the-counter medications within the prior 12
hours. Document vigorous physical activity and the presence of oral diseases or injury. Do
not eat a major meal within 60 minutes of sample collection. Rinse mouth with water to re-
move food residue and wait at least 10 minutes after rinsing to avoid sample dilution before
collecting saliva.

✓ After Saliva Collection

It is always best to freeze samples at or below -20ºC immediately after collection to preserve
the sample for possible use in future studies. If freezing is not possible, to minimize degrada-
tion of unstable analytes and to prevent bacterial growth (25), refrigerate immediately at 4°C
and maintain at this temperature for no longer than necessary (ideally less than 2 hours) be-
fore freezing at or below -20ºC (temperature of a regular household freezer). Samples stored
for more than 4 months should be frozen at -80ºC. Freezing and centrifugation also helps pre-
cipitate mucins in the samples, which makes pipetting easier. We recommend samples are ex-
pressed or centrifuged to remove saliva from swab collections as soon as possible and prior to

20
freezing at temperatures of -20ºC or below in order to minimize freeze-thaw cycles. How-
ever, samples can be frozen in the swab for up to 6 months.

4.2 PRESERVATION OF SAMPLE:

Saliva samples are often preserved to maintain sample integrity during storage and transpor-
tation, especially if immediate processing is not feasible.
Common preservation methods include:

Refrigeration:

Storing samples at temperatures between 2°C to 8°C (36°F to 46°F) to slow bacterial growth
and enzyme activity.

Freezing: Storing samples at -20°C (-4°F) or lower to prevent degradation of nucleic acids
and proteins.
Stabilization solutions: Adding chemical stabilizers to saliva samples to prevent degra-
dation of biomolecules.

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Drying: Lyophilization (freeze-drying) of saliva samples to remove moisture and preserve
biomolecules for long-term storage.

Oragene is a registered trademark of DNA Genotek Inc

➢ Long Term Sample Storage

Samples can be stored at -80ºC for several years; the exact time has not been determined and
may vary by analyte. However, many samples that have been stored properly for over four
years have shown little or no degradation. We recommend you consult the literature or con-
tact Salimetrics for details.

➢ Prior to Sample Testing

On the day samples are to be assayed, bring samples to room temperature, vortex, and then
centrifuge at 1500 x g for 15 minutes. If the samples appear viscous, centrifuge for a greater
amount of time, or break up the clot with a pipette tip and re-centrifuge. Assays should be
performed using only clear saliva, avoiding any sediment present in the bottom of the tube.
When pipetting viscous solutions such as saliva, greater accuracy in sample volume is ob-
tained by aspirating slowly, in order to avoid the formation of bubbles. Re-centrifuge tubes
following each freeze-thaw cycle since additional precipitates may develop upon refreezing.

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4.3. HANDELING OF SAMPLE:

Libelling: Clearly label the collection container with the participant’s information, includ-
ing name, date of birth, collection date, and unique identifier.

Storage: Store the collected saliva samples in a cool, dry place to prevent degradation. Avoid
exposure to extreme temperatures or direct sunlight.

Transportation: If the sample needs to be transported to a different location for analysis,

ensure that it is packaged securely to prevent breakage or leakage during transit. Follow any
instructions provided by the testing facility or healthcare professional regarding transporta-
tion protocols.

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 4.4. Extraction of DNA From saliva –

THROUGH INSTRUMENTATION METHOD:

❖ MATERIAL REQUIRE FOR INSTRUMENTION METHOD:

• Extraction buffer: it includes –

✓ Tris --------------0.0605g--------------0.121g
✓ NaCl --------------0.292g----------------0.584g
✓ EDTA-------------0.0185g---------------0.037g
✓ SDS----------------0.8g--------------------1.6g

• Proteinase k
• Thio glycerol
• Nuclear- free water
• Lysis buffer
• Elution buffer
❖ Instrument Specifications:

• Processing Time: 25–60 minutes

• Number of Samples: Up to 16

• Weight: 24.2lb (11kg)

• Dimensions: 13W × 13.6D × 11.8H inches (330.2 × 345.2 × 299.7mm)

• Power Requirements: 100–240 VAC (volts alternating current) ,50/60Hz (Hertz), 2.5A
(angstrom).

❖ Maxwell instrument

Automated Benchtop DNA Purification from Stabilized Saliva -

The Maxwell® RSC Stabilized Saliva DNA Kit is designed for automated DNA extraction
from stabilized saliva samples using the Maxwell® RSC Instruments. This kit is optimized
for maximum yield and purity. The Maxwell® RSC Stabilized Saliva DNA Kit delivers

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high-yield DNA from a non-invasive collection method. The Maxwell® RSC Instrument can
process from 1 to 16 samples and the Maxwell® RSC 48 can process from 1 to 48 samples
in a single run. The Maxwell® RSC Stabilized Saliva DNA Kit is also compatible for use on
the Maxwell® CSC Instrument in RUO Mode.

Application

This kit is designed for clinical research labs needing high-quality DNA from stabilized sa-
liva samples for molecular-based HLA typing and genetic testing.

• Automated DNA or RNA Extraction with Integrated Quantification.

• High-quality nucleic acid purification with minimal steps and less hands-on time.
• Sample are used semen stain, blood stain, hair, saliva, cigarette butts, tissue, skel-
eton remain etc.
• Intuitive software and touch screen interface.

PROMEGA MAXWELL INSTRUMENT

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 ❖ Casework kit for Maxwell / Maxwell® RSC Stabilized Saliva
DNA Kit -

• 48 Maxwell FSC cartridge

• 1 Maxwell FSC Plunger

• 50 Elution tube 0.5ml

• 20ml Elution buffer

• 32ml lysis buffer

• Case work extraction kit –

1. 50 ml: - casework extraction buffer

2. 2×10 mg: - Proteinase k

3. 900 microliters: - 1 Thioglycerol 1.25ml: - Nuclear free water

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 ❖ Process of extraction of saliva sample from maxwell:

4.5 Extraction of DNA From saliva –

THROUGH CHEMICAL METHOD:

MATERIAL REQUIRE FOR CHEMICAL METHOD:

• Saliva sample

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• 1.5ml fresh Eppendorf tube

• DNA Extracted buffer

• Proteinase k

• Vortex

• Dry bath

• Phenol chloroform isoamyl alcohol (25: 24:1)

• Centrifugation machine

• Sodium acetate

• Chilled ethanol

• Mili- Q water

❖ Phenol chloroform isoamyl method:

• Take 500 microliter of liquid sample of saliva in 1.5 ml fresh Eppendorf tube.

• Add 500 microliter DEB (DNA Extraction buffer)

• Add 10 microliter of Proteinases k

• Vortex the solution for few min

• Put on dry bath for cell lysis for overnight

• Now add 400-500 microliter Phenol chloroform isoamyl alcohol (25:24:1) to it.

• Spin for 10min

• Centrifuge the tube at 12000-14000rpm for 10min.

• Collect the supernatant

• Add 100microliter sodium acetate to supernatant

• Then add 700microliter chilled ethanol

• Mix by inverting and shaking

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• Centrifuge for 15min at 14000rpm

• Remove supernatant and add 100ml of 70% ethanol to pellets

• Again, Centrifuge at 10000 rpm for 5min

• Remove supernatant and add 100ml of 70% ethanol

• Centrifuge for 10000rpm for 5min

• Discard ethanol and allow the tube containing extracted DNA for dry bath for at least
half an hour to dry out the pellets.

• Now add 40-50microliter Milli –Q water

• Again, put it on dry bath for 10min and we will get DNA extracted

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5. RESULT:

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❖FACTOR – AFFECTING COMPOSITION AND SAM-
PLE OF SALIVA
The composition of saliva can be influenced by various factors, which include:

1. Diet:

- Consumption of different types of food and beverages can alter the pH, enzyme lev-
els, and other chemical components in saliva.
2. Hydration Status:
- Dehydration can reduce the volume and change the concentration of electrolytes
and other substances in saliva.
3. Medications:
- Certain medications, such as antihistamines, antidepressants, and diuretics, can af-
fect salivary flow and composition.
4. Health Conditions:
- Diseases and conditions such as diabetes, Sjögren's syndrome, and infections can
impact saliva production and its components.
5. Age:
- Salivary composition can change with age, often resulting in decreased saliva pro-
duction in older adults.
6. Hormonal Changes:
- Hormonal fluctuations, such as those occurring during pregnancy, menopause, or
menstrual cycles, can affect saliva composition.
7. Smoking and Alcohol Consumption:
- These habits can alter the chemical composition and decrease the protective com-
ponents of saliva.
8. Oral Hygiene and Health:
- Poor oral hygiene and the presence of dental diseases can influence the microbial
composition and overall chemistry of saliva.
9. Genetic Factors:
- Genetic predisposition can play a role in determining the baseline composition and
flow rate of saliva.

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 10. Time of Day:
- Circadian rhythms can cause variations in saliva composition, with different en-
zyme and hormone levels fluctuating throughout the day.
11. Stress and Emotions:
- Psychological stress and emotional states can impact saliva production and its bio-
chemical makeup.
12. Stimulus Type:
- The nature of the stimulus (e.g., chewing, taste, smell) can lead to differences in
the quantity and quality of saliva produced.

❖ PRECAUTION DURING COLLECTION PRESEVATION,

HANDLING, EXTRACTION OF DNA FROM SALIVA -
General Safety Precautions

1. Personal Protective Equipment (PPE):

-Wear appropriate PPE such as lab coats, gloves, safety goggles, and masks.

2. Hygiene Practices:

- Wash hands thoroughly before and after handling samples.

- Avoid touching face, especially eyes, nose, and mouth, while handling samples.

3. Work Area:

- Keep the work area clean and organized.

- Disinfect work surfaces before and after use.

- Use fume hoods or biosafety cabinets if working with volatile or biohazardous samples.

Handling Biological Samples

1. Biosafety Levels:

-Determine the appropriate biosafety level for the sample (BSL-1, BSL-2, BSL-3, BSL-4).

34
 - Follow specific protocols for each biosafety level.

2. Containment:

- Use proper containers for transporting and storing samples.

- Label samples clearly with necessary information (e.g., contents, date, and hazard level).

3. Disposal:

- Dispose of biological waste in designated biohazard containers.

- Autoclave or treat biological waste before disposal if required.

Handling Chemical Samples

1. Chemical Safety:

- Understand the properties** of the chemicals being handled (e.g., flammability, toxicity).

- Refer to Material Safety Data Sheets (MSDS) for specific handling and storage instruc-
tions.

2. Storage:

- Store chemicals in appropriate containers, clearly labelled.

- Separate incompatible chemicals to prevent reactions.

❖ FORENSC SIGNIFICANCE
Saliva samples are crucial in forensics for several reasons:

• DNA Analysis: Saliva contains epithelial cells that contain DNA. DNA extracted
from saliva can be compared with DNA from crime scenes, victims, or suspects to es-
tablish identity or exclusion.
Drug and Alcohol Testing: Saliva can be tested for the presence of drugs or al-
cohol, providing valuable information in cases involving impaired driving or drug-re-
lated offenses.
Trace Evidence: Saliva can contain trace amounts of other substances, such as
food particles or chemicals, which can provide additional clues or evidence in a case.

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 6. CONCLUSION:
Extracting DNA from saliva is a vital procedure in genetic analysis, offering a non-invasive,
straightforward, and efficient method to obtain genetic material.

1. Quality and Quantity: Saliva provides a sufficient quantity of high-quality DNA suitable
for various genetic analyses, including PCR, sequencing, and genotyping.

2. Non-Invasiveness: Compared to blood draws or tissue biopsies, saliva collection is painless

and less stressful for participants, enhancing compliance and participation rates in genetic
studies.

3. Stability: Saliva samples, when collected and stored properly using stabilization solutions,
maintain DNA integrity over time, facilitating long-term studies and biobanking.

4. Cost-Effectiveness: The process is generally cost-effective, as it reduces the need for spe-
cialized personnel and equipment for sample collection and handling.

5. Applications: DNA extracted from saliva is widely used in various fields, including medi-
cal diagnostics, ancestry testing, forensic investigations, and personalized medicine, showcas-
ing its versatility and utility.

In summary, DNA extraction from saliva is a reliable and practical method for obtaining ge-

netic material, supporting a wide range of scientific and clinical applications.

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 7. REFERENCES
Lindner et al. (et al., 20XX) discuss enhancements in forensic casework performance through
updates in extraction and isolation methodologies for the Maxwell® 16 instrument.

Pißarreck et al. (et al., 20XX) evaluate the maximum DNA yield achievable from a new non-
invasive buccal collection device across various extraction protocols.

Sweet et al. (et al., 20XX) present a modified Chelex method aimed at increasing DNA ex-
traction yield from saliva stains.

Goode et al. (et al., 20XX) describe methods for the collection and extraction of saliva DNA
specifically tailored for next-generation sequencing applications.

Methods for the isolation of DNA from saliva and forensic science samples containing saliva
are detailed in the work by various authors (et al., 20XX).

Janovičová et al. (et al., 20XX) investigate pre-analytical factors influencing extracellular
DNA in saliva, highlighting their impact on downstream genetic analyses.

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