MSC IV SEMESTER_DNA Profiling -DNA introduction and extraction.pdf

DNA Profiling -DNA introduction and extraction

1. DNA Profiling

2. Overview
Give a brief overview of what you’ll cover in your presentation.

3. 1.Sexual assault & homicide unit
2.Paternity unit
3.Human identification unit
4.Mitochondrial dnaunit
Profiling tools and techniques:
DNA Forensic Laboratory is estimated to increase the
capacity to examine to 2,000 cases per year
1.Disputed paternity/maternity
2.Criminal paternity
3.Rape and murder cases
4.Child abuse and sexual assault
cases (POCSO)
5.Human identification cases
6.Forensic challenged DNA cases:
charred bone , exhumed skeletal
remains, touch DNA, trace
elements etc.
7.Homicide cases
8.Kinship analysis.

16. Liquid form
Crime scene
(24 hours)
Collect in EDTA tube using
syringe or dropper Or Transfer
on gauze piece / FTA card. Air
dry
Fresh / Wet
clot Crime
scene
sterile tube and add equal
volume of normal saline /
PBSOr Transfer on gauze
piece / FTA card. Air dry it
and keep in paper packet /
envelope with desiccant.
Wet / damp
Crime scene,
clothing,
fabrics, Victim’s
clothing,
suspect’s
clothing etc
Thoroughly air dry at room
temperature. Roll it in clean in
paper or brown paper

17. Wet blood
on
Object
Thoroughly air dry at room
temperature. Collect the item as
it is. Pack in paper bag /
envelope , cardboard / shipping
boxes, depending upon the size
of object
Crust / stain
/Spatters
Crime scene,
or Unmovable
surface, floor,
concrete wall
etc
Moistened the dry blood stain
for 5-10 minutes with PBS /
distilled water. Collect the
moistened stain with foam
tipped swab / FTA card / gauze
piece and air dry the swab.
Stain
Weapon/firearm
/bullet Small
objects such as
household
utensils, stones,
bricks etc.
Allow the stains to dry. Collect
the item directly

18. SEMEN
Stain Vehicle upholstery,
carpet, wallpaper, wood
etc.
**Cut out the stained
area. Allow it to dry in
shade. Also collect an
unstained cutting as a
control from adjacent area.
Liquid form Object, crime
scene
** Collect the sample with
sterile gauze piece /
cotton swab / surface
swab. Air dry the swab
Wet / semi dry Mutilated
remains at crime scene or
place of recovery
**** Immediately store
parcel under freezing
conditionswithout any
preservative for DNA
analysis.

19. Bones / teeth
Wet / semi dry / dry Crime scene
or place of recovery
Clean and wash the bones and
teeth to remove any debris.
Allow it to dry completely in air.

20. Hair with root Dry or wet with
blood, semen,
saliva, Crime scene,
weapon, clothing
Collect the sample
with help of
tweezers / forceps
in white paper /
butter paper and
pack in paper
envelope. If found
attached in dry
blood, weapon etc.
do not remove

21. The following items may contain DNA
material
This Photo by Unknown Author is licensed under CC BY

22. ACTIVITY
Clothings, Pubic and head hair, evidence from body,
swab/smear (genitals : vulva, cervix, vaginal), vaginal
wash, oral swab
Penile swab, nail clippings, urine sample

23. GENERAL GUIDELINES FOR DNA
CASEWORK

24. LABORATORY ORGANIZATION (Equipment's/sterilization and bleaching/PPE for analyst, storage and
analysis area seperation
WORK PLACE PREPARATION (sterilization with 10% bleach and/or 70% ethanol, gloves)
LABORATORY WASTE DISPOSAL MANAGEMENT ( Human blood, other potential infectious body
fluid, Laboratory waste from infectious agents, Nucleic acid (natural & synthetic) containing
material,Sharps, Pipette tips)
SAMPLE HANDLING (DNA extraction and PCR in separate rooms, sterilization, sample handling
minimum, documentation.
BODY FLUID IDENTIFICATION ( in serious cases)

25. extraction negative
control
•2pg/microlitre
PCR controls
•A positive control: DNA
sample where the known
STR alleles of loci
•A negative control
Parallel (Extraction/
amplification)
CONCORDANT
ANALYSES AND
“DUPLICATE RULE”
EXOGENEOUS
POLICY
DNA EXTRACTION GUIDELINES

27. Forensic STR Analysis
DNA extraction
DNA quantitation
PCR amplification
(multiplex)
Electrophoresis
Interpretation of STR
profile

30. DNA EXTRACTION METHODOLOGIES
●reliable and efficient, it is also very time-consuming, uses hazardous chemicals, and, because of the
greater hands-on effort and multiple tube transfers involved, introduces increased opportunities for
contamination and sample mishandling
Phenol:chloroform:isoamyl alcohol (25:24:1)
ethanol precipitation or
through the use of a
centrifugal filter unit (i.e.,
Vivacon or Amicon devices),

31. Chelation Extraction
Chelex from Bio Rad (Fig.
21.4). Consisting of a
styrene divinylbenzene
copolymer containing paired
iminodiacetate ions
fast, can be easily automated, and require
minimal sample transfer.
inhibitory compounds (e.g., hematin and
immunoglobulin gamma in whole blood or
humic acid in soil-contaminated samples).

32. Solid Phase Extraction
More readily automatable extraction techniques
involve solid phase extraction methods
selectively bind DNA to a solid surface, such as silica in the presence of high
concentrations of chaotropic salts.
The bound nucleic acids and associated substrates are then separated from the
remaining cellular debris through the use of magnets or centrifugation.
Purified DNA is then readily eluted from the solid surface by the immersion of low
ionic strength or pH-adjusted buffers, allowing for nucleic acid recovery and
concentration.

33. QIAamp spin columns
QIAamp spin columns from
Qiagen selectively bind DNA to
silica in the presence of
chaotropic salts and elute DNA
under alkaline conditions

34. DNA IQ kit from the Promega Corporation
silica-based paramagnetic binding
resin that allows for the use of
magnets to isolate the silica-
bound DNA on the side of a
sample tube. This eliminates the
need for tube transfers during the
wash steps

35. The PrepFilersystem by ThermoFisher Scientific
https://www.thermofisher.com/in/en/home/industrial/forensics/human-identification/forensic-dna-
analysis/sample-preparation-extraction/prepfiler-forensic-dna-kits.html
paramagnetic resin,
but rather than
silica, it employs a
coating of a dextran
derivative, which
binds DNA in the
presence of alcohol

36. Differential Extraction
this is a pre-PCR approach that physically isolates
male cells for autosomal STR analysis, which offers a
superior power of discrimination.
This is in contrast to the use of Y-STRs, which simply
use male targeted genetic markers to selectively
amplify male DNA in the presence of excess female
DNA.

38. ISOLATION OF DNA FROM BLOOD/ SALIVA/
OTHER BODY FLUIDS ARCHIVED ON FTA
CARDS

39. ●WhatmanFTAproductsfacilitate
collection, transportation,
purificationandlong-termroom
temperaturestorageofnucleic
acids,allonasingledevice.
●FTAtechnologyhastheabilityto
lysecells,denatureprotein,
removalofcontaminants,and
protectsDNAfromdegradation.
●OnFTACardssmallareais
encircledforspottingof
blood/bodyfluidsandthismethod
isusedtoobtainDNAfromthe
followingtypesofsamples:
● PeripheralbloodBuccalcells
OtherbodyFluids

40. ●Reagents: FTA Purification Reagent TE Buffer

43. DIFFERENTIAL EXTRACTION OF DNA FROM
VAGINAL SWABS

44. ●Reagents:
●Proteinase K Solution: Dissolve 100 mg of Proteinase K in 5ml
sterile Milli-Q Grade water (final concentration 20mg/ml) Divide
into 1ml aliquots and store at –20oC
●96-100% Ethanol
●QIAamp Kit components: QIAamp spin column, Buffers ATL, AL,
AE, AW1 and AW2.
●Sterile Milli-Q Grade water
●20% Sarcosyl
●TNE (Tris/EDTA/NaCl) buffer
●0.39M DTT (Dithiothreitol)

46. Separation of Female and Male components

49. ChelexExtraction

50. Principle
The sample is boiled in a solution
containing minute beads of a chemical
called Chelex.
The boiling causes the cells to lyse,
releasing the DNA. The Chelexbinds to the
extraneous cellular material, and the entire
“complex” is removed by centrifugation,
leaving the DNA in the supernatant.
Since the high temperatures disrupt the
two strands of the DNA, generating single-
stranded molecules, this extraction process
is generally reserved for polymerase chain
reaction (PCR)-based typing techniques.

52. Procedure Collection of Cells (e.g., buccal cells, liquid blood)
Pipet 10 ml of liquid sample into the polypropylene test tube
For buccal cells, rinse your mouth with 10 ml of 1× PBS solution and vigorously
swish against your cheeks for 10 s. Expel the PBS solution back into the labeled
15 ml polypropylene test tube over the sink.
OR
If sterile swabs are available, place the swab inside your mouth and press it firmly
against the inside of your cheek. Roll the swab back and forth over the inside
surface of your cheek at least 10 times. Repeat on the other cheek. Place the
swab into a labeled 15 ml test tube containing 2 ml of 1× PBS solution. After
gentle swirling the cells will dislodge from the swab in 10–15 min.

53. STEP 1

54. STEP 2

55. Organic Extraction

56. Principle
❑Organic extraction is a general method
used for many situations when stained
fabric or clothing is suspected of
containing biological material.
❑The stain on the material is cut away from
the fabric, soaked in a warm solution
(stain extraction buffer)to release the
cells from the fabric, incubated with
proteinase K, and the DNA isolated using
organic solvents.
❑The organic extraction method maintains
the integrity of the DNA (i.e., large
segments are maintained) while
“cleaning” the DNA.

57. Stain extraction buffer: 10 mM
Tris-Cl/0.1 M NaCl/2% SDS/10
mM EDTA/39 mM DTT

58. STEP 1

61. QIAamp®DNeasyMINI KIT
ISOLATION OF DNA FROM
• LIQUID BLOOD
•DRIED BODY FLUID STAINS
•FRESH AND FROZEN TISSUES
•DNA FROM TRACE EVIDENCE
(CHEWING GUM)
•CIGARETTE BUTTS
Buffers ATL, AL, AE, AW1 and AW2.

62. QIAamp®DNeasyMINI KIT

63. QIAamp® DNA MICRO KIT: DNA extraction form NAIL
CLIPPINGS

64. REMOVAL OF DYES, IMPURITIES ETC.
FROM ISOLATED DNA
SEPHAROSE 6B Cleaning Treatment

65. BONE PROCESSING AND DNA ISOLATION
FROM TOOTH & BONE

66. FACTORS AFFECTING DNA ANALYSIS FROM
BONE
Morphology of bone
(spongy, brittle, non-
compact bones )
Quality of bone
(degraded, damaged,
burnt, charred,
exhumed, fragmented
bone )
Age of bone (fresh, old
or archived)

67. CLEANING AND DECONTAMINATION OF BONE SURFACE:
Cleaning
(remove soft
tissue/debris)
scraping, brushing, rinsing
with water or sonication
10% bleach
WASH
BUFFER (For
1ml wash
Buffer)
1% SDS 100 μl 25mM EDTA
50 μl 1μl Proteinase K
(20mg/ml) 5μl Sterile Milli-
Q Grade water 845 μl

68. SAMPLING METHODS FOR BONE
Cutting
(wearing PPE)
Sonication
(small bones)
Crushing/Grinding (motar
and pestle)
Milling/Processing with
Liquid Nitrogen
(cryogenic/grinds/
pulverize it by impacting a
magnetic based steel
impactor)

69. ISOLATION OF DNA FROM BONE (ORGANIC EXTRACTION)

71. ISOLATION OF DNA FROM TEETH (ORGANIC
EXTRACTION METHOD)
Larger teeth with no restorations should be chosen over smaller or restored teeth. Thus, non-restored
molars are the tooth of choice for DNA recovery.
Cleaning of Tooth Surface: 10% bleach

74. AUTOMATED DNA EXTRACTION
❑Automated DNA Extraction system is a Robotic technology to
isolate the DNA from various sources in an easiest way.
❑We can process a number of samples simultaneously in less
time as compared to manual procedures.
❑The system comes with different technologies i.e. Silica based
and magnetic beads-based technology.

75. EZ1® DNA INVESTIGATOR
Magnetic-particle technology combines the speed
and efficiency of silica-based DNA purification with
the magnetic particles.
DNA is isolated from lysates through its binding to
the silica surface of the particles in the presence of a
chaotropic salt.
The particles are separated from the lysates using a
magnet.
The DNA is then washed and eluted either in water
or TE buffer.
The user can choose elution volumes of 40 μl(EZ1
Advanced XL only), 50 μl, 100 μl, or 200 μl

77. The Main Features of the
EZ1 Instrument Include:
Purification of high-quality nucleic acids from 1–6
or 1–14 samples per run.
Small footprint to save laboratory space.
Preprogrammed EZ1 Cards containing ready-to-
use protocols for nucleic acid purification.
Prefilled, sealed reagent cartridges for easy, safe,
and fast setup of EZ1 instruments.
Complete automation of nucleic acid purification,
from opening of reagent cartridges to elution of
nucleic acids, with no manual centrifugation steps.

78. As
recomm
ended in
DFSS
DNA
profiling
manual

79. As
recomm
ended in
DFSS
DNA
profiling
manual

80. AUTOMATE EXPRESS DNA
ISOLATION SYSTEM
The Automate works on the chemistry of reagents. The
reagents used for DNA extraction are:
Prep Filer ExpressForensic DNA Extraction Kit (for
soft tissues)
Prep Filer Express BTAForensic DNA Extraction Kit (for
hard tissues)
The Prep Filer ExpressExtraction kit is based on the
property of magnetic particles which efficiently binds the
DNA and a multi-component surface chemistry.
During the washing steps the magnetic particles + DNA
complex remains stable, at the same time it removes
inhibitors and allows the efficient release of DNA during
elution to recover a highest amount of pure DNA.
Thirteen (13) samples can be isolated at one time

82. Body Fluids Protocol For processing the various body
fluids such as: Liquid samples (blood, saliva), Blood on
FTA paper or fabric, saliva or semen on any kind of
fabric, Buccal swabs, etc., the materials required for the
lysis
Bone and Tooth Protocol Sampling of Bone and Tooth
Trace Evidence Protocol Substances which are used for
the DNA isolationcould be chewing gum, cigarette butts
and adhesive tapes containing saliva or blood sample.

83. Rapid HIT
The Applied Biosystems RapidHITID System is a fast and simple-to-
use instrument that produces trusted lab-quality forensicDNA profiles in
as little as 90 minutes.
The system integrates sample preparation and capillary
electrophoresis to generate DNA profiles that are aggregated within
Applied Biosystems RapidLINK Software Software v1.3 and its v1.3.
Features of the Applied Biosystems RapidHIT ID System include:
• Sample processing 90 minutes
• 1-minute hands-on time with integrated sample cartridge
• Consumables tracking through radio frequency identification
(RFID)
• Intuitive touchscreen interface
• Facial recognition and barcode camera
• Fingerprint reader
• Up to twelve months of shelf life for both sample (with RapidHIT ID
ACE GlobalFiler Express Sample Cartridge )and primary cartridges
• FBI NDIS-approved rapid DNA system and booking station device*

84. https://www.youtube.com/watch?v=qSTwCwsc -mE

86. This Photo by Unknown Author is licensed under CC BY-SA