Procedures for

Forensic DNA
Analysis 1
Lucy Mwai - Gichuki, MBB,MLS
Lesson Obj. Steps Involved Steps in Forensic DNA Analysis
1 Collection Usually 1-2 day process (a minimum of ~5 hours)
Slot Blot
1 ng
2 Specimen Storage 0.3 ng
No DNA
0.5 ng
0.5 ng
3 Extraction 0.7 ng
1 ng
Blood Stain Buccal swab
1 ng
4 Quantitation Sample Collection DNA DNA

Biology
& Storage
Extraction Quantitation
5 Multiplex PCR

6 STR Typing Genetics If a match occurs, comparison of

DNA profile to population allele Multiplex PCR Amplification
7 Interpretation frequencies to generate a case
report with probability of a random
of Results match to an unrelated individual DNA separation and sizing

Technology
Database
8 Storage & Searching DNA
Database STR Typing
9 Calculation of Search
Match Probability Male: 13,14-15,16-12,13-10,13-15,16
6/28/2021 HFS 2321_Bsc. Forensic Science Year 3,
Semester 2_Forensic DNA AnalysisInterpretation of Results
 DNA extraction is the technique
used to isolate DNA in a biological
sample.
Definition
The first isolation of DNA was
done in 1869 by Friedrich
Miescher.
 What are the essential components of a
DNA extraction Procedure?
 Maximize DNA recovery
 Maximize the quality of DNA
Must be free from contamination with protein, carbohydrate,
lipids or other nucleic acids.
 Remove inhibitors like EDTA
 Remove or inhibit nucleases
 Double strand vs. Single strand (RFLP or PCR)
 1. Inorganic / Aqueous based (Proteinase K
What are the and Salting out) extraction
Most 2. Organic solvent-based (Phenol-Chloroform)
Commonly Extraction
used DNA 3. Solid phase isolation using spin columns
Extraction (Silica exchange resin- Qiagen,
Procedures in 4. Chelex (Ion Exchange Resin) Extraction
Forensic 5. FTA Paper (Collection, Storage, and
Science? Isolation)
 Target nucleic acid (ssDNA, dsDNA, Total
RNA, mRNA etc.)
Source organism (mammalian, lower
eukaryotes, plants, prokaryotes, Viruses etc)
What guides
which Starting material (whole organ, tissue, cell
extraction culture, blood)
method to Desired results (yield, purity, purification
use? time required)
Downstream applications(PCR, Cloning,
labelling, blotting, RT-PCR, cDNA synthesis,
RNase protection assays, etc)
 1. Aqueous solution-based
extraction / Inorganic Extraction
Step 1: Cell Lysis/ Membrane permeabilization: Breaking
cells open to release DNA into lysate
1. Mechanical/ Physical means
For more structured starting material (tissue pieces, plant material). No
heat as it would destroy Nucleic acids
Manual - Grinding (grinding under Liquid Nitrogen)
Automated single to multi sample processing - sonication, Bead beating,
tissue grinders
2. Chemical means -Lysis Buffer rapture cells by osmosis.
a) Detergents (NP-40 or Triton or SDS (Sodium Dodecyl Sulphate) Disrupt lipid
membranes’ Chelating agent like EDTA and EGTA also added to inhibit nuclease activity
b) Chaotropes (Guanidine Salts) - They interfere with OH groups to denature proteins.
GITC-Guanidinium thiocyanate or Guanidinium isothiocyanate
c) Salting out - Uses low pH and high salt conditions to selectively precipitate proteins.
E.g. NaOH alkaline salt

Precipitate out
DNA is left in DNA with
solution isopropanol
3. Enzymatic Disruption methods
For more structured starting material – Tissue pieces, Plant
materials, Bacteria and yeast
Proteases such as Proteinase K (digest proteins)- Bacteria
Collagenase – Plant material
Lipase – Yeast
Lysozyme, Zymolase & Lyticase – Tissue, Bacteria
Increases cost, this method not suitable for RNA extraction.
 The cell lysate after cell lysis contains;
Nucleic acids (DNA’s, RNA’s)
. *
Proteins .
*
*
.
Small molecules (Salts & Detergents) .*

Step 2: Separating DNA from Proteins and other cellular debris

Physical separation – Centrifugation, Filtering, Bead based clearing
Step 3: Precipitating the DNA with Alcohol
 Add ice-cold ethanol or isopropanol to DNA sample
 DNA is SOLUBLE in water, but INSOLUBLE in the presence of moderate conce. of
monovalent cations NA+ and alcohol, hence will come out of solution (precipitate)
Step 4: Cleaning the DNA

Wash DNA pellet with ice cold alcohol, and centrifuge

You can wash 2-3 times
Pour the alcohol off the pellet and dry
Step 5: Re-suspending the DNA in a slightly alkaline
buffer
DNA sample is re-suspended in a slightly alkaline buffer such as Tris or
Tris EDTA (TE) and is ready for use
 2. Organic Solvent Based Extraction

Step 1: Cell Disruption

Use SDS and Proteinase K. To avoid RNA contamination add RNAse, enzyme
that degrades RNA.

Step 2: Reagent addition

Add phenol:chloroform:isoamyl alcohol (25:24:1)
Phenol: Dissociates proteins bound to DNA, denature and solubilize them
Chloroform: denature proteins and lipids and stabilize the aq/organic boundary
Isoamyl alcohol: Prevent foaming and enables the separation of aq/organic
phases
Step 3: Centrifugation (3 phases are formed)
Top (Hydrophilic layer/ Aqueous Phase) has DNA
Middle: Interphase has proteins
Bottom (Hydrophobic layer/ Organic Phase) has lipids and cell debris
Step 4: Transfer aq. phase to a new tube
Step 5: Precipitation and recovery of
DNA
Add cold ethanol or isopropanol, DNA
precipitates out.
Pellet by centrifugation
Dissolve in buffer for downstream
processes such as PCR & Southern Blot
Analysis
3. Solid phase isolation using spin
columns
Step 1: Sample Lysis
Step 2: Clearing Debris
Step 3: Binding to purification matrix
a) Silica Binding Matrix – silica membrane, diatomaceous earth, slurries, particles).
Nucleic acid interacts with silica in the presence of chaotrophic salts (NaI,
Guanidine salts)
b) Ion exchange
c) Cellulose

Step 4: Washing
Using alcohol based wash buffers to remove proteins, salts and other
contaminants
Step 5: Elution
To elute the nucleic acid: Nuclease –free water, Buffered
solutions and TE
The choice is dependent on the downstream processes. The
higher the elution volume, the higher the yield and the lower the
concentration

More rapid
and effective
 4. Chelex Extraction
Chelex 100, Molecular Biology Grade resin from BioRad is a highly
pure, nuclease and ligase inhibitor-free chelating resin, certified not
to interfere with downstream PCR.
Specifically designed to complement the inherent requirements of
PCR, this pure, pipettable, small-scale resin is ready for downstream
use.
Ensuring the complete removal of PCR inhibitors, contaminating
metal ions that catalyze the digestion of DNA
 Chelex Extraction
Chelex 100 is an ion exchange resin
that is added as a 5% solution (wt/vol).
 Chelex is composed of styrene divinylbenzene copolymers
containing paired iminodiacetate ions that act as chelating
groups in binding polyvalent metal ions such as magnesium
(Mg2+).
 By removing the Mg2+ from the reaction, nucleases are
inactivated and the DNA is protected.
Chelex Extraction
 Chelex Extraction

 Step 1: A 5% solution of Chelex is added to a blood stain or liquid

blood and incubated at 56oC for 30 minutes. This step is used to
lyse red cells and remove contaminants and inhibitors such as heme
and other proteins.
 Step 2: The sample is then heated at 100oC for 8 minutes. This
causes the DNA to be denatured as well as disrupting membranes
and destroying cellular proteins.
 Step 3: The tube containing the Chelex is centrifuged, the resin is
pelleted, the supernatant containing the DNA is removed.
 Chelex Extraction
 The Chelex extraction process denatures double stranded DNA
and yields single stranded DNA, and thus cannot be used for the
RFLP procedure.
 It is advantageous for PCR-based typing methods because it
removes inhibitors of PCR and can be done in a single tube, which
reduces the potential for laboratory-induced contamination and
sample switching.
 Care should be taken not to have any residual Chelex with the DNA
extract, since Mg2+ is required for the Taq Polymerase.
 Pros:
 Relatively fast
 Can extract directly from cloth (stain)
 Minimizes contamination – uses only
Chelex a single tube
extraction  Removes PCR inhibitors
Con:
 Results in single-stranded DNA – not
useful for RFLP
 ®
5. FTA PAPER

A Unique Matrix For The Rapid

Preparation And Ambient Storage Of DNA
From Whole Blood And Other Biological
Samples

http://www.nfstc.org/pdi/Subject03/pdi_s03_m04_02_d.htm
 ®
FTA Paper
 Is a unique mixture of strong
buffers, protein denaturants,
chelating agents, and a UV
absorbing, free radical trap.
 The reagents are impregnated
into a cellulose-based filter
matrix such as Whatman
BFC180 or 31ET paper
 ®
FTA Gene Guard System
A novel system for the collection,
transport, storage and purification of DNA
 ®
What Does FTA Paper Do?
•kills blood borne pathogens on contact

•immobilizes DNA within the matrix

•protects DNA from degradation

•allows for long-term storage at room temp

 ®
Blood Samples Stored on FTA Paper Either
Dry or Wet for 6 Months in Barrier Pouch
PCR Template Concentration
Artifacts Minimized
No DNA Quantitation is Required
 ®
RFLP Analysis From Samples Stored on FTA
Paper

QuickTime™ and a

Photo CD Decompressor

are needed to use this picture

 FTA Untreated

Samples Stored
for 11 Months
Prior to RFLP
Analysis
Simple Method
For the
Application of
Blood
®
onto
FTA Paper
 Buccal Swab
Collection and
Direct
Transfer to
®
FTA Paper
 Pros:
 Very quick
FTA™  Useful for both storage and extraction
Paper Cons:
 Paper punched “jump” because of static
electricity – potential contamination
  Although DNA is relatively stable, it
does denature or get destroyed
through enzyme action, from
bacteria or through oxidation
 Therefore, samples should be
collected soon and preserved
Analyzing (usually in a buffer and by freezing)
if possible
the DNA  Care should also be taken not to
cross contaminate during collection
 Blood is also a potential pathogen, so
care must be taken to avoid exposing
yourself to blood borne viruses like
Hep B, tuberculosis or HIV
Thank You!
Questions?