Download Differential Extraction Separating the Weak from the Strong

Objectives
 Definition of differential extraction
 Anatomy of spermatozoa
 Concept of differential extraction
 Different methods used for differential extraction
 Questions/Comments
 f
Sperm
Cell
Definition
A procedure in which sperm are
separated, or extracted, from all
other cells in a sample using a
combination of phase separations
and differential centrifugations..
White
Blood
Cell
Epithelial
Cell
Anatomy of the Sperm Cell
Nucleus contains DNA
Plasma membrane contains disulfide bonds
 Single covalent bond
between thiol groups.
Disulfide Bonds
 Thiol groups –
functional group with
Sulfur atom and
Hydrogen atom.
 Functional group of
Cystine amino acid.
 Close proximity causes
oxidation reaction.
 Found in plasma
membrane of sperm
and gives strength.
Process for
Differential
Extraction
Evidence examined for stains
Stains presumptively tested for
seminal fluid
Sperm can be visualized under
microscope
Process for
Differential
Extraction
Portion of mixed stain removed
Sodium dodecyl sulfate (SDS),
Ethylenediametetraacitic acid
(EDTA), and Proteinase K added
Chemicals break up non-sperm
cells
Does not break down DNA
Heat
required for
cell break
down
Process for
Differential
Extraction
SDS – denatures proteins by
disrupting non-covalent bonds
EDTA – chelating agent, denature
nucleases and makes cell
membrane permeable
Proteinase K – hydrolyze histones,
inactivates nucleases and lyse cell
Process for Differential Extraction
Centrifugation separates by pelleting the sperm and leaving DNA in liquid
Process for
Differential
Extraction
Liquid (supernatant) removed and
sperm pellet washed
This is the non-sperm fraction aka
E-cell Fraction
Continue on through process
Process for
Differential
Extraction
Sperm cells still intact
Add SDS, EDTA, Proteinase K
PLUS DTT
Heat
required for
cell break
down
Process for
Differential
Extraction
Heating process repeated
DTT activated when heated
DTT – Dithiothreitol breaks disulfide
bonds in sperm membrane
Other chemicals work the same as
before
E-cell
Fraction
S-cell
Fraction
Process for
Differential
Extraction
Sperm DNA left known as S-cell
Fraction
Separation complete
Take both fractions onto next step
QIAGEN QIAcube
 Released in 2007
 Fully automated sample
preparation
 Purification of DNA, RNA
and protiens
 Lysis buffer  Incubate 
QIAcube
 Can vortex, centrifuge and
pipet reagents
Promega’s Differex
 Manual Method
 Commercial differential
separation technique
 Separation using
proprietary separation
solution
 Extract E-cell and S-cell
separately
Automated Differex
 Same as manual Differex
but sperm is pelleted
using magnetic beads
and magnet
 Automatable
Laser Microdissection
 Expedites separation by
simultaneously
identifying and excising
sperm and E-cell nuclei
 Preformed on specialized
microscope
 Laser cuts cells from
stain
 Very expensive
Erase Sperm Isolation Kit
 Nuclease used to destroy
epithelial DNA before
sperm is lysed
 Can be automated
References

U.S. Department of Justice. National Crime Victimization Survey. 2008-2012.

Voskoboinik L, Darvasi A. Forensic identification of an individual in complex DNA mixtures.
Forensic Science International – Genetics 2011.

Cupples CM, Champagne JR, Lewis KE, Cruz TD. STR Profiles from DNA samples with
“undetectable” or low Quantifiler results. Journal of Forensic Sciences 2009; 54:103-107.

Benschop CCG, Wiebosh DC, Kloosterman AD, Sijen T. Post-coital vaginal sampling with nylon
flocked swabs improves DNA typing. Forensic Science International – Genetics 2010; 4:115-121.

Vuichard, et. al. Differential DNA extraction of challenging simulated sexual-assault samples: A
Swiss collaborative study. Investigative Genetics 2011; 2:11.

Marrone A, Ballantyne J. Hydrolysis of DNA and its molecular components in the dry state. Forensic
Science International – Genetics 2010; 55(5):1331-1339.
Picture References

www.giantmicrobes.com

www.sco.ucsb.edu

www.b-daddy.org

www.eonexperience.com

graphicssoft.about.com

www.clker.com

www.dreamstime.com

Misty and Jason’s DNA Extraction powerpoint.
Special Thanks
 Dr. Staton
 Josh Stewart
 MUFSC 1st and 2nd
years
 Laura Kuyper
 Misty Marra
 Photoshop
 Jason Chute
 Kyo
 Steve Goff
 Stella
 Beckwith Thomson
 Gene
Questions/Comments