Objectives Definition of differential extraction Anatomy of - - PowerPoint PPT Presentation

Objectives

 Definition of differential extraction  Anatomy of spermatozoa  Concept of differential extraction  Different methods used for differential extraction  Questions/Comments

Definition

 f

A procedure in which sperm are separated, or extracted, from all

• ther cells in a sample using a

combination of phase separations and differential centrifugations.. Sperm Cell White Blood Cell Epithelial Cell

Anatomy of the Sperm Cell

Nucleus contains DNA Plasma membrane contains disulfide bonds

Disulfide Bonds

 Single covalent bond between thiol groups.  Thiol groups – functional group with Sulfur atom and Hydrogen atom.  Functional group of Cystine amino acid.  Close proximity causes

• xidation 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

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 Heat required for cell break down

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

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

Heat required for cell break down

Process for Differential Extraction

Sperm DNA left known as S-cell Fraction Separation complete Take both fractions onto next step E-cell Fraction S-cell Fraction

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  Misty Marra  Jason Chute  Steve Goff  Beckwith Thomson

 MUFSC 1st and 2nd years  Laura Kuyper  Photoshop  Kyo  Stella  Gene

Questions/Comments