Forensic Biology by Richard Li, with additions and edits by Ruth Ballard
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Transcript of Forensic Biology by Richard Li, with additions and edits by Ruth Ballard
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Forensic Biologyby Richard Li, with additions and edits by Ruth Ballard
Lecture 4: Identification of Semen
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Outline
Biological characteristics of semenSpermatozoaDetection of semen
Presumptive vs confirmatory tests Presumptive tests for semen
Detection of sperm “Christmas Tree” stain Confirmatory test for semen
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Biological Characteristics of semenTypical ejaculation
2-5 ml of semen, 160 million sperm▪ 3 pg DNA/sperm = 480,000 ng DNA/ejaculate▪ Only 1 ng DNA needed for STR typing!
Seminal fluid▪ Medium for ejaculation▪ Enzymes and other proteins
▪ Acid Phospahatase (AP), Prostate Specific Antigen (PSA), and semenogelin
Sperm cells- Spermatozoa3
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Biological Characteristics of semen
Semen is an extremely good source of DNA The best! BUT…
Not all semen stains contain sperm Vasectomy- blocks sperm from being
ejaculated▪ Semen still produced▪ DNA typing probably not possible
Infertility▪ Depending on severity, DNA typing may be
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Spermatozoa
Three distinct regions: Head – acrosome and nucleus (with
haploid DNA) Middle Piece (mitochondria) Tail (flagella; mobility)
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Presumptive vs Confirmatory TestsPresumptive tests
Fast, easy, inexpensive Great for screening evidence to find possible
stains Usually detect enzymes specific to the body fluid False positives (hence “presumptive”)
▪ Open to attack in courtConfirmatory tests
Not available for most body fluids▪ Main exceptions are semen and blood
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Presumptive Tests for Semen
Semen fluoresces under ALS UV light
▪ long-wave = “Woods Lamp” = 365 nm
Crime Lite (500 nm)Lots of false
positives8
Alternative light source
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Presumptive Tests for SemenAcid phosphatase enzyme
Advantages▪ High levels in fresh semen stains▪ Very fast, inexpensive▪ Can be done in the field
Limitations▪ Activity may be weak or absent in older stains▪ Also present at low levels in vaginal fluid and
bacteria▪ Not species-specific
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Presumptive Tests for SemenAP assay
AP liberates naphthol from alpha-naphthol and the naphthol then reacts with brentamine to form a purple-colored dye
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sodium phosphate + naphtholα-naphthyl acid phosphate monosodium salt Acid
phosphatase
Coupling reaction
Purple azo dyenapthol + Brentamine
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Overlay method▪ Spray a Whatman paper
circle with distilled water▪ Lay the paper down over
the suspected semen stain▪ Leave in contact with stain
30-60 seconds▪ Remove paper circle from
stain and spray with AP spot solution
▪ Look for a rapid color change to purple
Presumptive Tests for Semen
Positive acid phosphatase overlay
assay
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Presumptive Tests for SemenSpot test method
▪ Wet sterile cotton swab with distilled water▪ Roll swab across stain▪ Saturate swab with AP solution
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Presumptive Tests for Semen MUP
More sensitive than acid phosphatase AP catalyzes the removal of the phosphate
residue on the substrate 4-methylumbelliferone phosphate (MUP), which generates fluorescence under UV light
Filter paper overlay▪ Filter paper placed in contact with putative semen
stain and then removed and taken to dark room▪ Sprayed with MUP ▪ Fluorescence detected with UV lamp
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Presumptive Tests for Semen Prostate-specific antigen (PSA)
Major protein in seminal fluid Also detected in urine, fecal matter,
sweat, milk but at much lower levels Half-life of dried stain: 3 years Hydrolyzes semenogelins (seminal vesicle
specific antigens) Detected with immunochromatographic
test strip assay15
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Presumptive Tests for SemenSemenogelins
Higher concentration in seminal fluid than PSA
Not found in urine, milk, sweat Greater specificity for semen than PSA Detected with immunochromatographic
test strip assay
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Immunochromatographic test strip assay for semenogelin Rapid and simple Specificity still under debate Rapid Stain Identification (RSID-Semen)
▪ Independent Forensics
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Presumptive Tests for Semen
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T
Positive RSID™ semen test
human semenogenlin
monoclonal gold-labeled murine anti human semenogelin antibody to epitope 1
monoclonal unlabeled murine anti human semenogelin antibody to epitope 2
polyclonal unlabeled goat anti murine antiglobulin
T C
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T C
Negative RSID™ semen test
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Detection of SpermMicroscopic examination
“Christmas Tree” stain▪ Nuclear Fast Red stains nuclei red▪ Picroindigocarmine stains tails green
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Acrosomes don’t stain well in primate sperm
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