BIOLOGY. FORENSIC MOLECULAR BIOLOGY Forensic labs are inundated with requests for forensic biology cases due to the power of DNA technology. DNA: Deoxyribonucleic

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Forensic labs are inundated with requests for forensic biology cases due to the power of DNA technology.

DNA: Deoxyribonucleic acid is a double-stranded helix of nucleotides, which carries the genetic information of a cell.

Traditional ABO blood typing methods have evolved to DNA analysis, with high certainties of identity.


Double helix: A structure that can be compared to a spiral staircase, consisting of two polynucleotide chain wound around each other.


Multiplex polymerase chain reaction short tandem repeat (PCR-STR) – is capable of producing sole source attribution probabilities of one in a trillion or more.

The double-stranded DNA molecule was first described by Watson and Crick in the 1950s.


DNA typing technology was first used in 1985 by Dr. Alec Jeffreys.

Jeffreys recognized that certain regions of DNA contain repeats of the same sequences; these repeat regions vary in length in different individuals.


Jeffreys named the process for isolating and reading these DNA markers “DNA fingerprinting”

Jeffreys used a molecular biology technique known as restriction fragment length polymorphism (RFLP) to compare molecules.


RFLP provided a very powerful tool for forensic DNA typing. However, the process was costly and time consuming, taking six to eight weeks to develop.

The use of radioactive probes presented a safety hazard.


At least .05 micrograms of intact DNA was required to successfully analyze an RFLP-generated DNA profile.

This limitation was solved in 1986, when Kari Mullis developed a molecular DNA technique known as polymerase chain reaction (PCR).


Polymerase chain reaction (PCR) – A technique for replicating or copying a portion of a DNA strand outside a living cell.

PCR revolutionized forensic DNA typing, because it allowed very small amounts of DNA recovered from the crime scene to be amplified.


The PCR technique allows the forensic scientist to replicate a DNA molecule many times.

In a three-hour run (28 cycles), millions of copies of specific target DNA can be produced.

PCR technology meets the needs of the forensic crime lab, in that it is sensitive, safe, fast, robust, and economical.


The forensic biology laboratory is divided into two sections, serology and DNA.

– The serology laboratory is the first section to open a biology case and begin analysis.

Serology : The science that deals with the properties and reaction of blood serum.


The forensic scientist opens the seals of the case and checks the inventory of the evidence against the evidence submission form.

Analyses of samples from victims and suspects must be separated by time and space.


The serology laboratory screens all incoming evidence for human semen, blood, and saliva stains.

Stains and liquids identified by these screening techniques are forwarded to the DNA unit for analysis.

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Blood screening methods are widely used by all laboratories and some crime scene units to exclude items in order to increase the efficiency of the DNA laboratory.

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At the scene of a violent crime, investigators often discover large quantities of blood on the victim, the walls, the floors, and the furniture.

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Investigators at the crime scene want to answer three questions:– Is this blood?

– Is it from a human?

– How closely does it match the blood of the victim or suspect?

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Sometimes the blood sample is small, perhaps because the perpetrator attempted to clean up the blood at the crime scene.

In this case the forensic examiner needs to detect the presence of blood that might otherwise be overlooked, either because it is a very small drop or because the background it has landed on hides the blood from the naked eye.

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The presumptive tests for blood rely on hemoglobin’s ability to catalyze the oxidation of certain reagents.

– Catalysis – The causing or accelerating of a chemical change by the addition of a substance that is not permanently affected by the reaction.

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Leucomalachite green (LMG) is a color change reagent oxidized by peroxide that yields a colored reaction product.

If blood is present, LMG produces a blue-green color when the peroxide is added.

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Strong oxidizers and other iron-containing compounds can result in false positives using these techniques.

The screening test may consume the entire sample, leaving none for DNA analyses.

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The scientist must evaluate the benefits of the screening process to decide if screening may be an unnecessary risk to subsequent DNA analyses.

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It is important to note that blood isn’t the only material that will cause LMG to change color.

Vegetable matter, such as horseradish and potatoes, can give false-positive results (although they are unlikely to be present at a crime scene, of course).

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Immunochromatographic test devices used in the medical community to detect human hemoglobin are now widely used in forensic laboratories to screen for human blood prior to DNA analysis.

The OneStep ABAcard HemaTrace test device provided by Abacus Diagnostics is used for human blood screening tests.

SEMINAL FLUID AND SEMINAL FLUID AND SPERMSPERM

The prosecution of sexual offense cases relies heavily on the confirmed presence of seminal fluid and semen.

Semen is fluid produced by the testes and accessory sex glands, which includes the prostate gland, bulbourethral glands, and seminal vesicles.


Semen is more than 90% water and has a pH in the range of 7.2 to 7.4.

One milliliter of semen contains 20 million to 100 million sperm cells.

A normal male ejaculates 2 to 6 mL of seminal fluid.


A DNA profile can be developed from many other human tissues and fluids.

However, the defense can claim that the resulting DNA profile was from a source other than seminal fluid or semen, and not the result of sexual assault.


There are several methods used to collect and screen for the presence of seminal fluid and semen.


Often, the crime scene may include a large number of items that might have been stained by seminal fluid.

For example, multiple garments, bed clothing, rugs, drapes, and solid surfaces.


The forensic examiner often searches the crime scene with an ultraviolet (UV) light to detect the presence of seminal stains.

The UV light makes the search of the scene go quickly, and semen fluoresces under UV light, making stains easy to locate.


Other body fluids such as urine also fluoresce in UV light, however, so this test is always considered presumptive for semen.

Another presumptive test often used at a crime scene to detect seminal fluid is the acid phosphatase test.


The prostate gland secretes the enzyme acid phosphatase, and its concentration is much higher in seminal fluid than in any other body fluid.

This compound can be easily detected with a simple color test.


If the stain is semen, the reagent will produce a rapid color change, with a purple color appearing in less than 1 minute.

In the presence of diazotized o-dianisidine, acid phosphatase will react with naphthyl phosphate to produce a purple color.


Prostate-Specific Antigen (PSA)

– Analysts often examine stains or swabs that they suspect contain semen (because of the presence of acid phosphatase), but that yield no detectable spermatozoa.


Prostate-Specific Antigen (PSA)

– How, then, can one unequivocally prove the presence of semen?

– The solution to this problem came with the discovery in the 1970s of a protein called p30 or prostate specific antigen (PSA)


Once the material is proven to be semen, the next task is to associate the semen as closely as possible with an individual.

Forensic scientists can link seminal material to an individual by DNA typing.

SEMINAL FLUID AND SPERM SEMINAL FLUID AND SPERM COLLECTION AND SCREENING COLLECTION AND SCREENING

METHODSMETHODSVisual examination of stain– Dried, coarse, and crusted materials

– Noticeable stains on clothing, bedding, and other substrates

– Alternate light source creating fluorescence of stain or substrate


METHODSMETHODSSample collection

– Cutting from clothing or other fabrics

– Swabs from surface of nonporous materials

– Patting the surface of stains with moistened filter paper or swabs

– Lifting small amounts of material from large stains


METHODSMETHODSBrentamineColor test for seminal fluid

Prostate specific antigen (PSA)Visualization of PSA/antibody interface


METHODSMETHODS

Acid phosphatase Color test for seminal fluid

Microscopic visualization of sperm

SEXUAL ASSAULT SEXUAL ASSAULT EVIDENCE KITSEVIDENCE KITS

Historically, emergency room personnel have used collection items that were readily available, such as swabs and blood tubes, to collect evidence for sexual assault investigation.

SEXUAL ASSUALT SEXUAL ASSUALT EVIDENCE KITSEVIDENCE KITS

The victim’s clothing and control samples of blood are placed large bags or boxes and submitted to the forensic laboratory for analysis.

To protect this kind of evidence, all the outer garments and undergarments from the people involved should be carefully removed and packaged separately in paper (not plastic) bags.


Forensic laboratories have standardized the process by developing collection kits specifically for sexual assault evidence.

The kit contains all collection tools, seals, and containers needed for blood, secretions, and trace evidence.


Training programs have also been developed for emergency room personnel; this specialization is entitled sexual assualt nurse examiner (SANE).


The rape victim must undergo a medical examination as soon as possible after the assault.

At this time, the appropriate items of physical evidence are collected by trained personnel.


Evidence collectors should have an evidence-collection kit from the local crime laboratory.


The following items of physical evidence are to be collected:

1. Pubic combings. Place a paper towel under the buttocks and comb the

pubic area for loose or foreign hairs.

SEXUAL ASSUALT SEXUAL ASSUALT EVDENCE KITSEVDENCE KITS

2. Pubic hair standard/reference samples.

– Cut fifteen to twenty full length hairs from the pubic area at the skin line.


3. External genital dry-skin areas.

– Swab with at least one dry swab and one moistening swab.


4. Vaginal swabs and smear.

– Using two swabs simultaneously, carefully swab the vaginal area and let the swabs air-dry before packaging. Using two additional swabs, repeat the swabbing procedure and smear the swab onto separate microscope slides, allowing them to air-dry before packaging.


5. Cervix swabs.

– Using two swabs simultaneously, carefully swab the cervix area and let the swabs air-dry before packaging.

SEXUAL ASSUALT SEXUAL ASSUALT EVIDENCE KITS.EVIDENCE KITS.

6. Rectal swabs and smear.

– To be taken when warranted by case history. Using two swabs simultaneously, swab the rectal canal, smearing one of the swabs onto a microscope slide. Allow both samples to air-dry before packaging.


7. Oral swabs and smear.

– To be taken if oral-genital contact occurred. Use two swabs simultaneously to swab the cheek area and gum line. Using both swabs, prepare one smear slide. Allow both swabs and the smear to air-dry before packaging.


8. Head hairs.

– Cut at the skin line a minimum of ten full-length hairs from each of the following scalp locations: center, front, back, left side, and right side. A total of at least fifty hairs should be cut and submitted to the laboratory.


9. Blood sample.

– Collect at least 7 milliliters in a vacuum tube containing the preservative EDTA. The blood sample can be used for DNA typing as well as for toxicological analysis if required.


10. Fingernail scrapings

– Scrape the undersurface of the nails with a dull object over a piece of clean paper to collect debris. Use separate paper, one for each hand.


11. All clothing.

– Package as described earlier.


12. Urine specimen.

– Collect 30 milliliters or more of urine from the victim for analysis for Rohypnol, GHB, and other substances associated with drug-facilitated sexual assualts.


If the suspect is apprehended, the following items are routinely collected:

1. All clothing and any other items believed to have been worn at the time of assault.

2. Pubic hair combings.


3. Pulled head and pubic hair standard/reference samples.

4. A penile swab taken within twenty-four hours of the assault, when appropriate to the case history.


5. A blood sample or buccal swab for DNA typing purposes.

The forceful physical contact between victim and assailant, may result in a transfer of physical evidence – blood, semen, hairs, and fibers.

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BIOLOGY. FORENSIC MOLECULAR BIOLOGY Forensic labs are inundated with requests for forensic biology cases due to the power of DNA technology. DNA: Deoxyribonucleic