Enhanced Genetic Analysis of Single Human Bioparticles Recovered by Simplified Micromanipulation from Forensic ‘Touch DNA’ Evidence

The overall goal of this procedure is to recover individual or agglomerated cells termed putative bio particles from forensic touch DNA evidence and obtain a DNA profile of the donor. During the commission of a violent crime, a small number of cells are transferred from an individual to another individual, such as when an assailant grabs a victim’s wrist. In order to recover the small number of cells first, use a low adhesion gel film attached to a microscope slide to sample the contact area.

The second step is to stain the transferred bio particles for easier viewing under the microscope. Next, isolate the bio particles from the gel film using micro-manipulation. Collect the water soluble adhesive at the tip of a tungsten needle and use it to remove bio particles of interest from the gel film surface.

The final step is to carefully transfer the individually collected bio particles to a PCR amplification mix at the bottom of A PCR collection to ultimately enhanced DNA typing methods are used to obtain a DNA profile of the donor of trace DNA evidence. Okay, in my lab, we focus on the genetic analysis of the small numbers of cells that are transferred during the commission of a violent crime against the person between one individual and another individual, or between an individual and, and an object. Traditional methods often fail to recover the DNA profile from such small numbers of cells.

Here we have developed a simplified method for the physical recovery and genetic analysis of such small numbers of cells, either individual cells or agglomerates of cells that can be incorporated into any forensic laboratory. Demonstrating the procedure will be Katie Fairish, a grad student in my lab. To begin this procedure, prepare the gel film that will be used for collecting bio particles.

Cut the gel film to the desired size appropriate for the glass microscope slide being used as the solid support. For example, for a three inches by one inch glass slide, use the gel film size of two inches by 0.75 inches. Remove the white backing from the gel film and attach the gel film to the microscope slide pressing down firmly to ensure a complete attachment.

Note that there is a clear top protective layer that should not be removed to allow pressure to be applied along the piece of gel film without contaminating the gel film surface prepared gel film slides with the protective layer attached can be stored at room temperature until needed. When ready to use the gel film, use sterile tweezers to remove the clear top protective layer. Place the gel film surface in direct contact with a desired touched object or surface.

For example, human skin from the wrist. Apply a small amount of pressure to ensure efficient transfer of bio particles to the gel film. Do not apply too much pressure because the glass slide could break multiple samplings from the same object or surface can be collected onto the same piece of gel film In the same manner, bio particles can be collected from objects and clothing items such as a shirt collar.

To confirm the transfer of bio particles onto the gel film, examine the slide on a stereo microscope using trans or epi illumination. A magnification of approximately 20 x is best for viewing larger areas of the gel film, while a magnification of approximately 200 x is best for viewing individual bio particles. Store the gel film sample slide at room temperature in a covered slide box.

Until ready for staining and or bio particle collection, bio particles can be stained to aid in visualization. To accomplish this first place the gel film sample slide prepared as shown earlier onto a slide staining rack over a sink. Using a disposable transfer pipette cover the entire gel film surface with trian blue stain incubate at room temperature for one to two minutes.

Remove excess stain by tilting the slide to allow the stain to drain off into the sink if needed. The slide can be rinsed by gentle flooding with sterile water using a disposable transfer pipette. After the slide has been aired dried, view the slide using a stereo microscope to ensure proper staining.

The stained slide can be stored at room temperature in a covered slide box. Begin this procedure by placing the appropriate number of 0.2 milliliter PCR tubes in a rack and labeling the tubes appropriately. Prepare an appropriate volume of STR amplification mix for the desired number of samples.

Vortex and master mix well and briefly centrifuge in a mini centrifuge PI path. 3.5 microliters of amplification. Mix into each 0.2 milliliter tube cap each tube loosely.

Place a piece of double-sided sticky tape directly onto a glass block. This will be used to hold the 0.2 milliliter tube in place during sample collection. Place another piece of double-sided sticky tape onto a clean glass microscope slide.

Then place a piece of water soluble wave shoulder tape on top of the double-sided sticky tape. Secure the tape in position by applying pressure along the length of the tape. The protective top white layer can then be removed.

Next place the first 0.2 milliliter tube onto the double-sided sticky tape On the glass block. Set this tube aside until the sample is collected. Place the water soluble wave soldier tape slide under the microscope while viewing at a low magnification.

Use the tip of a tongues stand needle to gently scrape the surface of the tape in order to collect the small amount or ball of adhesive at the end of the needle. The size of the ball of adhesive will depend on the number of bio particles to be collected Carefully. Remove the tongues.

Stand needle with adhesive from under the microscope without disturbing the tip of the needle. Place the needle in a rack if needed. The most critical aspect of the procedure is the collection and the transfer of the bio particles.

To ensure success, the techniques must be practiced until the analyst is proficient at them. Place a previously prepared gel film sample on the microscope stage using a glass block to support the slide. Adjust the focus and magnification until the bio particles can be easily viewed.

Identify the bio particles that will be collected. Retrieve the tungsten needle with the adhesive ball and place the needle over the gel film surface where the bio particles of interest are located. Press the tip of the needle down so that it is in contact with the bio particle.

Lift the needle up to ensure that the bio particle has been collected. Repeat this process until the desired number of bio particles has been collected. Place a prepared 0.2 milliliter PCR tube on the microscope stage.

Adjust the magnification so that the bottom of the tube containing the amplification mix is in focus. While viewing through the microscope, carefully insert the tungsten needle into the 0.2 milliliter PCR tube until the tip of the needle is in the amplification mix. Hold the needle in the amplification mix until the adhesive dissolves and the release of bio particles into the solution is observed.

Remove the needle. Place the 0.2 milliliter tube upright and loosely cap it. Clean the tungsten needle with a pre-moistened alcohol wipe and repeat this collection procedure for additional samples.

To begin the enhanced amplification protocol for autosomal STR profiling of the collected bio particles, add 1.5 microliters of lysis buffer to each 0.2 milliliter tube containing amplification mix and the bio particles close the lids of the tubes tightly. Place the samples in the thermal cycler and amplify the samples using the cycling conditions described in the protocol text When the amplification is complete. Store the amplification products at four degrees Celsius until ready for detection by capillary electrophoresis.

No representative images of individual bio particles in a shirt collar sample are shown. The bio particles collected are circled. Red circles indicate that a profile was recovered.

The percent allele recovery is included. Black circles indicate that a profile was not recovered. Similarly, these images are of clumped bio particles in a shirt collar sample shown here is the STR profile obtained from one of the individual bio particle samples from the shirt collar.

Its accuracy was determined by comparison to a reference profile. Allele numbers are designated below each peak at each locus. The x axis represents fragment size and the Y axis represents signal intensity.

This profile is of high quality with reasonably balanced inter locus peak heights and no allelic dropout. This next profile is a full STR profile obtained from one of the clumped bio particle samples from the shirt collar. Lastly, this figure shows an example of unsuccessful DNA profiling of a single bio particle with a 3%allele recovery.

This method should promote the recovery of single source DNA profiles from assailants in physical assault cases. It hasn’t escape our attention that this method also removes some of the problems associated with the interpretation and deconvolution of mixed DNA profiles.