ROUTINE DNA ANALYSIS

• Jurisdiction: Australia

ROUTINE DNA ANALYSIS

DNA extraction .............................................................................................. [80.600]

DNA Quantitation .......................................................................................... [80.620]

DNA amplification .......................................................................................... [80.640]

Capillary electrophoresis............................................................................... [80.660]

DNA typing .................................................................................................... [80.680]

DNA profile interpretation .............................................................................. [80.700]

[80.600] DNA extraction

Overview

As mentioned, nuclear DNA is present in the nuclei of cells contained in biological material (such as blood, semen and saliva). Inside the cells other cellular material such as mitochondria may be found. The DNA extraction process liberates the DNA from the cell and removes all other cellular material and fluid, leaving the purified DNA in solution.

There are a number of methods for extracting DNA which can generally be separated into organic and non-organic methods. The type of biological material and the surface on which it is deposited will determine the best technique for extraction. Most laboratories however favour (but are not be limited to) a particular method for routine analysis and will modify this depending on the biological material or item on which it is deposited. For example, extraction of DNA from spermatozoa cells will first require steps to separate spermatozoa cells from non-spermatozoa cells. Likewise a cigarette butt with saliva deposited may require special attention due to chemicals in the cigarette which inhibit the PCR process. Techniques described in this chapter have been classified as either current or legacy. This classification was determined via a questionnaire sent to Australian and New Zealand forensic laboratories (2016). Legacy techniques may be in routine use in some laboratories and may be relevant for historical cases (ie, cold cases).

Current techniques

FTA® paper analysis

FTA® paper was developed by Lee Borgoyne at Flinders University, South Australia. It is a cellulose-based paper which is impregnated with chemicals that lyse (break open) the cells on contact, bind the DNA to the paper and prevent the growth of mould and fungus which would otherwise degrade the DNA (Burgoyne, 1996; Rogers and Burgoyne, 1997). FTA® paper is most useful in the collection of reference person samples and is used in most of the jurisdictions in Australia for the collection of reference samples (saliva or blood) permitted under forensic procedures type legislation.

The FTA® paper is particularly suited to Australian conditions as it provides a stable method for storing and transporting the DNA over long and often environmentally harsh distances. The paper also lends itself towards automation. Blood can be placed directly onto the card or saliva can be transferred via a foam swab. The paper is then washed to remove inhibitors and other contaminants and placed directly into the PCR reaction tube (see Direct PCR at [80.640]).

There is no requirement to quantitate the sample due to the punching of a predetermined amount of card providing an approximate working range quantity of DNA. The end product of this process is amplified DNA in a PCR tube, not purified genomic DNA as with other extraction methodologies. Therefore, a sample must be re-punched if a re-amplification is required. DNA can also be extracted from the FTA® paper via other methods as described here (eg, magnetic bead extraction) (Stangegaard, 2013). This approach results in genomic DNA and requires quantitation; however, it permits the re-amplification of a sample without re-punching.

Magnetic bead extraction

Commonly used in many Australian jurisdictions since 2011, magnetic beads can be used to bind DNA by exploiting the negative charge of the DNA molecule. An initial extraction process is used which is specifically tailored for the type of biological material to be extracted; this improves the amount of DNA to be yielded from the biological material by lysing the cells to release the DNA. The magnetic bead resin is then added to the extraction solution which then binds the DNA. Tubes containing the DNA in solution are located next to a magnet to allow washing of the samples to remove any inhibitors and unwanted cellular material. With the use of chemicals, the purified DNA can then be removed from the beads into solution ready for the PCR. The advantages of magnetic bead technology include that it can be used in automated systems. The system is highly efficient at binding small amounts of DNA and uses no harmful organic solvents.

DNA purification

After the initial extraction process has been completed, the DNA may be further purified and concentrated to increase the chance of obtaining a DNA profile. This is particularly useful in LTDNA samples where there may be few copies of the DNA in a large volume and where there may be an increased number of inhibitors not fully removed by the initial extraction process. The extraction solution containing the DNA is placed into a small column/tube containing a membrane. A buffer solution is added in order to create optimal conditions for PCR, and the column is centrifuged. The membrane allows inhibitors to pass whilst retaining the DNA. In this way, the DNA can be cleaned and a set volume retained for further analysis.

Differential extraction

A modified version of the standard extraction process can be used for sexual assault cases where samples are thought to contain mixtures of semen and other biological material. This process is designed to exploit the difference in structure and density between spermatozoa and other biological material in order to separate out the spermatozoa from the remainder of the biological material. The sample is subjected to incubation in buffer containing an enzyme called proteinase K, followed by centrifugation; as a result of this process spermatozoa, if present in the sample, form a pellet at the base of the tube with the remainder of the biological material remaining in solution. The spermatozoa-enriched pellet and spermatozoa-depleted solution are subsequently separated and the remaining steps in the DNA process performed separately on both samples; the result of this analysis is that two separate DNA profile results are obtained, one from the spermatozoa-enriched sample (male, typically offender sample) and the other from the spermatozoa-depleted fraction of the sample (female, typically victim sample). This technique is employed to minimize mixed DNA profiles (combination of male DNA and female DNA) thereby avoiding complex profile interpretations.

Legacy techniques

Phenol-chloroform method

The basic organic extraction procedure uses a detergent (sodium dodecyl sulphate) to rupture the cells' membranes and a protease (proteinase K) which denatures (breaks down) the proteins to elicit the DNA from the nucleus of the cell. The organic solvent phenol-chloroform then removes the proteins and other cellular material, leaving the soluble DNA in the organic aqueous portion of the extraction solution. This technique is particularly beneficial in the removal of organic inhibitors and degraded samples found in tissue (eg, muscle) from burnt remains, leaving good-quality, clean DNA. The procedure, however, uses hazardous chemicals (phenol-chloroform) and is time-consuming.

Chelex method

The basic chelex method consists of boiling the sample in a 5% chelex solution. Cellular material, including proteins, is broken down during the boiling process and the DNA is released, denatured (made single stranded) in solution (Walsh et al, 1991). Chelex is a commercially available product made up of chelating resin beads (chelate is derived from the Greek word "claw" due to its ability to grasp (bind) metal ions). The beads have a high affinity for polyvalent metals ions (eg, magnesium) that would otherwise degrade the DNA. The chelex method is simple and quick and is more cost-effective than organic extractions. There are also fewer steps required, which reduces the chance of contamination or transfer errors. However, the chelex resin beads must be completely removed...