High Sensitivity DNA Analysis

• DNA Enhancement
• Additional Amplification Cycles
• High Sensitivity Analysis - A Staged Approach
• Reporting

SGMplus

SGMplus is the type of DNA analysis routinely used in forensic casework. SGMplus® isthe name of a commercially available (Applied Biosystems) set of ten genetic markers (known as Short Tandem Repeat or STR markers) plus a marker to determine the individual's gender.

Samples submitted for DNA profiling first undergo a procedure to extract the DNA before the amount of DNA is measured, using a highly sensitive 'quantitation' technique. This measurement guides the laboratory procedures which are followed and influences the interpretation of the profile results.

Obtaining an SGMplus profile involves the amplification, or copying, of sections of the extracted DNA. Routinely, 28 amplification cycles are undertaken to obtain a standard SGMplus profile.

Standard SGMplus (28 cycle) is an extremely sensitive technology which can be applied to very small amounts of body fluids and other cellular material, including items that have only been handled.

Due to the sensitivity of SGMplus, specially designed 'clean-room' laboratories and procedures are used to minimise the possibility of contamination. Cellmark's 28 cycle SGMplus technique is accredited to ISO17025 and resulting profiles that meet the required criteria can be loaded to the UK National DNA Database® (NDNAD).

DNA Enhancement

Cellmark's 'DNA Enhancement' has been developed to improve the detection of 28 cycle SGMplus results when very low levels of DNA are present. There is no increase in the number of PCR cycles.

It involves two stages which can be summarised as follows:

• Further Purification - additional purification allows the results to be seen more clearly thereby increasing the amount of DNA information that can be obtained.
• Analysing more amplification product - more DNA is tested so the sensitivity of the process is increased.

Cellmark carried out initial validation of these techniques in 2004 and a further extensive validation was completed in 2007. The technical processes involved in Cellmark's DNA Enhancement technique have been widely used in other, non-forensic areas of DNA analysis for many years. The use of these processes has been published in peer reviewed scientific publications (Simplified Low-Copy-Number DNA Analysis by Post-PCR Purification. P.J.Smith, M.S and J. Ballantyne, J Forensic Sci, 2007, 52, 820-829) and the scientific basis by which they increase sensitivity is well understood and accepted within the general scientific community.

Cellmark's DNA Enhancement service is not currently included within Cellmark's ISO17025 accreditation but the procedures have been validated and are delivered within quality systems certified to ISO9001:2000. DNA profiles resulting from DNA Enhancement cannot be retained on the NDNAD although profiles can be the subject of 'one-off' speculative searches and results can and have been presented in court.

Validation data for DNA Enhancement was submitted to the UK Accreditation Service (UKAS) at the end of 2007 with an application for the inclusion of this service within Cellmark's ISO17025 accreditation.

Additional Amplification Cycles

Using additional amplification cycles can provide increased sensitivity because more copies of the DNA are made. Cellmark has validated a 34 PCR cycle SGMplus approach which is delivered within quality systems certified to ISO9001:2000. This process is not currently included within Cellmark's ISO17025 accreditation, and DNA profiles resulting from this 34 PCR cycle analysis cannot be loaded to the NDNAD, although profiles can be speculatively searched. The 34 PCR cycle analysis is always preceded by DNA quantitation, 28 cycle SGMplus analysis and consideration of 'DNA enhancement'. Recourse to 34 cycles is therefore only occasionally required.

High Sensitivity Analysis - A Staged Approach

All samples submitted for DNA analysis, even when high sensitivity analysis is requested, will undergo testing with standard SGMplus analysis. We take a staged approach:

1. Specialist DNA extraction
2. DNA quantitation - to accurately measure how much DNA is present
3. SGMplus 28 cycle analysis
4. Evaluation leading to:

(a) Reporting of the 28 cycle SGMplus result

or

(b) DNA Enhancement if required/suitable

or

(c) Additional PCR cycle analysis as appropriate

Reporting

The amount of detected DNA determines how the results will be analysed and reported. It is not always possible to determine the type of body fluid from which the DNA originated although this may sometimes be inferred by the circumstances of the case. The DNA result does not itself assess how the DNA came to be found in a particular location, or when it was deposited there.

Cellmark's staged approach to the forensic analysis of small amounts of DNA is designed to ensure that crime scene samples benefit from the use of higher sensitivity techniques, applied only when standard 28 cycle SGMplus is insufficient for the low level of DNA present.

For further information on Cellmark's procedures for high sensitivity DNA analysis:

Email: info@cellmarkforensics.co.uk

Website: www.cellmarkforensics.co.uk