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DNA: Low Copy Number DNA Testing in the Criminal Justice System

 

Updated 23/01/08

Testing samples containing very small amounts of DNA

  • The standard DNA test (SGM+) is used where an identifiable stain such as blood or semen is found. The smallest bloodstain visible with the naked eye (c. 50 - 100 cells) contains enough DNA for this test.
  • This analyses eleven areas (also called markers or loci) of DNA, consisting of ten variable areas and a sex test.
  • These areas are copied 28 times and instrumental analysis used to capture and analyse the result.
  • This test is used to produce DNA profiles for adding to the National DNA Database (NDNAD).
  • This type of test is used worldwide, and a variety of commercial systems looking at different areas (markers, loci) of DNA are available.
  • Some samples are invisible to the naked eye, in poor condition due to external factors e.g. fire and water, or have been retained from crimes that occurred many years ago. These may have too little DNA for the standard test to be successful.

How the Low Copy Number (LCN) technique works

  • The LCN test is based upon the same scientific principles as the standard SGM+
  • test, with variations designed to increase the sensitivity of the process, including copying the DNA sample 34 times rather than the standard 28.
  • The test can obtain a profile from as few as 5 - 10 cells, or from DNA that is in poor condition. This could be the amount of DNA left on a cup by drinking from it or on a pen by writing with it.
  • This increased sensitivity means ultra-clean laboratories are needed for the testing to minimise contamination of the sample by DNA from any other source.
  • Rather than performing a separate quantitation stage, a dilution stage is now included routinely to ensure that nothing else present in the sample has caused the result to be lost.

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Special DNA precautions

  • All staff examining items that may need any DNA analysis are required to take special precautions and wear protective clothing to minimise the chance of their own DNA coming into contact with the sample.
  • All the solutions and laboratory equipment used in the process are specially pre-treated to destroy DNA.
  • A database of DNA profiles from all FSS scientific staff and many manufacturers is used to screen results before they are searched against the NDNAD.
  • When investigators and scientists collect samples from the scene of a crime, precautions should be taken to ensure that their DNA does not come into contact with the sample.

Interpretation of results

  • The standard test produces reproducible and repeatable results when the quantity of DNA is not a limiting factor. This is like casting a net into a well-stocked lake with a variety of fish; the catch will represent the different types of fish.
  • The LCN test is more like casting a net into a lake with few fish; the catch may not represent all the types of fish and a second attempt may contain different types. The individual catches may not fully represent all the types but repeat analysis may assist.
  • In LCN testing, each sample is divided into three parts or aliquots, and two of these are tested. The third is retained for further testing in the event of a failure or to confirm the presence of a mixture.
  • Only those DNA components that are seen twice are included in any calculation, to show that the result is reproducible.
  • If the result is not reproducible or is a complex mixture then it is disclosed but no calculation is carried out.
  • Statistical interpretation of the results allows for the possibility that some of the DNA may be due to contamination, or other effects caused by working with such low level samples.

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Strengths and limitations

  • The LCN test enables profiles to be obtained from samples where the quantity or quality of DNA present is inadequate to give a result from the standard SGM+ test
  • The statistical calculation, as for all DNA results, compares the likelihood of obtaining that result given the two competing accounts of the defence and prosecution about the person or people who may have left the DNA
  • The LCN result does not in itself provide information about the type of body fluid the DNA came from, although there may be circumstances that enable this to be inferred. For example, a profile obtained from the rim of a cup may be inferred to have come from saliva.
  • The LCN result does not itself provide information about the actions that caused this DNA to be deposited on an item.

International use of LCN

  • The FSS LCN test requires an ultra-clean laboratory and so is more expensive and less widely offered than the standard test.
  • The site of this bespoke laboratory is remote from other DNA Units, operates stringent entry requirements, is fitted with positive air pressure and specialist lighting and chemical treatments to minimise DNA contamination.
  • LCN methods have been used as evidence in a number of countries, ie; United States (New York), New Zealand, Holland, Italy, Germany, Croatia, Austria and Switzerland. Other countries including Belgium, Sweden, United States, Australia, Canada, Cayman Islands and Bermuda have requested this type of analysis from the FSS, who provided statements and scientists to attend Court.
  • Appendix B lists the internal validation and peer review process LCN has undergone within the FSS. Various methods are being developed to profile and interpret small quantities of DNA. The FSS process is accepted by the international, operational forensic community, some of whom are developing other ways to address these issues.

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Annex A

Separate Supplementary Information for Operation Cube Cases:

Use Of LCN

  • LCN testing has been used since 1999, and currently represents about 3.3% of all DNA tests.
  • Some of these are 'cold cases' which were not solved at the time where samples have been logged and saved awaiting improvements in analysis that may yield a result some time in the future.
  • If these samples pre-date the special DNA precautions that are now taken, their suitability for testing is reviewed in advance and any limitations on the interpretation of the result due to this are made clear in the statement.

Operation Cube

  • During 2005 it became apparent that some of the samples where little or no profile was obtained could be retested and improved by diluting the sample. It is thought that an inhibition may be present in these, but the cause is not known.
  • All the affected samples are being retested in a project called Operation Cube; c2500 samples have been completed, with 314 enhanced results and 16 cases have been progressed as a result.

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Annex B

Validation and Accreditation of the Technique

  • Two principal Forensic Science Service papers about LCN have been published in peer-reviewed journals.
  • Further extensive validation covering all the stages of the technique and interpretation of results includes eleven more papers, forty-three internal FSS Research Reports, contributions to several textbooks and a European collaborative exercise.
  • A list of the externally published FSS papers, internal research reports produced and validation that was carried out to support all the stages of the LCN process is available from FSS Headquarters on request.
  • Further work is not 'publishable' in scientific journals since it will no longer be novel - eg; once Dolly the sheep had been cloned, the next cloned sheep was no longer news!
  • The Custodian of the NDNAD sets the standards for DNA profiles that are acceptable for adding to the NDNAD. LCN has been accepted by the Custodian for this purpose since 2002, following successful completion of proficiency tests.
  • In 2007, an external review was commissioned by an ACPO chaired working group. The review accepted that the FSS LCN method was validated according to the processes that were in force within the FSS at the time, although currently not against any internationally or nationally recognised technical standard.
  • The FSS processes have been accredited for LCN to ISO17025 by UKAS (www.ukas.com) since 2001 and ISO9001 by BSI since 2005. Information about these standards can be obtained from (www.iso.org).
  • The guidelines set by the European Network of Forensic Science Institutes (ENSFI) (www.enfsi.eu/get_doc.php?u) and the Scientific Working Group on DNA Analysis methods (SWGDAM) have been met:
    (www.fbi.gov/hq/lab/fsc/backissu/july2004/standards/2004_03_sta ndards02.htm)
  • A collaborative study with other European forensic laboratories and the US Armed Forces DNA Identification Laboratory reported that LCN compared favourably against other methods for testing poor quality samples: (www.cstl.nist.gov/biotech/strbase/pub_pres/EDNAPdegradedDNAstudy.pdf)

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