Title: APPENDIX 1: ANALYTICAL STEPS FOR GENETIC FINGERPRINTING

Appendix 1: Analytical Steps for Genetic Fingerprinting

1. Taking the sample; this step is essential for the quality of the genetic fingerprint 

2. Isolating DNA from the cells; the procedures used are e.g.:

1. A stain extraction buffer composed of water, salts and an enzyme called Proteinase K dissolves dried stains and lyses cell membranes to get the DNA into solution. 

2. The extract is spun high to coagulate the DNA molecules; the resulting DNA pellets are rinsed several times with buffer solutions followed by organic extraction to remove soluble proteins and carbohydrates. 

3. The DNA is isolated on a filter and reconstituted in a buffer. 

1. Enzymatic restriction: to cut the DNA into specific fragments. Various enzymes, depending on the type of tandem repeats to be analysed (RFLP or VNSTR), are used. For these tandem repeats the following procedure is slightly different: 

1. Using RFLP: no further preparation is carried out at this point.

2. Using VNSTR and AmpFLP: if there is insufficient DNA for an appropriate analysis, polymerase chain reaction (PCR) can be used to amplify the DNA; this makes the VNSTR method usable for small samples with small amount of DNA. The PCR procedures are carried out repeatedly in the following steps:

1. 1. 1. Denaturation: the double helix is split into single-stranded DNA at a temperature of 94°C. 

      2. Annealing: a primer is added at 54°C to mark the DNA parts (called templates, in this case the STR-single strands) to be copied. Polymerase, bases and other chemicals are added. 

      3. Extension: at 72°C the polymerase works best and copies the STR. 

1. Separation of the fragments: to separate the fragments gel electrophoresis or ion exchange chromatography is used. This separates the fragments by charge and bulk. 

2. Detection; to detect the fragments two main techniques are used: 

1. Fluorescent colours in connection with lasers to stimulate the fluorescence and photographic paper (used with RFLP and VNSTR)

2. With RFLP after fixing it to a nylon membrane (so called Southern Blot) radioactive marked testing probes in connection with X-ray paper (autorad detection).

1. Analysis: there are different ways to analyse the resulting printouts:  

1. Direct comparison of patterns (bars / signals) from different sources and against references; one example is given in where three samples (1 and 2 are suspects, 3 is a sample from the site of the crime) and four loci are analysed. The comparison shows that the pattern of suspect no. 2 matches with the patterns of the sample from the crime scene. The patterns of suspect no. 1 do not match.

2. Absolute determination of the alleles of different analysed loci; storage of the results in a database for comparison with later analysed sources.

3. Further statistical analysis based on b.: The occurrence of each allele on each locus has a certain likeliness with respect to a specific population. For comparison purposes a lot of statistical data on different populations with respect to the most common analysed loci are available. The comparison of the found alleles with the appropriate reference table allows for statements how common the observed allele patterns are in the reference population. Analysing five loci (e.g. FIBRA, SE33, THO1, FGA and VWA/VWF) the result from a given example can be 1 : 25000000000 (25 billions) [BEN01] within a middle European population.