Fragment analysis
The dried DNA was dissolved in 2 μl of loading buffer (7 M Urea, 5 mM EDTA, 0.5% Blue Dextrane, pH 8.0) containing a ROX-labelled size marker derived from specifically designed λ-phage PCR fragments (45 bp, 80 bp, 120 bp, 160 bp, 200 bp, 240 bp, 280 bp, 320 bp, 360 bp, 400 bp, 450 bp, 500 bp, 550 bp, 600 bp, 650 bp, 700 bp, 750 bp, 800 bp, 850 bp, 900 bp). The fragments were denaturated at 90°C for 6 minutes, loaded onto a 36 cm long, 5% (29:1) polyacrylamid, 7 M urea gel, and separated at 2000 V, 50 mA, and 51°C in a 96 lane ABI377 DNA analyzer. The fragment sizes of the FAM, JOE, and NED labeled fragments between 45 and 900 bp were calculated from the raw data relative to the ROX size marker by the GeneScan program (Applied Biosystems) and exported as text files. The data pertaining to the peaks (lane number, height, area, colour, calculated length of fragments) were imported into a relational database scheme (Oracle 8.01 relational database management system) and integrated with the information on the cDNA library, the location of the colonies on the microtiterplates, the colony composition of the DNA preparation, the labelling reaction, and the gel conditions.
Analysis of restriction fingerprints derived from single clones – single clone analysis (SCA) A computer program (written with the Microsoft Visual Studio 6 software suite) was designed that extracted fingerprints from the raw data of single clone analysis. In one lane, the peak for each restriction enzyme was chosen, that had the maximum height (NewHeight) after applying the following empirically determined formula in order to account for the broadening in peak shape and the resulting lower peak height that goes along with increasing numbers of base pairs: NewHeight = MeasuredHeight / ((CalculatedBasePairs / - Denominator) + Addend), with Denominator = 3.0, Addend = 500. The corresponding calculated lengths of the fragments were stored as fingerprints in the database.