After hybridisation, washing and scanning, Cy5 and Cy3 fluorescence intensities are measured for each feature on the array, normalised, and log2 ratios of the test DNA (for example, Cy5) divided by the reference DNA (for example, Cy3) are then plotted against chromosome position.
The sensitivity of the methods was estimated by analyzing the reference DNA fragment containing 5hmC provided by the kit.
Afterwards, 40 µl of the DRB containing proteinase K were added to each well and samples were incubated at 60°C for 15 min, followed by an incubation at 95°C for 3 min. The sensitivity of the methods was estimated by analysing the reference DNA fragment containing 5hmC provided by the kit.
In every sample, for every probe, a tumour to normal DNA copy number ratio was calculated by dividing the median area under the peak for the 12p12.1 probe by the value for the reference DNA.
Each T/S value was later converted to number of base pairs (bp) by multiplying the T/S value by the known LTL of the reference DNA, which is a pooled sample of DNAs from several normal Utah whites aged 65 years and older.
The quantities of telomeric product (T) and single copy gene (S) were determined relative to the reference DNA by the standard curve method.
Mutations were detected by comparing these samples to the reference DNA sequence (GenBank accession number: HSU94788).
Genomic DNA from HeLa cells was used as the reference to quantify the T and S values relative to the reference DNA sample by the standard curve method.
Wells containing reference whole-mouse DNA (strain C57BL/6J, Jackson Laboratory) diluted over 16-fold range for T PCR (10, 5, 2.5, 1.25 and 0.625 ng per well) and for S PCR (5, 2.5, 1.25, 0.625 and 0.312 ng per well) were included in each PCR run so that the quantity of targeted templates in each research sample could be determined relative to the reference DNA sample by the standard curve method.
Interestingly, more than 25% of the RRAs are private/rare variants of individuals from African populations, and this is in accordance with the fact that the majority of the reference DNA used by the human genome project was from donor RP11, an individual likely of African-American ancestry [ 23, 24].
These detection rates for the 500 K GeneChip® were similar to those from our previous work using pooled DNA on the 10 K and 100 K GeneChip® platforms [ 29, 32, 33] and only slightly less than for individual genotyping of the reference DNA sample provided by Affymetrix (99.3% for Sty, 98.9% for Nsp).
