Table 6.

Methods used in screening mutations of the F8 and F9 genes [49]

Stage of analysis	Special procedures	Reagents and methods
Multiplex PCR-amplification of the Factor VIII gene, F8 (26 exons) and Factor IX, F9 (8 exons), both of which are located on the X-chromosome	Extracts which were shown to be free of contamination by foreign samples based on the analyses of mtDNA and STR-markers were used for analyzing the nuclear genes. Just as in the previous analyses, negative controls were used: PCR-amplification of "empty" extracts (obtained by performing all of the extraction procedures, but without adding the bone sample) and PCR amplification without the addition of any DNA.	~ 210 pairs of primers were designed for multiplex PCR amplification of short overlapping sequences (< 200 bp), that would cover all the exons and the intron-exons boundaries of the F8 and F9 genes. The primers were grouped into 14 sets for the F8 gene and 3 sets for the F9 gene, with each set consisting of 7 to 30 pairs of primers. About 100 pg of human DNA (~ 16-17 diploid genomes) were used for the initial multiplex PCR.
Sequencing	Sequencing of the blood clotting factor genes F8 and F9 was done in parallel with the mitochondrial genome to have a control for contamination and unequivocal identification of the sample.	Individual PCR-fragments were excised and purified from a 2.5 % agarose gel and then sequenced using two strategies. One involved mixing the PCR products in equimolar amounts and using them for sequencing (Illumina GA). The other approach involved the direct sequencing of individual PCR-products on a 96-capillary sequencer 3730xl DNA analyzer (Applied Biosystems).
Genotyping of the identified F9 gene mutation	8 independent amplifications of the N7 sample (Empress Alexandra) were performed. For other bone samples, 2 to 7 independent extracts were analyzed from each sample.	The mutation which was initially found during the DNA analysis of skeleton N7 was verified by sequencing ultra-short amplicons (63 b.p. and 83 b.p.), which were obtained with specially developed primers. The same primers were used to amplify the DNA from bone fragments N146 (Prince Alexei) and N3, N5, N6, N147 (Nicolas the Second's and Alexandra's daughters).
Analysis of splicing products		The amplified fragment which bore the mutated region of the F9 gene was cloned into the pET01 Exontrap vector (MoBiTec). After verifying the structure of the recombinant molecule by sequencing, it was used to transfect a cell culture. cDNA was obtained from the spliced DNA by using RT-PCR. The cDNA was then amplified, and the PCR product library was sequenced (Illumina GA).The spliced sequences were identified in 812,114 reads. 99.982 % of the transcripts were spliced at the mutant site, and only 0.018 % were spliced at the wild-type site.