3.4. Alternative Splicing Validation HLA-DPA1 (Experiment 3)
Gel electrophoresis of amplicons from cDNA for HLA-DPA1 exon 2–4 amplification showed the predominant expected band size of 538 bp. However, in many brain samples, distinct smaller bands were seen (Figure 7). These gel-purified bands were directly sequenced and the results confirmed alternative splicing of these smaller bands as containing parts of exons 2, 3, and 4 (Table 10). The HLA-DPA1 sequencing primers are located in exon 2 and exon 4 of NM_033554 as:
Forward5’-TGGACAAGAAGGAGACCGTCT-3’(positon307/327);
Reverse5’-TTTATGATGAGGACGGTGCC-3’(position 844/825). All the sequences were in the range from 307 to 844 of NM_033554.
The cDNA alignment to HLA-DPA1 RefSeq showed that differential splicing of exon 2–3 and 3–4 was involved, although did not involve exon skipping, tending rather toward a novel splice site extended into adjacent exons (Figure 8). The sequence of four subjects shows full-length amplicon of exons 2, 3, and 4 (512–515 bp), and truncated versions of exons 2, 3, and 4 (215–400 bp). The UCSC sequence tracks show truncation of exon 2, 3, and 4, thereby suggesting different cryptic splice sites that occur (Figure 8).
Total sequences from forward primer to reverse primer are 538 bp, and in position 307−844 of NM_033554.3. Exon Bound1aries 2 (264–453) 3 (454–735) 4 (736–902) representative samples from four different subjects using anterior cingulate cDNA. The forward primer sequences (-f.ab1 are matched to reverse primer sequence nomenclature (-r.ab1).
For more details, the next section shows the sequencing results for forward and reverse primers for HLA-DPA1_07 splicing site position from 427 to 641 in NM_033554 and the HLA-DPA1_01 splicing site at position 478 to 643 in NM_033554.
Box 1  Sequencing Result of Bands from HLA-DPA1-07 Gel.  Sequencing of cDNA HLA-DPA1_ (07-f.ab1 with Forward primer)   350AGCCTTTTCCTTTGAGGCTCAGGGCGGGCTGGCTAACATTGCTATATTGAACAACAACTTGAATACCTTGATCCAGCG427 (spliced out region) ---------------------------------------------------------------------------------------------------------------------------------------------642TTCCATTACCTGACCTTTGTGCCCTCAGCAGAGGACTTCTATGACTGCAGGGTGGAGCACTGGGGCTTGGACCAGCCGCTCCTCAAGCACTGGG735 736AGGCCCAAGAGCCAATCCAGATGCCTGAGACAACGGAGACTGTGCTCTGTGCCCTGGGCCTGGTGCTGGGCCTAGTCGGCATCATCGTGGGCACCGTCCTCATCATAAA844   Sequencing of cDNA HLA-DPA1_(07-r.ab1 with Reverse primer)   801GCACCnGGCCCAGGGCACAGAGCACAGTCTCCGTTGTCTCAGGCATCTGGATTGGCTCTTGGGCCT736 735CCCAGTGCTTGAGGAGCGGCTGGTCCAAGCCCCAGTGCTCCACCCTGCAGTCATAGA AGTCCTCTGCTGAGGGCACAAAGGTCAGGTAATGGAA642 (spliced out region) ---------------------------------------------------------------------------------------------------------------------------------------------427CGCTGGATCAAGGTATTCAAGTTGTTGTTCAATATAGCAATGTTAGCCAGCCCGCCCTGAGCCTCAAAGGAAAAGGCTTGGCCAAACTCCTCCAGATGCCAGACGGTCTCCTTCTTGTCCA307  The sequencing result for both forward and reverse primers are consistent to show a novel splice site for HLA-DPA1 located in exons 3 and 4.
Box 2  Sequencing Result of Bands from HLA-DPA1-01 Gel.  cDNA HLA-DPA1_(01-f.ab1 with Forward primer). 360TTTGAGGCTCAGGGCGGGCTGGCTAACATTGCTATATTGAACAACAACTTGAATACCTT GATCCAGCGTTCCAACCACACTCAGGCCGCCAATGATCCCCCTGAGGTGACCGTGTTTC478 (spliced out region)---------------------------------------------------------------------------------------------------------------------------------------------644CATTACCTGACCTTTGTGCCCTCAGCAGAGGACGTCTATGACTGCAGGGTGGAGCACTGGGGCTTGGACCAGCCGCTCCTCAAGCACTGGGAGGCCCAAGAGCCAATCCAGATGCCTGAGACAACGGAGACTGTGCTCTGTGCCCTGGGCCTGGTGCTGGGCCTAGTGGGCATCATCGTGGGCACCGTCCTCATCATAAA844   cDNA HLA-DPA1_(01-r.ab1 Reverse primer)   796GGCCCAGGGCACAGAGCACAGTCTCCGTTGTCTCAGGCATCTGGATTGGCTCTTGGGCCTCCCAGTGCTTGAGGAGCGGCTGGTCCAAGCCCCAGTGCTCCACCCTGCAGTCATAGACGTCCTCTGCTGAGGGCACAAAGGTCAGGTAATG644 (spliced out region)------------------------------------------------------------------------------------------------------------------------------------478 GAAACACGGTCACCTCAGGGGGATCATTGGCGGCCTGAGTGTGGTTGGAACGCTGGATCAAGGTATTCAAGTTGTTGTTCAATATAGCAATGTTAGCCAGCCCGCCCTGAGCCTCAAAGGAAAAGGCTCGGCCAAACTCCTCCAGATGCCAGACGGTCTCCTTCCTTGTCC307  The sequencing result for both the forward and reverse primer shows a consistent alternative splice site in exon 3.