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DNA typing of HLA-A using polymerase chain reaction (PCR) and sequence-specific oligonucleotide probes (SSOP)
151
Abstracts
7.4 #216
DNA TYPING OF HLA-A USING POLYMERASE CHAIN REACTION (PCR) AND SEQUENCE-SPECIFIC OLIGONUCLEOTIDE PROBES (SSOP) K Cao, WX Shi, J Nielsen, J Stearman, and M Chopek, American Red Cross, National HLA Reference Laboratory, Rockville, MD We developed a PCR-SSOP typing system for HLA-A. We chose several HLA-A locus specific primers from published intronic sequences and performed empirical pairwise tests. The best 5' primer was similar to that used by TL Bugawan et al (Tissue Antigens 1994,44:137-147). For optimal amplification the 3' primer was taken from a region of intron 3 that is deleted in both HLA-B and HLA-C. These primers gave a product of 1185 bp that included all of exons 1,2, and 3. Using 10% DMSO and annealing at 65° C, these primers were specific for HLA-A as seen with a battery of different class I probes. (14 bp of 5' UT + 7 bp of exon 1) 5' primer: CC CAg ACg CCg Agg ATg gCC g 3' primer: gCA ggg Cgg AAC CTC AgA gTC ACT CTC T (intron 3, bp 155 - 182) A panel of 55 DIG-11-ddUTP labeled probes were detected with Lumiphos chemiluminescence. One hundred and six (106) cell lines of the 10th International Histocompatibility Workshop (IHW) and 494 random, healthy unrelated donors were typed. All HLA-A locus group specificities and most HLA-A alleles were observed. The 55 SSOPs could distinguish all but two pairs ofHLA-A alleles (A*0201 :::: A*0209 and A*31011 :::: A*31012). We found that cell line IHW 9057 was A*6601 instead of A26 as noted in the supporting data for the 12th IHW cell panel. All494 donors gave DNA typings that were consistent with the A locus serologic typing and defined more clearly several difficult typings. Some new reaction patterns have been observed and this implies that there may be novel alleles. These will be confirmed by molecular cloning of the PCR product and automated fluorescent sequencing.
7.4 #217
PATTERN MATCHING SOFTWARE ACHIEVES MORE RELIABLE AUTOMATED HLA SEQUENCING-BASED TYPING. MP ConradI, CL Brown, RB Chadwick I , LA Johnston-Dow l , MD McGinnis l , EH Rozernuller2, MGJ Tilanus2, and M Kronicki. IPerkin Elmer, Applied Biosystems Division, Foster City CA. 2University Hospital Utrecht, The Netherlands. Many groups use solid-phase sequencing protocols employing T7 (Sequenase™) polymerase for HLA sequencing-based typing (SBT) because these sequencing protocols generally yield uniform base incorporation patterns which can be readily analyzed for heterozygozity with current software tools. The alternative Taq cycle-sequencing protocols require less template and are easier to perform, however. they suffer from a higher degree of sequence-dependent base incorporation. This results in uneven band intensities which can be difficult to interpret in an automated fashion and must be carefully examined by a trained eye for proper typing. Fortunately. it has been shown that these uneven patterns are reproducible, using either AmpliTaq or AmpliTaq CS+. To facilitate HLA-SBT, we have developed software which exploits the base incorporation reproducibility of Taq. In a preliminary step. the software "learns" typical peak patterns from a reference panel of well characterized and suitably varied samples. These statistical characteristics of the reference panel are then used in the interpretation of new sequences. This allows for reliable base calls even when the base incorporation is relatively uneven. We have applied this general principle to the specific case of DRBI SBT. Using AmpliTaq CS+ protocols with dye-labelled primers, we find that polymorphic base positions which cannot be reliably called by a simple threshold technique can be called properly using this new algorithm. This allows accurate, automated typing results to be obtained from a simpler chemistry protocol.
Abstracts
7.4 #216
DNA TYPING OF HLA-A USING POLYMERASE CHAIN REACTION (PCR) AND SEQUENCE-SPECIFIC OLIGONUCLEOTIDE PROBES (SSOP) K Cao, WX Shi, J Nielsen, J Stearman, and M Chopek, American Red Cross, National HLA Reference Laboratory, Rockville, MD We developed a PCR-SSOP typing system for HLA-A. We chose several HLA-A locus specific primers from published intronic sequences and performed empirical pairwise tests. The best 5' primer was similar to that used by TL Bugawan et al (Tissue Antigens 1994,44:137-147). For optimal amplification the 3' primer was taken from a region of intron 3 that is deleted in both HLA-B and HLA-C. These primers gave a product of 1185 bp that included all of exons 1,2, and 3. Using 10% DMSO and annealing at 65° C, these primers were specific for HLA-A as seen with a battery of different class I probes. (14 bp of 5' UT + 7 bp of exon 1) 5' primer: CC CAg ACg CCg Agg ATg gCC g 3' primer: gCA ggg Cgg AAC CTC AgA gTC ACT CTC T (intron 3, bp 155 - 182) A panel of 55 DIG-11-ddUTP labeled probes were detected with Lumiphos chemiluminescence. One hundred and six (106) cell lines of the 10th International Histocompatibility Workshop (IHW) and 494 random, healthy unrelated donors were typed. All HLA-A locus group specificities and most HLA-A alleles were observed. The 55 SSOPs could distinguish all but two pairs ofHLA-A alleles (A*0201 :::: A*0209 and A*31011 :::: A*31012). We found that cell line IHW 9057 was A*6601 instead of A26 as noted in the supporting data for the 12th IHW cell panel. All494 donors gave DNA typings that were consistent with the A locus serologic typing and defined more clearly several difficult typings. Some new reaction patterns have been observed and this implies that there may be novel alleles. These will be confirmed by molecular cloning of the PCR product and automated fluorescent sequencing.
7.4 #217
PATTERN MATCHING SOFTWARE ACHIEVES MORE RELIABLE AUTOMATED HLA SEQUENCING-BASED TYPING. MP ConradI, CL Brown, RB Chadwick I , LA Johnston-Dow l , MD McGinnis l , EH Rozernuller2, MGJ Tilanus2, and M Kronicki. IPerkin Elmer, Applied Biosystems Division, Foster City CA. 2University Hospital Utrecht, The Netherlands. Many groups use solid-phase sequencing protocols employing T7 (Sequenase™) polymerase for HLA sequencing-based typing (SBT) because these sequencing protocols generally yield uniform base incorporation patterns which can be readily analyzed for heterozygozity with current software tools. The alternative Taq cycle-sequencing protocols require less template and are easier to perform, however. they suffer from a higher degree of sequence-dependent base incorporation. This results in uneven band intensities which can be difficult to interpret in an automated fashion and must be carefully examined by a trained eye for proper typing. Fortunately. it has been shown that these uneven patterns are reproducible, using either AmpliTaq or AmpliTaq CS+. To facilitate HLA-SBT, we have developed software which exploits the base incorporation reproducibility of Taq. In a preliminary step. the software "learns" typical peak patterns from a reference panel of well characterized and suitably varied samples. These statistical characteristics of the reference panel are then used in the interpretation of new sequences. This allows for reliable base calls even when the base incorporation is relatively uneven. We have applied this general principle to the specific case of DRBI SBT. Using AmpliTaq CS+ protocols with dye-labelled primers, we find that polymorphic base positions which cannot be reliably called by a simple threshold technique can be called properly using this new algorithm. This allows accurate, automated typing results to be obtained from a simpler chemistry protocol.