the hla - a locus exon 2 region from position 73346 , allele a*01011 , was used for the design of consensus oligos ( figure 1 ) . allele - specific ( variant ) oligos were designed based on single or double nucleotide variants according to alignment to the arbitrarily selected consensus sequence ( hla - a*0201 ) . variant and consensus oligos consisted of 18-mers with a 5 ' amino - modifier having a six - carbon spacer for immobilization ( operon technologies , inc , alameda , ca ) . melting temperatures of oligo probes were maintained as close as possible to a range of 5660c . 350400 pmol/ l oligo probes synthesized in 96-well format were dried and re - suspended in 3  ssc for printing . probes were spotted onto a 3d - link activated slide for covalent immobilization ( motor roller ) using omnigrid robotic printer ( genemachine ) with four printing pins picking up 0.25 l of probe solution and depositing 0.6 nl per spot ( telechem international , inc . ) . spot diameter was 90100 m , spaced at 250 m to prepare 4  16  6 spot / arrays . after printing , slides were kept in a sealed humidifier chamber at room temperature overnight and blocked with 50 mm ethanolamine , 0.1 m tris , ph9 and 0.1% sds at 50c for 15 minutes followed with rinsing in water twice , washing with 4  ssc/0.1% sds 50c for 45 minutes , rinsing with water briefly and centrifuging at 800 rpm for 3 minutes with micro - plate carriers . 12 heterozygous or homozygous samples were tested for oligo nucleotide array hybridization and confirmed by sequence - based typing using the abi prism 3700 - 96 analyzer . in order to generate single strand dna , pcr products from exon 1 to exon 5 of the hla - a locus were amplified with an attachment of t7 promoter to the 5 ' end using 5 ' t7-ex1a-6 primer 5'aaacgacggccagtgaatacgactcactataggcgccagacgccgaggatggcc3 ' ) and three 3 ' primers ( 3 ' ex 5-a 993 - 1 cat tgc tgg cct ggt tct cc ; 3 ' ex 5-a 993 - 2 cat tgc tgg cct ggt tct ctt ; 3 ' ex 5-a 993 - 3 cat tgc tgg cct agt tct ctt ) . pcr reaction was mixed with 25 l of hotstart pcr reagents ( qiagen , ca ) , 5 ng-0.5 g of genomic dna , 5 l of 15 m 5'primer , 5 l of 15 m 3'primer mix and h2o to a 50 l final volume . the reaction was cycled at 95c for 10 minutes , 96c for 35 seconds , 65c for 45 seconds , 72c for 3 minutes , 4 cycle ; 96c for 30 seconds , 60c for 40 seconds , 72c for 3 minutes , 19 cycles and 96c for 30 seconds , 55c for 40 seconds , 72c for 2 minutes , 9 cycles . one l of pcr product from each sample was analyzed using a bioanalyzer on dna7500 chip ( agilent biotechnology ) . the pcr products were then precipitated with etoh at room temperature and re - suspended in depc - treated h2o at 0.1 g/l concentration . in vitro transcription ( ivt ) for each sample , the following reaction mixture was made : 4 l of each 75 mm ntp ( a , g , c and utp ) , 4 l reaction buffer , 4 l enzyme mix ( rnase inhibitor and t7 phage polymerase ) and 1 g purified pcr product in 16 l depc - treated h2o . amplified rna was then purified using trizol reagent according to manufacture instruction ( gibcobrl ) and re - suspended in 40 l of depc water . rna concentrations were estimated by using a bioanalyzer on rna 6000 chip ( agilent biotechnology ) . fluorescence - labeled single strand cdna was generated by reverse transcription ( rt ) and used for hybridization . in the rt reaction , 4 l of first strand buffer , 1 l random hexmer ( 8 g/l ; boehringer mannheim ) , 2 l 10  low t - dntp ( 5 mm a , c and gtp , 2 mm dttp ) , 2 l 1 mm cy - dutp ( cy3 for reference sample or cy5 for test sample unless otherwise specified ) , 2 l 0.1 m dtt , 1 l rnasin and 1.2 g amplified rna in 8 l depc h2o were mixed and heated for five minutes at 65c . this was followed by addition of 1 l superscript ii ( life technology , ) , 40 minutes of incubation at 42c , another 1 ul of superscript ii and 50 more minutes continued incubation at 42c . reactions were stopped by addition of 2.5 l 500 mm edta , 5 l 1 m naoh and heated to 65c for 15 minute to hydrolyze the rna . tris buffer ( 12.5 l of 1 m ) was added immediately to neutralize the ph , and the volume risen to 70 l by adding 35 l of 1  te . the flow through mixed with 200 l 1  te was concentrated to 2040 l using microcon ym-30 column ( millipore ) and further concentrated to 8 ul using speed - vacuum . cy3- and cy5-labeled probes were combined ( 1:1 ratio ) and 5 l 20  ssc , 0.5 l 10% sds and 0.5 l of 4 mg / ml salmon sperm dna were added to the probe for hybridization . prepared probe mixture was applied to an array slide with cover slid and hybridized at 47c for different amounts of time as described in the text . slides were washed with 4  ssc , 2  ssc with 0.1 % sds , 1  ssc , 0.2  ssc and 0.05  ssc sequentially for one minute each step and dried by centrifugation at 800 rpm for 3 minutes . the slides were then scanned for fluorescent signal using a genepix 4000b scanner and the results analyzed using genepix pro3 software ( axon instruments , inc . ) . the hla - a locus exon 2 region from position 73346 , allele a*01011 , was used for the design of consensus oligos ( figure 1 ) . allele - specific ( variant ) oligos were designed based on single or double nucleotide variants according to alignment to the arbitrarily selected consensus sequence ( hla - a*0201 ) . variant and consensus oligos consisted of 18-mers with a 5 ' amino - modifier having a six - carbon spacer for immobilization ( operon technologies , inc , alameda , ca ) . melting temperatures of oligo probes were maintained as close as possible to a range of 5660c . 350400 pmol/ l oligo probes synthesized in 96-well format were dried and re - suspended in 3  ssc for printing . probes were spotted onto a 3d - link activated slide for covalent immobilization ( motor roller ) using omnigrid robotic printer ( genemachine ) with four printing pins picking up 0.25 l of probe solution and depositing 0.6 nl per spot ( telechem international , inc . ) . spot diameter was 90100 m , spaced at 250 m to prepare 4  16  6 spot / arrays . after printing , slides were kept in a sealed humidifier chamber at room temperature overnight and blocked with 50 mm ethanolamine , 0.1 m tris , ph9 and 0.1% sds at 50c for 15 minutes followed with rinsing in water twice , washing with 4  ssc/0.1% sds 50c for 45 minutes , rinsing with water briefly and centrifuging at 800 rpm for 3 minutes with micro - plate carriers . 12 heterozygous or homozygous samples were tested for oligo nucleotide array hybridization and confirmed by sequence - based typing using the abi prism 3700 - 96 analyzer . in order to generate single strand dna , pcr products from exon 1 to exon 5 of the hla - a locus were amplified with an attachment of t7 promoter to the 5 ' end using 5 ' t7-ex1a-6 primer 5'aaacgacggccagtgaatacgactcactataggcgccagacgccgaggatggcc3 ' ) and three 3 ' primers ( 3 ' ex 5-a 993 - 1 cat tgc tgg cct ggt tct cc ; 3 ' ex 5-a 993 - 2 cat tgc tgg cct ggt tct ctt ; 3 ' ex 5-a 993 - 3 cat tgc tgg cct agt tct ctt ) . pcr reaction was mixed with 25 l of hotstart pcr reagents ( qiagen , ca ) , 5 ng-0.5 g of genomic dna , 5 l of 15 m 5'primer , 5 l of 15 m 3'primer mix and h2o to a 50 l final volume . the reaction was cycled at 95c for 10 minutes , 96c for 35 seconds , 65c for 45 seconds , 72c for 3 minutes , 4 cycle ; 96c for 30 seconds , 60c for 40 seconds , 72c for 3 minutes , 19 cycles and 96c for 30 seconds , 55c for 40 seconds , 72c for 2 minutes , 9 cycles . one l of pcr product from each sample was analyzed using a bioanalyzer on dna7500 chip ( agilent biotechnology ) . the pcr products were then precipitated with etoh at room temperature and re - suspended in depc - treated h2o at 0.1 g/l concentration . in vitro transcription ( ivt ) for each sample , the following reaction mixture was made : 4 l of each 75 mm ntp ( a , g , c and utp ) , 4 l reaction buffer , 4 l enzyme mix ( rnase inhibitor and t7 phage polymerase ) and 1 g purified pcr product in 16 l depc - treated h2o . amplified rna was then purified using trizol reagent according to manufacture instruction ( gibcobrl ) and re - suspended in 40 l of depc water . rna concentrations were estimated by using a bioanalyzer on rna 6000 chip ( agilent biotechnology ) . fluorescence - labeled single strand cdna was generated by reverse transcription ( rt ) and used for hybridization . in the rt reaction , 4 l of first strand buffer , 1 l random hexmer ( 8 g/l ; boehringer mannheim ) , 2 l 10  low t - dntp ( 5 mm a , c and gtp , 2 mm dttp ) , 2 l 1 mm cy - dutp ( cy3 for reference sample or cy5 for test sample unless otherwise specified ) , 2 l 0.1 m dtt , 1 l rnasin and 1.2 g amplified rna in 8 l depc h2o were mixed and heated for five minutes at 65c . this was followed by addition of 1 l superscript ii ( life technology , ) , 40 minutes of incubation at 42c , another 1 ul of superscript ii and 50 more minutes continued incubation at 42c . reactions were stopped by addition of 2.5 l 500 mm edta , 5 l 1 m naoh and heated to 65c for 15 minute to hydrolyze the rna . tris buffer ( 12.5 l of 1 m ) was added immediately to neutralize the ph , and the volume risen to 70 l by adding 35 l of 1  te . the flow through mixed with 200 l 1  te was concentrated to 2040 l using microcon ym-30 column ( millipore ) and further concentrated to 8 ul using speed - vacuum . cy3- and cy5-labeled probes were combined ( 1:1 ratio ) and 5 l 20  ssc , 0.5 l 10% sds and 0.5 l of 4 mg / ml salmon sperm dna were added to the probe for hybridization . prepared probe mixture was applied to an array slide with cover slid and hybridized at 47c for different amounts of time as described in the text . slides were washed with 4  ssc , 2  ssc with 0.1 % sds , 1  ssc , 0.2  ssc and 0.05  ssc sequentially for one minute each step and dried by centrifugation at 800 rpm for 3 minutes . the slides were then scanned for fluorescent signal using a genepix 4000b scanner and the results analyzed using genepix pro3 software ( axon instruments , inc . ) .