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 . ) .
 detection of unknown single nucleotide polymorphism ( snp ) relies on large scale sequencing expeditions of genomic fragments or complex high - throughput chip technology . 
 we describe a simplified strategy for fluorimetric detection of known and unknown snp by proportional hybridization to oligonucleotide arrays based on optimization of the established principle of signal loss or gain that requires a drastically reduced number of matched or mismatched probes . 
 the array consists of two sets of 18-mer oligonucleotide probes . 
 one set includes overlapping oligos with 4-nucleotide tiling representing an arbitrarily selected " consensus " sequence ( consensus - oligos ) , the other includes oligos specific for known snp within the same genomic region ( variant - oligos ) . 
 fluorescence - labeled dna amplified from a homozygous source identical to the consensus represents the reference target and is co - hybridized with a differentially - labeled test sample . 
 lack of hybridization of the test sample to consensus- with simultaneous hybridization to variant - oligos designates a known allele . 
 lack of hybridization to consensus- and variant - oligos indicates a new allele . 
 detection of unknown variants in heterozygous samples depends upon fluorimetric analysis of signal intensity based on the principle that homozygous samples generate twice the amount of signal . 
 this method can identify unknown snp in heterozygous conditions with a sensitivity of 82% and specificity of 90% . 
 this strategy should dramatically increase the efficiency of snp detection throughout the human genome and will decrease the cost and complexity of applying genomic wide analysis in the context of clinical trials . 
Material and methods
Oligo nucleotide probe design
Oligonucleotide array printing and post process of slides
Test and consensus reference samples
Preparation of target nucleic acids
Target labeling and hybridization