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A new, sensitive and simple method for HLA class II DNA typing: Sequence specific oligonucleotide probe RNA hybridization (SSOP-RHI)
Abstracts
37
1.1
INTERNATIONAL CELL EXCHANGE OF CLASS I DNA TYPING. M Lau, MS Park, and PI Terasaki, UCLA Tissue Typing Laboratory, UCLA School of Medicine, Los Angeles, CA. Introduction of Class I DNA typing into the International Cell Exchange during the past year provided the first opportunity to check serologic assignments by molecular biology methods. There were 12 cell typed by DNA for Class I specificities. The participants grew from 7 to the current 20 laboratories, and the methods used were PCR-SSP, PCR-SSOP, ARMS-SSP, PCR-SSCP, RFLP, and direct sequencing. Among the 47 HLA-A, B antigens in which serological typing had 70% or greater concordance, DNA typing confirmed all the serologic assignments. However, when the serology labs had obvious differences, with no ready consensus, DNA typing was invaluable in resolving the differences. Examples given below are the difficult B15 and B70 splits which were convincingly resolved by DNA typing: Assignments by Serology (% detection) B·1502 (B75) B·1503. (li721 B·151O (B71) B·1513 (B77) B·1518 (B71var)
B75 (62%) B72{21o/<>t B7l ( 7%) B77 (46%) B71 ( 6%)
B62 (20%) B70 (69%) B70 (74%) B63 (16%) B70 (69%)
B15 ( 9%) B7l ( 2%) B72 (10%) B15 (13%) B72 (10%)
B70 ( 5%)
B53 (10%) B75 ( 4%)
DNA typing results also differentiated A·6801 and A·6802 for 2 cells assigned A68 by only 56% and 20%, respectively, by serologic methods. Every month 4 cells typed serologically are being examined by DNA typing. In conclusion, Class I DNA typing was useful in reconfirming 38 specificities which had 70%-100% agreement by serolOgic typing and in establishing the correct answer for 9 specificities for which serologic agreement could not be reached.
7.4
A NEW. SENSITIVE AND SIMPLE METHOD FOR HLA CLASS II DNA TYPING: SEQUENCE SPECIFIC OLIGONUCLEOTIDE PROBE RNA HYBRIDIZATION ISSOp·RH!. K Matsubara 1, S Takeda 1, V Iwaki 2, J Cicciarelli 2, S Musa 2, J Garcia·Gomez 2. Diagnostics Technology Labs, Chugai Pharmaceutical Co. LTO , Tokyo, Japan 1 and USC School of Medicine, Metic Transplantation Labs, Los Angeles, California, USA2 We have developed a new technique, SSOP·RH, for rapid and accurate detection of HLA Class II DNA polymorphism. The advantages over other ONA typing methods are a non·isotopic format, simple handling, short assay time, mass screening, and the detection of single·base differences. The SSOP·RH procedure involves isolating DNA and synthesizing complementary RNA using reverse transcriptase' mediated amplification ITMA}, a method whereby a newly synthesized DNA double·strand molecule becomes the template of RNA synthesis by RNA polymerase under isothermal conditions. The newly synthesized RNA molecules also become the template for DNA synthesis using reverse transcriptase. DNA extension is achieved through the DNA polymerase activity of the enzyme reverse·transcriptase. RNA molecules are used as targets for the Hybridization Protection Assay (HPA), an SSO procedure utilizing oligonucleotide probes attached to acridinium esters, a chemiluminescent agent. The probes are hybridized in solution with amplified RNA. When probe and target are completely base-paired, the acridinium ester molecule attached to the probe intercalates between the hybridized strands_ If probe and target are not matched perfectly, the acridinium ester molecule will not intercalate_ When a hydrolysis reagent is added, acridinium ester molecules attached to unhybridized or partially matched probes are hydrolyzed and thus, inactivated. The molecules that have intercalated between perfectly-matched probes are protected from hydrolysis. The protected acridinium ester will emit chemiluminescence in the presence of base and hydrogen peroxide. The resulting flash reaction is then read on a chemiluminescent reader, a quantitative analysis is made by computer and results are double·checked by hand. SSOP-RH is a simple, fast, and accurate method that does not require thermocycling. The procedure is less tedious to perform than other SSO methods because washing steps are eliminated, which also shortens the time it takes to perform the test. Elimination of radioactivity makes the assay safe to handle. SSOP·RH can be completely automated, allowing for mass ORB 1 screening. These advantages give SSDP·RH a decided edge over conventional HLA Class II DNA typing methods.
37
1.1
INTERNATIONAL CELL EXCHANGE OF CLASS I DNA TYPING. M Lau, MS Park, and PI Terasaki, UCLA Tissue Typing Laboratory, UCLA School of Medicine, Los Angeles, CA. Introduction of Class I DNA typing into the International Cell Exchange during the past year provided the first opportunity to check serologic assignments by molecular biology methods. There were 12 cell typed by DNA for Class I specificities. The participants grew from 7 to the current 20 laboratories, and the methods used were PCR-SSP, PCR-SSOP, ARMS-SSP, PCR-SSCP, RFLP, and direct sequencing. Among the 47 HLA-A, B antigens in which serological typing had 70% or greater concordance, DNA typing confirmed all the serologic assignments. However, when the serology labs had obvious differences, with no ready consensus, DNA typing was invaluable in resolving the differences. Examples given below are the difficult B15 and B70 splits which were convincingly resolved by DNA typing: Assignments by Serology (% detection) B·1502 (B75) B·1503. (li721 B·151O (B71) B·1513 (B77) B·1518 (B71var)
B75 (62%) B72{21o/<>t B7l ( 7%) B77 (46%) B71 ( 6%)
B62 (20%) B70 (69%) B70 (74%) B63 (16%) B70 (69%)
B15 ( 9%) B7l ( 2%) B72 (10%) B15 (13%) B72 (10%)
B70 ( 5%)
B53 (10%) B75 ( 4%)
DNA typing results also differentiated A·6801 and A·6802 for 2 cells assigned A68 by only 56% and 20%, respectively, by serologic methods. Every month 4 cells typed serologically are being examined by DNA typing. In conclusion, Class I DNA typing was useful in reconfirming 38 specificities which had 70%-100% agreement by serolOgic typing and in establishing the correct answer for 9 specificities for which serologic agreement could not be reached.
7.4
A NEW. SENSITIVE AND SIMPLE METHOD FOR HLA CLASS II DNA TYPING: SEQUENCE SPECIFIC OLIGONUCLEOTIDE PROBE RNA HYBRIDIZATION ISSOp·RH!. K Matsubara 1, S Takeda 1, V Iwaki 2, J Cicciarelli 2, S Musa 2, J Garcia·Gomez 2. Diagnostics Technology Labs, Chugai Pharmaceutical Co. LTO , Tokyo, Japan 1 and USC School of Medicine, Metic Transplantation Labs, Los Angeles, California, USA2 We have developed a new technique, SSOP·RH, for rapid and accurate detection of HLA Class II DNA polymorphism. The advantages over other ONA typing methods are a non·isotopic format, simple handling, short assay time, mass screening, and the detection of single·base differences. The SSOP·RH procedure involves isolating DNA and synthesizing complementary RNA using reverse transcriptase' mediated amplification ITMA}, a method whereby a newly synthesized DNA double·strand molecule becomes the template of RNA synthesis by RNA polymerase under isothermal conditions. The newly synthesized RNA molecules also become the template for DNA synthesis using reverse transcriptase. DNA extension is achieved through the DNA polymerase activity of the enzyme reverse·transcriptase. RNA molecules are used as targets for the Hybridization Protection Assay (HPA), an SSO procedure utilizing oligonucleotide probes attached to acridinium esters, a chemiluminescent agent. The probes are hybridized in solution with amplified RNA. When probe and target are completely base-paired, the acridinium ester molecule attached to the probe intercalates between the hybridized strands_ If probe and target are not matched perfectly, the acridinium ester molecule will not intercalate_ When a hydrolysis reagent is added, acridinium ester molecules attached to unhybridized or partially matched probes are hydrolyzed and thus, inactivated. The molecules that have intercalated between perfectly-matched probes are protected from hydrolysis. The protected acridinium ester will emit chemiluminescence in the presence of base and hydrogen peroxide. The resulting flash reaction is then read on a chemiluminescent reader, a quantitative analysis is made by computer and results are double·checked by hand. SSOP-RH is a simple, fast, and accurate method that does not require thermocycling. The procedure is less tedious to perform than other SSO methods because washing steps are eliminated, which also shortens the time it takes to perform the test. Elimination of radioactivity makes the assay safe to handle. SSOP·RH can be completely automated, allowing for mass ORB 1 screening. These advantages give SSDP·RH a decided edge over conventional HLA Class II DNA typing methods.