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Novel RFLP pattern of DR2 and DR-DQ linkage in three unrelated chinese families
Abstracts
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NOVEL RFLP PATTERN OF DR2 AND D R - D Q LINKAGE IN THREE UNRELATED CHINESE FAMILIES. ~ B. Colombe, M. Garovoy. Immunogcnetcs and TransplantationLaboratory, Deparunem of SurgciT,Universityof California,San Franicsco. In tissue typing potential bone marrow donors by RFLP, we f~md a v~riant pauern for DR2 in three unrelated Chinese families. Using a TaqI digest and RTV-I(DRB) hybridization, DRwI5 normally exhibits 12.Skb, 1.6kb and 1.3kb bands while DRwl6 has 14.8kb, 1.5kb and 1.45kb bands. In this vatiam, the individuals have hh¢ 1.5 and 1.45kb bands but lack tlm 14.Skb band of a DRwl6. However, h~y have the 12.5kb band which is characteristic of the DRwlS. This novel Imttem segregatesas predicted in the three families. To investigate this further, we used PCR amplification of the DRB, DQB and DQA genes and sequence specific olig~mucleodde hybrkfizaton to more clearly define the HLA Class lI specificites. Using the primers and SSOs of the 1lth International Workshop, all members of these families having this unique RFLP pattem type as DRB 1* 1602, DQB 1*0502 and DQA 1*0102. This isin conw~ to cell line RML (American indian) and a Caucasian individual in our siudy which both type as DRBl*1602, DQBI*0201 and DQAI*0501 and show the nommI DRwl6 RFLP Imttem. DQB 1*0502-DQA 1"0102 is asualiy found in linkage with DRB 1* 1601 rather than the 1602 found in our family members. These results suggest that this novel DR2 molecule consists of the 1st exon of the DRBI geno of DRBI*1602 with differences at the second exert or introaie regioas and the appea~a~ of a new ~nkage pattern between DRBI and DQBI.
A CHEMILUMINg:SCENTTECHNIQUE FOR DETECTION OF HLA POLYMORPHISM USING PCR AMPLIFICATION AND SEQUENCE SPECIFIC OLIGONUCLEOTIDE HYBRIDIZATION. ~ . £ , B. Hney, B. Colombe, M. Garovoy, Immunogenetcs & Transplantation Laboratory, Department of Surgery, University of Califo~ia, San Francisco In an attempt to minimize the use of isotopes in the laboratory, we have investgated the use of non-isotopic methods for the detection of SSO probes hybridized to PCR amplified product. Among several methods commercially available, the Genius Nonradioactive Nucleic Acid Detection System offered by Bochringcr Mannheim Biechemicals used in conjunction with Lumi-Phos 530 was most useful. The SSO°s are first tailed with digoxigenin-11-dUTP using terminal transferase which adds 4-6 dUTP's per oligo. The labeled SSO is then hybridized to PCR amplified product which has been immobilized on a nylon membrane. After appropriate washes, Ihe membranes are blocked and then incubated with a high affinity sheep anri-digoxigenin Fab fragment conjugated to alkaline phosphamse. Lumi-Plu~ ¢~ntalns Lunfigen PPD which upon dephosphorylation by alkaline phosphatase produces an unstable intermediate which decomposes, emitting light. Detection is perfo~ned by reacting the membrane with the Lumi-Phos and exposure to X-ray film for 5 minutes to I hour. The labeled probes c,,n he st~red at -20°{2 and we have been able to re-use the hybridization mixture containing the probe for up to 5 hybridizations without much loss of signal. The advantage of this technique over a detection system using X-phosphate and NBT is that results can he stored on X-ray film and it also allows for multiple exposures over a 24 hour period. Membranes can easily be stripped and re-probed multiple times. We performed the I lth International Workshop DNA Component using this detection system and found the results to be as sensitive and reliable as P-32. The obvious environmental advantages coupled with the ease of use has made this chemilunfinescem approach oar technique of ch~ce.
57
#220 4.3
#221 5.4
NOVEL RFLP PATTERN OF DR2 AND D R - D Q LINKAGE IN THREE UNRELATED CHINESE FAMILIES. ~ B. Colombe, M. Garovoy. Immunogcnetcs and TransplantationLaboratory, Deparunem of SurgciT,Universityof California,San Franicsco. In tissue typing potential bone marrow donors by RFLP, we f~md a v~riant pauern for DR2 in three unrelated Chinese families. Using a TaqI digest and RTV-I(DRB) hybridization, DRwI5 normally exhibits 12.Skb, 1.6kb and 1.3kb bands while DRwl6 has 14.8kb, 1.5kb and 1.45kb bands. In this vatiam, the individuals have hh¢ 1.5 and 1.45kb bands but lack tlm 14.Skb band of a DRwl6. However, h~y have the 12.5kb band which is characteristic of the DRwlS. This novel Imttem segregatesas predicted in the three families. To investigate this further, we used PCR amplification of the DRB, DQB and DQA genes and sequence specific olig~mucleodde hybrkfizaton to more clearly define the HLA Class lI specificites. Using the primers and SSOs of the 1lth International Workshop, all members of these families having this unique RFLP pattem type as DRB 1* 1602, DQB 1*0502 and DQA 1*0102. This isin conw~ to cell line RML (American indian) and a Caucasian individual in our siudy which both type as DRBl*1602, DQBI*0201 and DQAI*0501 and show the nommI DRwl6 RFLP Imttem. DQB 1*0502-DQA 1"0102 is asualiy found in linkage with DRB 1* 1601 rather than the 1602 found in our family members. These results suggest that this novel DR2 molecule consists of the 1st exon of the DRBI geno of DRBI*1602 with differences at the second exert or introaie regioas and the appea~a~ of a new ~nkage pattern between DRBI and DQBI.
A CHEMILUMINg:SCENTTECHNIQUE FOR DETECTION OF HLA POLYMORPHISM USING PCR AMPLIFICATION AND SEQUENCE SPECIFIC OLIGONUCLEOTIDE HYBRIDIZATION. ~ . £ , B. Hney, B. Colombe, M. Garovoy, Immunogenetcs & Transplantation Laboratory, Department of Surgery, University of Califo~ia, San Francisco In an attempt to minimize the use of isotopes in the laboratory, we have investgated the use of non-isotopic methods for the detection of SSO probes hybridized to PCR amplified product. Among several methods commercially available, the Genius Nonradioactive Nucleic Acid Detection System offered by Bochringcr Mannheim Biechemicals used in conjunction with Lumi-Phos 530 was most useful. The SSO°s are first tailed with digoxigenin-11-dUTP using terminal transferase which adds 4-6 dUTP's per oligo. The labeled SSO is then hybridized to PCR amplified product which has been immobilized on a nylon membrane. After appropriate washes, Ihe membranes are blocked and then incubated with a high affinity sheep anri-digoxigenin Fab fragment conjugated to alkaline phosphamse. Lumi-Plu~ ¢~ntalns Lunfigen PPD which upon dephosphorylation by alkaline phosphatase produces an unstable intermediate which decomposes, emitting light. Detection is perfo~ned by reacting the membrane with the Lumi-Phos and exposure to X-ray film for 5 minutes to I hour. The labeled probes c,,n he st~red at -20°{2 and we have been able to re-use the hybridization mixture containing the probe for up to 5 hybridizations without much loss of signal. The advantage of this technique over a detection system using X-phosphate and NBT is that results can he stored on X-ray film and it also allows for multiple exposures over a 24 hour period. Membranes can easily be stripped and re-probed multiple times. We performed the I lth International Workshop DNA Component using this detection system and found the results to be as sensitive and reliable as P-32. The obvious environmental advantages coupled with the ease of use has made this chemilunfinescem approach oar technique of ch~ce.