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Incorporating class II analysis by PCR based oligotyping into the clinical HLA lab
142
Abstracts
C-7.4 #271
DIRECT PCR ON WASHED BLOOD CELLS OR WHOLE BLOOD Lin Wu, Bridget McCarthy, James Kadushin, and Cindy Nuss Gen Trak Inc., 5100 Campus Drive, Plymouth Meeting, PA 19462
The Polymerase Chain Reaction (PCR) is a powerful technology for HLA DNA typing. However, its full potential in clinical laboratories has been hindered partially by the tedious requirement of DNA preparation. We have developed a simple, rapid and user-friendly method for direct PCR on washed blood cells or whole blood. In this method, only 1 ul of blood is required as a starting sample. One ul of blood was washed twice in NaCI-EDTA buffer and boiled in water for 10 min before PCR was performed. We demonstrated that using washed blood cells was as sensitive and specific as using purified DNA as the starting material for PCR. Further modifications of this method utilized whole blood, without washing in NaCI-EDTA buffer, for boiling before PCR was performed. Equally efficient amplification was demonstrated with this modified protocol. Direct PCR was successfully performed on blood treated with different anti-coagulants. In conclusion, we have established a time- and money-saving method to perform PCR directly on washed blood cells or whole blood. This method should be useful in facilitating HLA DNA typing, particularly in a clinical laboratory.
C-7.4 #272
I N C O R P O R A T I N G CLASS 11 ANALYSIS BY PCR BASED O L I G O T Y P I N G I N T O T H E C L I N I C A L HLA LAB. LK G a u l RP Montero, Puget Sound Blood Center, 921 Terry Ave, Seattle WA. In recent years the molecular analyses of HLA class II has identified numerous allelic variants to which serological reagents are not available. Hence molecular typing methods such as, RFLP, PCR based oligotyping, PCR-RFLP, PCR-SSP and so forth are being applied in various labs. All these methods are well worked out, but do require some technical skills in molecular biology. In our lab the regular dot blot system was not opted as it requires numerous time consuming hybridizations. For a trained serologist, using a reverseblot kit is probably the best alternative. However, there are no commercial kits available for all the class II loci. It is possible to prepare ones own oligotyping kit, which is both affordable and less intimidating to a trained serologist. We prefer oligobloting, where allele specific oligonucleotides are immobilized onto a nylon membrane (similar to sera on a tray), and the labeled PCR products are used in individual hybridziatons. This requires careful selection of oligonucleotides, to satisfy uniform wash conditions. We use oligos varying in length, with T d at 60"C. Thus, the first step is to select allele specific oligonucleotides, tail them, and immobilizing them on the nylon membranes. To achieve uniform tailing conditions, we prepare tailing premixes in batches; one simply adds the oligonucleotide to be tailed to the tube with tailing premix, and incubates at 37"C. The second step is to amplify crude lysate or DNA samples. This step has been simplified by preparing premixes made in batches. At the time of the reaction, one simply adds the test DNA sample to the premix tubes and amplifies. We have also simplified the labeling procedure by incorporating digoxigenin labeled dUTP into the PCR premix. The incorporation has yielded highly consistent signals. Incorporation of label during the PCR cycles may be superior to using labeled primers. The last step is hybridization and color detection.
Abstracts
C-7.4 #271
DIRECT PCR ON WASHED BLOOD CELLS OR WHOLE BLOOD Lin Wu, Bridget McCarthy, James Kadushin, and Cindy Nuss Gen Trak Inc., 5100 Campus Drive, Plymouth Meeting, PA 19462
The Polymerase Chain Reaction (PCR) is a powerful technology for HLA DNA typing. However, its full potential in clinical laboratories has been hindered partially by the tedious requirement of DNA preparation. We have developed a simple, rapid and user-friendly method for direct PCR on washed blood cells or whole blood. In this method, only 1 ul of blood is required as a starting sample. One ul of blood was washed twice in NaCI-EDTA buffer and boiled in water for 10 min before PCR was performed. We demonstrated that using washed blood cells was as sensitive and specific as using purified DNA as the starting material for PCR. Further modifications of this method utilized whole blood, without washing in NaCI-EDTA buffer, for boiling before PCR was performed. Equally efficient amplification was demonstrated with this modified protocol. Direct PCR was successfully performed on blood treated with different anti-coagulants. In conclusion, we have established a time- and money-saving method to perform PCR directly on washed blood cells or whole blood. This method should be useful in facilitating HLA DNA typing, particularly in a clinical laboratory.
C-7.4 #272
I N C O R P O R A T I N G CLASS 11 ANALYSIS BY PCR BASED O L I G O T Y P I N G I N T O T H E C L I N I C A L HLA LAB. LK G a u l RP Montero, Puget Sound Blood Center, 921 Terry Ave, Seattle WA. In recent years the molecular analyses of HLA class II has identified numerous allelic variants to which serological reagents are not available. Hence molecular typing methods such as, RFLP, PCR based oligotyping, PCR-RFLP, PCR-SSP and so forth are being applied in various labs. All these methods are well worked out, but do require some technical skills in molecular biology. In our lab the regular dot blot system was not opted as it requires numerous time consuming hybridizations. For a trained serologist, using a reverseblot kit is probably the best alternative. However, there are no commercial kits available for all the class II loci. It is possible to prepare ones own oligotyping kit, which is both affordable and less intimidating to a trained serologist. We prefer oligobloting, where allele specific oligonucleotides are immobilized onto a nylon membrane (similar to sera on a tray), and the labeled PCR products are used in individual hybridziatons. This requires careful selection of oligonucleotides, to satisfy uniform wash conditions. We use oligos varying in length, with T d at 60"C. Thus, the first step is to select allele specific oligonucleotides, tail them, and immobilizing them on the nylon membranes. To achieve uniform tailing conditions, we prepare tailing premixes in batches; one simply adds the oligonucleotide to be tailed to the tube with tailing premix, and incubates at 37"C. The second step is to amplify crude lysate or DNA samples. This step has been simplified by preparing premixes made in batches. At the time of the reaction, one simply adds the test DNA sample to the premix tubes and amplifies. We have also simplified the labeling procedure by incorporating digoxigenin labeled dUTP into the PCR premix. The incorporation has yielded highly consistent signals. Incorporation of label during the PCR cycles may be superior to using labeled primers. The last step is hybridization and color detection.