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Automated typing of fluorescent microsatellite markers: technical aspects
Abstracts
147
B-7.4 #177
PCR-SSP: AN EASY METHOD OF GENERIC AND ALLELE TYPING FOR HLA-A2 AND A28. C Grania. M Salazar, S Alosco, M Chopek and EJ Yunis. American Red Cross, Northeast Region, Dedham, MA. A2 and A28 specificities are very polymorphic within the HLA-A locus and represents a major portion of HLA-A alleles in many populations. Currently there are 9 alleles of A2 and 4 alleles of A28. As these alleles are not determined by serology several molecular typing approaches have been proposed for their identification, including PCR-SSO and reverse dot blot. The PCR-amplification with sequence specific primers (PCR-SSP) method has been extensively used for class II and increasingly being used class I typing. Here we have used specific primer combinations to achieve amplifications of the exon 2 and 3 of the HLA-A2 and A28 alleles. The generic typing was performed with two PCR reactions. Based on these results a second set of amplifications (two reactions for A28 samples and nine reactions for A2 samples) was utilized for characterization of the alleles. The methodology was successfully tested on 50 serologically determined HLA-A2 and 10 A28 samples. It represents an alternative to replace serological methods and has the added advantage of subtyping the alleles of these two specificities.
B-7.4 #178
AUTOMATED TYPING OF FLUORESCENT MICROSATELLITE MARKERS: TECHNICAL ASPECTS. MM Barmada, A Colomar, WB Bias, BJ Schmeckpeper, Immunogenetics Laboratories, Department of Medicine, Johns Hopkins University, Baltimore, MD. There is a need for faster, more accurate and more sensitive methods for parentage testing and for monitoring engraftment after bone marrow transplantation. Such methodology, driven by the goals of the Human Genome Project, includes automated DNA sequencers, capable of distinguishing fluorescently labeled DNA fragments on DNA sequencing gels. We have examined several parameters related to the efficiency and reproducibility of automated genotyping schemes using fluorescently labeled microsatellite markers. In particular, parameters related to the amplification of these markers using the polymerase chain reaction (PCR), such as magnesium chloride concentration, primer concentration, type of DNA prep used, and final volume of reaction, were examined in order to increase the efficiency of amplification while simultaneously reducing the cost per reaction. Also, parameters related to the resolution of these markers using the Applied Biosystems model 373A automated DNA sequencer were examined in order to reduce the amount of sample handling. We found that: (1) optimal PCR parameters varied from marker to marker, but could be limited to one of four sets of conditions developed, (2) no purification (desalting) of samples was required prior to mixing and loading, and (3) new loading buffers greatly improved our ability to load quickly all lanes of a sequencing gel and improved the resolution of small size differences. The Mendelian inheritance of several markers has also been verified using our new techniques in both Amish and non-Amish pedigrees. These technical modifications should enhance the use of these markers clinically as a quick and efficient method of monitoring engraftment following bone marrow transplantation. In addition, we are developing a small panel of such markers for parentage testing.
147
B-7.4 #177
PCR-SSP: AN EASY METHOD OF GENERIC AND ALLELE TYPING FOR HLA-A2 AND A28. C Grania. M Salazar, S Alosco, M Chopek and EJ Yunis. American Red Cross, Northeast Region, Dedham, MA. A2 and A28 specificities are very polymorphic within the HLA-A locus and represents a major portion of HLA-A alleles in many populations. Currently there are 9 alleles of A2 and 4 alleles of A28. As these alleles are not determined by serology several molecular typing approaches have been proposed for their identification, including PCR-SSO and reverse dot blot. The PCR-amplification with sequence specific primers (PCR-SSP) method has been extensively used for class II and increasingly being used class I typing. Here we have used specific primer combinations to achieve amplifications of the exon 2 and 3 of the HLA-A2 and A28 alleles. The generic typing was performed with two PCR reactions. Based on these results a second set of amplifications (two reactions for A28 samples and nine reactions for A2 samples) was utilized for characterization of the alleles. The methodology was successfully tested on 50 serologically determined HLA-A2 and 10 A28 samples. It represents an alternative to replace serological methods and has the added advantage of subtyping the alleles of these two specificities.
B-7.4 #178
AUTOMATED TYPING OF FLUORESCENT MICROSATELLITE MARKERS: TECHNICAL ASPECTS. MM Barmada, A Colomar, WB Bias, BJ Schmeckpeper, Immunogenetics Laboratories, Department of Medicine, Johns Hopkins University, Baltimore, MD. There is a need for faster, more accurate and more sensitive methods for parentage testing and for monitoring engraftment after bone marrow transplantation. Such methodology, driven by the goals of the Human Genome Project, includes automated DNA sequencers, capable of distinguishing fluorescently labeled DNA fragments on DNA sequencing gels. We have examined several parameters related to the efficiency and reproducibility of automated genotyping schemes using fluorescently labeled microsatellite markers. In particular, parameters related to the amplification of these markers using the polymerase chain reaction (PCR), such as magnesium chloride concentration, primer concentration, type of DNA prep used, and final volume of reaction, were examined in order to increase the efficiency of amplification while simultaneously reducing the cost per reaction. Also, parameters related to the resolution of these markers using the Applied Biosystems model 373A automated DNA sequencer were examined in order to reduce the amount of sample handling. We found that: (1) optimal PCR parameters varied from marker to marker, but could be limited to one of four sets of conditions developed, (2) no purification (desalting) of samples was required prior to mixing and loading, and (3) new loading buffers greatly improved our ability to load quickly all lanes of a sequencing gel and improved the resolution of small size differences. The Mendelian inheritance of several markers has also been verified using our new techniques in both Amish and non-Amish pedigrees. These technical modifications should enhance the use of these markers clinically as a quick and efficient method of monitoring engraftment following bone marrow transplantation. In addition, we are developing a small panel of such markers for parentage testing.