Formamide improves HLA-DR low resolution typing by sequence-specific PCR
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136 Abstracts B-7.4 #259 FORMAMIDE IMPROVES HLA-DR LOW RESOLUTION TYPING BY SEQUENCE- SPECIFIC PCR DB Bellissimo and LA Baxter-Lowe, The Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin. PCR amplification with sequence-specific primers (PCR-SSP) as described by Olerup and Zetterquist (Tissue Antigens 39: 225-235) is a rapid and sensitive method for DR low resolution typing. In this method, the assignment of the alleles is based on the presence or absence of amplified product after using sequence-specific primers. Nonspecific amplifications or low signal intensity in some PCR reactions make some samples difficult to type. We have observed this problem with primer mixes 14.2, 52 and 53. We tested whether the addition of formamide in the PCR reaction would improved the typing results. DNA was purified using the QIAamp blood kit and amplified in the presence of 1-5% formamide. Primer mix 14.2 has a GC-rich primer that causes a background problem in negative samples. The addition of 3 % formamide into the PCR reaction reduced the background without affecting the signal intensity of a positive sample. Weak amplifications sometimes observed with primer mix 52 were significantly improved with 2% formamide in the PCR reaction. Nonspecific amplification was often seen with primer mix 53. The addition of 4% formamide reduced the background from a negative sample without affecting the signal intensity of a positive sample. Although the formamide improved the typing with these three primer mixes, it decreased or abolished the signal from human growth hormone (HGH) control primers. By increasing the length of the HGH primers and increasing the concentration of the primers in the PCR from 0.2 #M to 0.4-0.6 #M, amplification of the HGH gene in the presence of formamide was achieved. We have demonstrated that the quality of problematic amplification reactions used for DR low resolution typing by PCR-SSP can be improved by including formamide in the PCR reaction. B-7.4 #260 HLA SEQUETYPING - STRATEGIES FOR FLEXIBLE SOLID PHASE SEQUENCING TO PROVIDE DEFINITIVE HLA TYPING LA Baxter-Lowe~ T Daniels, M Eberle, J Pentek, S Szmania, and C Keever. The Blood Center of Southeastern Wisconsin, Milwaukee, WI. The extreme polymorphism of HLA genes has provided an interesting challenge for development of molecular HLA typing methods. Frequent heterozygosity is complicated by the fact that the composite sequence of two alleles may be identical for many different pairs of alleles. This study examines a rapid sequencing method, sequetyping, to deter- mine the exact sequence of individual alleles. Solid phase sequencing is achieved by selective amplification of individual alleles using affinity labelled primers and capture of amplified DNA on magnetic beads. Panels of biotinylated primers have been designed to achieve selective amplification of individual HI_A-A, -B, -C, and -DR alleles in heterozygotes. Amplified DNA is captured on streptavidin-coated beads, DNA is melted and complementary strands are recovered using ethanol precipitation or capture with a second affinity molecule. The DNA bound to beads is used as a template for sequencing reactions using dideoxy terminators and the sequences are determined using an automated sequencer. If ambiguities exist in the initial sequence, the sequence of the complementary strand is determined. The feasibility of this approach has been demonstrated using a wide range of samples. Certain infrequent combinations of alleles are not selectively amplified using the panel of primers. In this circumstance the composite sequence of two alleles can be determined and used to predict the individual alleles in most samples. After optimization is complete it will be possible to routinely determine the sequences of selectively amplified HLA genes in less than 24 hours. Major advantages of solid phase sequencing are: (1) it overcomes problems related to heterozygotes (2) it avoids cloning methods which are labor intensive and introduce artifacts (3) it provides the most definitive information (actual sequence), and (4) it is amenable to automation. These studies have revealed novel HLA alleles and demonstrated the feasibility of HLA sequetyping which is likely to be the typing method of choice in the future.
Transcript of Formamide improves HLA-DR low resolution typing by sequence-specific PCR
136 Abstracts
B-7.4 #259
FORMAMIDE IMPROVES HLA-DR LOW RESOLUTION TYPING BY SEQUENCE- SPECIFIC PCR DB Bellissimo and LA Baxter-Lowe, The Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin.
PCR amplification with sequence-specific primers (PCR-SSP) as described by Olerup and Zetterquist (Tissue Antigens 39: 225-235) is a rapid and sensitive method for DR low resolution typing. In this method, the assignment of the alleles is based on the presence or absence of amplified product after using sequence-specific primers. Nonspecific amplifications or low signal intensity in some PCR reactions make some samples difficult to type. We have observed this problem with primer mixes 14.2, 52 and 53. We tested whether the addition of formamide in the PCR reaction would improved the typing results. DNA was purified using the QIAamp blood kit and amplified in the presence of 1-5% formamide. Primer mix 14.2 has a GC-rich primer that causes a background problem in negative samples. The addition of 3 % formamide into the PCR reaction reduced the background without affecting the signal intensity of a positive sample. Weak amplifications sometimes observed with primer mix 52 were significantly improved with 2% formamide in the PCR reaction. Nonspecific amplification was often seen with primer mix 53. The addition of 4% formamide reduced the background from a negative sample without affecting the signal intensity of a positive sample. Although the formamide improved the typing with these three primer mixes, it decreased or abolished the signal from human growth hormone (HGH) control primers. By increasing the length of the HGH primers and increasing the concentration of the primers in the PCR from 0.2 #M to 0.4-0.6 #M, amplification of the HGH gene in the presence of formamide was achieved. We have demonstrated that the quality of problematic amplification reactions used for DR low resolution typing by PCR-SSP can be improved by including formamide in the PCR reaction.
B-7.4 #260
HLA SEQUETYPING - STRATEGIES FOR FLEXIBLE SOLID PHASE SEQUENCING TO PROVIDE DEFINITIVE HLA TYPING LA Baxter-Lowe~ T Daniels, M Eberle, J Pentek, S Szmania, and C Keever. The Blood Center of Southeastern Wisconsin, Milwaukee, WI.
The extreme polymorphism of HLA genes has provided an interesting challenge for development of molecular HLA typing methods. Frequent heterozygosity is complicated by the fact that the composite sequence of two alleles may be identical for many different pairs of alleles. This study examines a rapid sequencing method, sequetyping, to deter- mine the exact sequence of individual alleles. Solid phase sequencing is achieved by selective amplification of individual alleles using affinity labelled primers and capture of amplified DNA on magnetic beads. Panels of biotinylated primers have been designed to achieve selective amplification of individual HI_A-A, -B, -C, and -DR alleles in heterozygotes. Amplified DNA is captured on streptavidin-coated beads, DNA is melted and complementary strands are recovered using ethanol precipitation or capture with a second affinity molecule. The DNA bound to beads is used as a template for sequencing reactions using dideoxy terminators and the sequences are determined using an automated sequencer. If ambiguities exist in the initial sequence, the sequence of the complementary strand is determined. The feasibility of this approach has been demonstrated using a wide range of samples. Certain infrequent combinations of alleles are not selectively amplified using the panel of primers. In this circumstance the composite sequence of two alleles can be determined and used to predict the individual alleles in most samples. After optimization is complete it will be possible to routinely determine the sequences of selectively amplified HLA genes in less than 24 hours. Major advantages of solid phase sequencing are: (1) it overcomes problems related to heterozygotes (2) it avoids cloning methods which are labor intensive and introduce artifacts (3) it provides the most definitive information (actual sequence), and (4) it is amenable to automation. These studies have revealed novel HLA alleles and demonstrated the feasibility of HLA sequetyping which is likely to be the typing method of choice in the future.
