Heterozygote and mutation detection by direct automated fluorescent DNA sequencing using a mutant Taq DNA polymerase.

We describe a method for direct cycle sequencing of PCR fragments amplified from genomic DNA or cDNA. DNA sequencing template is amplified using PCR and oligonucleotide primers flanking the region of interest. The amplified fragment is directly cycle sequenced using fluorescent sequencing primers, Sanger dideoxy sequencing chemistry and an enzyme mixture of a mutant Taq DNA polymerase and thermostable pyrophosphatase. The sequence ladders produced are analyzed on a real-time, automated four-color sequencing system. The method produces sequence ladders from unpurified PCR fragments of sufficiently high quality such that heterozygotes can be reproducibly detected and identified by software that recognizes signal-strength patterns indicative of mixed-base positions.

Ancient, highly polymorphic human major histocompatibility complex DQA1 intron sequences.

A 438 basepair intron 1 sequence adjacent to exon 2 in the human major histocompatibility complex DQA1 gene defined 16 allelic variants in 69 individuals from wide ethnic backgrounds. In contrast, the most variable coding region spanned by the 247 basepair exon 2 defined 11 allelic variants. Our phylogenetic human intron 1 tree derived by the Bootstrap algorithm reflects the same relative allelic relationships as the reported DQA1 exon 2 tree [Gyllensten and Erlich, Hum Immunol 36:1-10, 1989]. Thus 3' DQA1 intron 1 and exon 2 have cosegregated since divergence of the human races. Comparison of human alleles to a Rhesus monkey DQA1 first intron sequence found only 10 nucleotide substitutions unique to Rhesus, with the other 428 positions (98%) found in at least one human allele. This high degree of homology reflects the evolutionary stability of intron sequences since these two species diverged over 20 million years ago. Because more intron 1 alleles exist than exon 2 alleles, these polymorphic introns can be used to improve tissue typing for transplantation, paternity testing, and forensics and to derive more complete phylogenetic trees. These results suggest that introns represent a previously underutilized polymorphic resource.

Strategy for definition of DR/DQ haplotypes in the 4AOHW cell panel using noncoding sequence polymorphisms.

Our previously described intron-based DQA1-typing method provides 11 allelic and suballelic groups, including the eight alleles encoded at the second exon. Concurrent testing for the presence of the DRB3, DRB4, and DRB5 loci and the Rsa I pattern of the DRw52 group simplifies the typing requirements for allele assignment at the highly polymorphic DRB1 locus. The DRB1-allele-shortlisting process relies on known DR/DQ haplotypes. In addition to reducing the testing requirements for definitive DRB1 allele assignment, this strategy allows inference of the DR/DQ haplotype and assists in recognition of novel and/or unusual associations.

HLA-DQA1 allele and suballele typing using noncoding sequence polymorphisms. Application to 4AOHW cell panel typing.

HLA-DQA1 typing of the 4AOHW cell panel is presented using a novel strategy that exploits both intron and exon polymorphisms. Intron sequences adjacent to the variable HLA-DQA1 second exon exhibit stable polymorphisms that are specific for locus alleles and certain suballelic DR/DQ haplotypes. A PCR-RFLP method has been developed that is based on amplification of a 780-bp segment extending from intron 1 through exon 2 to intron 2. Stable sequence polymorphisms provide restriction enzyme sites and confer mobility variations detected on polyacrylamide minigel electrophoresis. Direct band comparison of amplified products and restriction fragments with known standards facilitates pattern comparison, obviating the requirement for accurate molecular weight determination. This method, using only two enzymes, identifies a total of 11 allelic and suballelic groups, including all eight DQA1 alleles encoded at the second exon.