R. Bruce Wallace

The ligation amplification reaction (LAR)--amplification of specific DNA sequences using sequential rounds of template-dependent ligation.

A novel DNA sequence detection method that utilizes the ligation of oligonucleotide pairs that are complementary to adjacent sites on appropriate DNA templates is described. The product is increased by either linear or exponential amplification using sequential rounds of template-dependent ligation. In the case of linear amplification, a single pair of oligonucleotides is ligated, the reaction is heated to dissociate the ligation product, and an additional round of ligation is performed. After n rounds there is a (1 + x) X n-fold amplification of product, where x is the efficiency of the ligation reaction. Exponential amplification utilizes two pairs of oligonucleotides, one complementary to the upper strand and one to the lower strand of a target sequence. The products of the ligation reaction serve as templates for subsequent rounds of ligation. In this case there is (1 + x)(n-1)-fold amplification of product after n rounds. A single base-pair mismatch between the annealed oligonucleotides and the template prevents ligation, thus allowing the distinction of single base-pair differences between DNA templates. At high template concentrations, the ligation reaction has an efficiency approaching 100%. In this report, we demonstrate the use of the ligation amplification reaction (LAR) to distinguish the normal from the sickle cell allele of the human beta-globin gene. We also report the use of LAR as a detection system for polymerase chain reaction-enriched DNA sequences.

A set of synthetic oligodeoxyribonucleotide primers for DNA sequencing in the plasmid vector pBR322

Seven oligonucleotide primers complementary to the plasmid vector pBR322 at positions adjacent to five of the unique restriction endonuclease cleavage sites (EcoRI, HindIII, BamHI, SalI and PstI) have been chemically synthesized. The polarity of the primers is such that any DNA inserted at one or a combination of two of the above restriction sites may be sequenced by the chain termination method using one of the synthetic DNA primers. One of the primers for sequencing inserts at the PstI site of pBR322 is also complementary to the M13 phage vector designated bla6. This set of universal primers is useful for rapid sequence determination of DNA cloned into pBR322 or M13bla6.

Simultaneous analysis of multiple polymorphic loci using amplified sequence polymorphisms (ASPs)

In this paper we present a systematic approach to gene mapping and genotyping based on the simultaneous analysis of multiple amplified sequence polymorphisms (ASPs). These genetic markers measure variation in DNA sequences which have been amplified by a polymerase and/or a ligase. The amplified sequence lengths are determined by appropriate choice of oligonucleotides used in the amplification reaction. We describe three classes of ASPs: restriction site polymorphisms, sequence length polymorphisms, and DNA base pair changes not associated with restriction sites. Simultaneous analysis of multiple ASPs using a modified automated DNA sequencing apparatus should be possible because amplification with oligonucleotides provides control over the fragment lengths generated. Development of an automated ASP technology is therefore the next logical step for efficient gene mapping and genotyping of individuals. With this technology, one gel would be sufficient to indicate the most probable locations of a gene and a second gel would permit the selection of the correct location while simultaneously providing a fine structure map.

Application of an allele-specific polymerase chain reaction to the direct determination of ABO blood group genotypes

The allele-specific polymerase chain reaction (ASPCR) procedure has proven a powerful tool for the detection and analysis of known genetic polymorphisms. Here, we present a novel application of the ASPCR technique to determine the ABO genotypes of individuals without the need of family analysis. The method introduces a new strategy for primer design that permits the identification of the different ABO genotypes according to the molecular size of allele-specific amplification products. Four primer sets, each specific for a different set of ABO alleles, are mixed in one reaction and the amplification products are resolved on a polyacrylamide gel. Forty-one individuals belonging to various families, whose ABO phenotypes were previously determined serologically, were typed with this new variation of the ASPCR technique. A 100% correlation between the serology and the ASPCR data was found. The Mendelian segregation of ABO alleles was also demonstrated in families. The method is rapid, simple, reproducible, and specific. Potential applications include gene mapping, genetic disease diagnosis, HLA typing, paternity testing, and forensic science.

Isolation of a cDNA clone from the B-G subregion of the chicken histocompatibility (B) complex

The B-G antigens are highly polymorphic antigens encoded by genes located within the major histocompatibility complex (MHC) of the chicken, the B system. The B-G antigens of the chicken MHC are found only on erythrocytes and correspond to neither MHC class I nor class II antigens. Several clones were selected from a λgt11 erythroid cell expression library by means of rabbit antisera prepared against a purified, denatured B-G antigen. One clone chosen for further study, λbg28, was confirmed as a B-G subregion cDNA clone by the results obtained through using it as a nucleic acid hybridization probe. In Northern hybridizations λbg28 anneals specifically with erythroid cell mRNA. In Southern blot analyses the λbg28 clone could be assigned to the B system-bearing microchromosome of the chicken karyotype on the basis of its hybridization to DNA from birds disomic, trisomic, and tetrasomic for this microchromosome. The cDNA clone was further mapped to the B-G subregion on the basis of its pattern of hybridization with DNA from birds of known B region recombinant haplotypes. Southern blot analyses of the hybridization of λbg28 with genomic DNA from birds of known haplotypes strongly suggest that the B-G antigens are encoded by a highly polymorphic multigene family.

Identification of the cloned gene for the murine transplantation antigen H-2Kb by hybridization with synthetic oligonucleotides.

The H-2K(b) gene is a member of the large major histocompatibility complex class I gene family. Since many members of this family cross-hybridize with class I cDNA probes, the cloned H-2K(b) gene was identified by hybridization with specific oligonucleotide probes. This clone was definitively shown to encode the H-2K(b) polypeptide by partial DNA sequencing and by serological and tryptic peptide analyses of the expressed product.

Discrimination among the transcripts of the allelic human β-globin genes βA, βS and βC using oligodeoxynucleotide hybridization probes

Abstract Three nonadecadeoxynucleotides complementary to the sense strand of the normal human β-globin gene, βA, and to the two allelic genes βS and βC were synthesized. The βS and βC globin genes both differ from the βA gene by a single nucleotide substitution in the sequence coding for codon 6. The oligodeoxynucleotides are complementary to the genes in the region of the mutations and are therefore allele-specific. When radiolabeled and used as hybridization probes, the oligodeoxynucleotides are found to hybridize specifically to the mRNA transcribed from each allele.

Identification of an H-2Kb-related molecule by molecular cloning

Based on the published amino-acid sequence of H-2Kb, we synthesized a mixture of eight 16-base long oligodeoxyribonucleotides representing all possible coding sequences for residues 51–56 (Trp-Met-Glu-Gln-Glu-Gly). The hexadecanucleotide mixture was used as a probe to screen recombinant DNA clones constructed from cytoplasmic PolyA+ RNA isolated from the murine thymoma cell line EL4 (b haplotype). Of the 30 000 independent clones screened, one clone was found to hybridize with the probe. DNA sequence analysis showed that the cDNA clone was derived from a portion of an H-2Kb-related mRNA. The clone encodes a protein sequence identical with a region of H-2Kb in 42 consecutive residues (50 through 91). The sequence then diverges from the H-2Kb sequence and, after a single Glu codon, a termination codon is encountered. It is possible that this mRNA codes for a small 92 amino-acid protein with a sequence identical (except for a carboxy-terminal Glu residue) with the amino terminus of H-2Kb It is further speculated that this mRNA is coded for by the H-2Kb gene and differs from the H-2Kb mRNA in the pattern of posttranscriptional splicing.

Enzymatic synthesis of DNA probes complementary to a human variable number tandem repeat locus

Both cloned and synthetic DNA probes complementary to human variable number tandem repeat (VNTR) loci have been used to detect restriction fragment length polymorphism. In this report, we describe an approach for the enzymatic synthesis of a DNA probe complementary to one VNTR locus. The probe is produced by annealing short synthetic oligonucleotides comprising single repeat units and enzymatically ligating them into a polymeric DNA probe. In HinfI digests of human genomic DNA separated by agarose gel electrophoresis, this ligated oligonucleotide probe (LOP) detects multiple polymorphic loci in the range of 3-23 kb producing highly informative DNA fingerprint patterns when different individuals are compared. The hybridization pattern is very stable even under high-stringency wash conditions. The LOP is more easily generated than cloned VNTR probes and is totally synthetic, avoiding problems associated with cloned probes including bacterial growth and maintenance as well as in vitro labeling.

Genotype and phenotype: A practical approach to the immunogenetic analysis of lymphoproliferative disorders☆

Determination of cell lineage and clonality in lymphoproliferative disorders (LPD) is greatly enhanced by molecular genetic analysis in conjunction with morphologic and immunologic techniques. We now report on a technique in which we used cryostat-cut, fresh-frozen sections (CCFFS) prepared from tissues in a manner that allows DNA hybridization studies to be coordinated readily with routine morphologic and immunohistologic studies. Thirty-seven cases representing a broad spectrum of reactive and malignant LPD were examined with this method. Samples of DNA were extracted from frozen sections, subjected to Southern blot hybridization, and probed for rearrangements of the immunoglobulin (Ig) heavy-chain and the kappa and lambda light-chain genes, as well as for the T-cell receptor beta-chain gene. We also evaluated the effects of (1) diagnostic category of LPD, (2) volume of the tissue sample, and (3) fibrosis, necrosis, and ice crystal artifacts in the sample on the recovery of DNA. Ice artifact and sample size had the greatest negative impacts on the quantity and condition of DNA recovered. Of 19 samples involved by B-cell LPD, the results of immunogenetic studies were consistent with the immunophenotypes in all but one case. Of the T-cell lymphomas from which sufficient DNA was available (three out of five of the T-cell cases), all showed rearrangements of the T-cell beta-chain gene. In order to reduce sample processing time, we evaluated alternate blot hybridization methods, rapid alkaline transfers, and direct hybridization of synthetic oligonucleotides in dried agarose gels, and found that they decreased the time required for hybridization studies. In summary, the use of CCFFS as the source of DNA allows study of gene rearrangements and, at the same time, preserves frozen-tissue blocks in tumor banks for further immunologic studies. The development of time-effective methods will make the routine use of molecular-genetic analysis more practical in the diagnostic hematopathology laboratory.