Abstract: A novel method of pyrophosphorolysis activated polymerization (PAP) has been developed. In PAP, pyrophosphorolysis and polymerization by DNA polymerase are coupled serially for each amplification by using an activatable oligonucleotide P* that has a non-extendible 3′-deoxynucleotide at its 3′ terminus. PAP can be applied for exponential amplification or for linear amplification. PAP can be applied to amplification of a rare allele in admixture with one or more wild-type alleles by using an activatable oligonucleotide P* that is an exact match at its 3′ end for the rare allele but has a mismatch at or rear its 3′ terminus for the wild-type allele. PAP is inhibited by a mismatch in the 3′ specific sequence as far as 16 nucleotides away from the 3′ terminus. PAP can greatly increase the specificity of detection of an extremely rare mutant allele in the presence of the wild-type allele.

Abstract: Truncated amplification (TA) provides high fidelity, template driven amplification of target nucleic acid molecules in which truncating oligonucleotides are used as primers to produce truncated terminal products that are in most, if not all, cases no more than three rounds of replication from the original template. In certain embodiments, TA can amplify target nucleic acids quadratically or geometrically, depending on the number of truncating oligonucleotides used for amplification.

Abstract: pK-matched buffers, each containing two effective buffering components: one weak base and one weak acid which have similar pKa at 25° C. (within 0.3 pK units). On agarose gels, the buffers in various concentrations were tested for separation of double-stranded DNA fragments with various DNA markers, agarose gel concentrations, and field strengths. Mobility was inversely proportional to the logarithm of molecular weight. The buffers provided high resolution without smearing at more dilute concentration than is possible with standard TAE (Tris/Acetate, pH 8.0) or TBE (Tris/Borate, pH 8.3) buffers. The buffers were also tested in 7M urea denaturing LongRanger™ sequencing gels and in non-denaturing polyacrylamide SSCP gels. The pK-matched buffers provide good separation and high resolution, at a broad range of potential pH values.

Abstract: pK-matched buffers, each containing two effective buffering components: one weak base and one weak acid which have similar pKa at 25° C. (within 0.3 pK units On agarose gels, the buffers in various concentrations were tested for separation of double-stranded DNA fragments with various DNA markers, agarose gel concentrations, and field strengths. Mobility was inversely proportional to the logarithm of molecular weight. The buffers provided high resolution without smearing at more dilute concentration than is possible with standard TAE (Tris/Acetate, pH 8.0) or TBE (Tris/Borate, pH 8.3) buffers. The buffers were also tested in 7M urea denaturing LongRanger™ sequencing gels and in non-denaturing polyacrylamide SSCP gels. The pK-matched buffers provide good separation and high resolution, at a broad range of potential pH values.

Abstract: A novel method of pyrophosphorolysis activated polymerization (PAP) has been developed. In PAP, pyrophosphorolysis and polymerization by DNA polymerase are coupled serially for each amplification by using an activatable oligonucleotide P* that has a non-extendable 3′-deoxynucleotide at its 3′ terminus. PAP can be applied for exponential amplification or for linear amplification. PAP can be applied to amplification of a rare allele in admixture with one or more wild type alleles by using an activatable oligonucleotide P* that is an exact match at its 3′ end for the rare allele but has a mismatch at or near its 3′ terminus for the wild type allele. PAP is inhibited by a mismatch in the 3′ specific subsequence as far as 16 nucleotides away from the 3′ terminus. PAP can greatly increase the specificity of detection of an extremely rare mutant allele in the presence of the wild type allele.

Abstract: A novel method of pyrophosphorolysis activated polymerization (PAP) has been developed. In PAP, pyrophosphorolysis and polymerization by DNA polymerase are coupled serially for each amplification by using an activatable oligonucleotide P* that has a non-extendable 3′-deoxynucleotide at its 3′ terminus. PAP can be applied for exponential amplification or for linear amplification. PAP can be applied to amplification of a rare allele in admixture with one or more wild type alleles by using an activatable oligonucleotide P* that is an exact match at its 3′ end for the rare allele but has a mismatch at or near its 3′ terminus for the wild type allele. PAP is inhibited by a mismatch in the 3′ specific subsequence as far as 16 nucleotides away from the 3′ terminus. PAP can greatly increase the specificity of detection of an extremely rare mutant allele in the presence of the wild type allele.

Abstract: Methods are presented for determining the presence of an inversion in the factor VIII gene which cause hemophilia A. The methods encompass long distance, multiplex PCR (including overlapping PCR). The use of deaza-dGTP, high levels of DNA polymerases and high levels of DMSO aid in successfully performing the PCR. The use of a novel technique called subcycling PCR can also be applied as part of the methods. The technique allows for the determination of whether a person is homozygous or hemizygous for the inversion and has hemophilia A or whether a person is heterozygous for the inversion and is a carrier. The technique of long distance, multiplex PCR including use of deaza-dGTP, high levels of DNA polymerases and high levels of DMSO are applicable to the determination of the presence of other gross chromosomal aberrations such as deletions/inversions, translocations and inversions.

Abstract: A novel method of pyrophosphorolysis activated polymerization (PAP) has been developed. In PAP, pyrophosphorolysis and polymerization by DNA polymerase are coupled serially for each amplification by using an activatable oligonucleotide P* that has a non-extendable 3′-deoxynucleotide at its 3′ terminus. PAP can be applied for exponential amplification or for linear amplification. PAP can be applied to amplification of a rare allele in admixture with one or more wild type alleles by using an activatable oligonucleotide P* that is an exact match at its 3′ end for the rare allele but has a mismatch at or near its 3′ terminus for the wild type allele. PAP is inhibited by a mismatch in the 3′ specific subsequence as far as 16 nucleotides away from the 3′ terminus. PAP can greatly increase the specificity of detection of an extremely rare mutant allele in the presence of the wild type allele.

Abstract: A denaturation fingerprinting method (dnF) involves subjecting a nucleic acid segment of interest to bidirectional cycle sequencing using oppositely oriented primers and incorporating two different dideoxynucleotides (ddNTPs) in the sequencing reaction. The resulting fragments are separated by denaturing electrophoresis. In one embodiment, designated dnF2R, reactions and electrophoretic separation using the two ddNTPs are conducted separately. In an alternative embodiment, designated dnF1R, one of the ddNTPs has a mobility altering modification such that electorphoretic separation occurs when both ddNTPs are employed in the same reaction. The methods are useful for detecting genetic mutations.

Abstract: Methods are presented for determining the presence of an inversion in the factor VIII gene which cause hemophilia A. The methods encompass long distance, multiplex PCR (including overlapping PCR). The use of deaza-dGTP, high levels of DNA polymerases and high levels of DMSO aid in successfully performing the PCR. The use of a novel technique called subcycling PCR can also be applied as part of the methods. The technique allows for the determination of whether a person is homozygous or hemizygous for the inversion and has hemophilia A or whether a person is heterozygous for the inversion and is a carrier. The technique of long distance, multiplex PCR including use of deaza-dGTP, high levels of DNA polymerases and high levels of DMSO are applicable to the determination of the presence of other gross chromosomal aberrations such as deletions/inversions, translocations and inversions.

Abstract: A denaturation fingerprinting method (dnF) involves subjecting a nucleic acid segment of interest to bidirectional cycle sequencing using oppositely oriented primers and incorporating two different dideoxynucleotides (ddNTPs) in the sequencing reaction. The resulting fragments are separated by denaturing electrophoresis. In one embodiment, designated dnF2R, reactions and electrophoretic separation using the two ddNTPs are conducted separately. In an alternative embodiment, designated dnF1R, one of the ddNTPs has a mobility altering modification such that electorphoretic separation occurs when both ddNTPs are employed in the same reaction. The methods are useful for detecting genetic mutations.

Abstract: A denaturation fingerprinting method (dnF) involves subjecting a nucleic acid segment of interest to bidirectional cycle sequencing using oppositely oriented primers and incorporating two different dideoxynucleotides (ddNTPs) in the sequencing reaction. The resulting fragments are separated by denaturing electrophoresis. In one embodiment, designated dnF2R, reactions and electrophoretic separation using the two ddNTPs are conducted separately. In an alternative embodiment, designated dnF1R, one of the ddNTPs has a mobility altering modification such that electorphoretic separation occurs when both ddNTPs are employed in the same reaction. The methods are useful for detecting genetic mutations.

Abstract: Experiments were performed to test for a set of SSCP conditions that would detect virtually all mutations in a nucleic acid being analyzed. The effects of buffer, gel matrix, temperature, and additive were all tested. Dideoxy fingerprinting was used as a tool to generate a large statistical sample (about 1,500) of mutation-containing single-stranded segments in order to evaluate adequately the sensitivity under a given condition. Mutations in exons H and B/C of the factor IX gene were utilized. SSCP sensitivity, as conveniently measured by the average SSCP efficiency, varied with conditions. Correlation coefficients (R) identified pairs of conditions that were either close to independent or complementary. Five conditions were selected with sufficient redundancy to detect all the mutations in the set tested. The sensitivity of multi-conditional SSCP (SSCP5) was determined by blinded analyses on samples containing mutations in all the eight exon regions in the factor IX gene.

Abstract: Bi-directional polymerase chain reaction (PCR) amplification of specific alleles (Bi-PASA). Two outer primers (P and Q) and two inner primers (A and B) are used. A and B are each specific for different alleles. In heterozygotes, three segments are amplified: a segment of size AQ resulting from one allele, another segment of size PB resulting from the second allele, and a combined segment of size PQ. In homozygotes, segment PQ and either segments AQ or PB amplify.

Abstract: The present invention relates to DNA sequences coding for genes which participate in a yeast cell wall .beta.-glucan assembly pathway. These genes are essential for normal cell growth and are tools whicn enable the `in vitro` or `in vivo` screening for differential inhibition of fungi pathogenic to plants and animals, including man by specific antifungal agents. Further, a method which allows one to produce structurally modified glucans by using the DNA sequences of the present invention is also provided. Such modified glucans can be used to facilitate protein extraction from a microorganism; as food additives or as immunomodulators. Also, a method for selecting yeast mutants resistant to killer toxins from other yeasts or fungi which comprises exposing sensitive yeast cells to such killer toxins on agar plates, and selecting for yeast cells that survived such exposure by their ability to grow and form colonies.