City of Hope

Baylor College of Medicine

Steve S Sommer

Qiang Liu

Arthur D Riggs

Jinong Feng

Jin Yan

John J Rossi

Yun Yen

Kevin Ferreri

Fouad Kandeel

Carolyn Buzin

Jeffrey Towbin

Rasha Shehatta

The present invention provides a method for detecting transplantation failure of a transplanted organ or cells which comprises detecting a donor-positive but recipient-negative DNA marker in the recipient's plasma using pyrophosphorolysis activated polymerization. Because of the high sensitivity, specificity and selectivity of pyrophosphorolysis activated polymerization, transplantation failure can be detected at early stages and treatment can be initiate earlier.

Ultra-high sensitive monitoring of early transplantation failure

A method of diagnosing, assessing susceptibility, and/or treating schizophrenia involving the identification and/or observation of microRNAs (miRNA) and variant miRNA are provided. Micro RNAs alleles associated with schizophrenia and schizophrenia spectrum disorders were identified and ultra-rare variants in the precursor or mature miRNA were identified. Functional analyses of ectopically expressed copies of the variant miRNA precursors demonstrate loss of function, gain of function and altered expression levels. The present invention also provides methods for selecting a preferred therapy for a particular subject or group of subjects or individuals at risk for or suffering from schizophrenia or psychosis by use of miRNAs.

Micro RNAs and their methods of use for the treatment and diagnosis of schizophrenia and schizophrenia spectrum disorders

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&#x2032; 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&#x2032; end for the rare allele but has a mismatch at or near its 3&#x2032; terminus for the wild-type allele. PAP is inhibited by a mismatch in the 3&#x2032; specific sequence as far as 16 nucleotides away from the 3&#x2032; terminus. PAP can greatly increase the specificity of detection of an extremely rare mutant allele in the presence of the wild-type allele. Specificity results from both pyrophosphorolysis and polymerization since significant nonspecific amplification requires the combination of mismatch pyrophosphorolysis and misincorporation by the DNA polymerase, an extremely rare event. Using genetically engineered DNA polymerases greatly improves the efficiency of PAP.

Serial coupling of nucleic acid metabolism and extension for nucleic acid amplification

Reaction mixture for pyrophosphorolysis activated polymerization (PAP)

The three &#x3b2;-neurexins have similar roles in synaptogenesis and interact with the neuroligins. Mutations located within the gene encoding neurexin 1 have been identified as molecular markers associated with autism and autism-related disorders. The estimated attributable risk is 2%. The invention provides methods of diagnosing or predicting susceptibility to developing autism in an individual by determining the presence or absence of one or more genetic variant of a neurexin 1 gene in an individual.

High frequency of neurexin 1&#x3b2; signal peptide structural variants in patients with autism

Pyrophosphorolysis activated polymerization (PAP)

The present invention relates generally to the field of human genetics. Specifically, the present invention relates to methods and materials used to detect a human sporadic DCM predisposing gene, specifically the dystrophin gene, some mutant alleles of which cause susceptibility to sporadic DCM. More specifically, the invention relates to germline mutations in the dystrophin gene and their use in the diagnosis of predisposition to sporadic DCM. The invention also relates to the prophylaxis and/or therapy of sporadic DCM associated with a mutation in the dystrophin gene. The invention further relates to the screening of drugs for sporadic DCM therapy. Finally, the invention relates to the screening of the dystrophin gene for mutations/alterations, which are useful for diagnosing the predisposition to sporadic DCM.

Alterations in the dystrophin gene associated with sporadic dilated cardiomyopathy

pK-matched buffers, each containing two effective buffering components: one weak base and one weak acid which have similar pKat 25&#xb0; 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&#x2122; 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. In comparison to TAE and TBE, pK-matched buffers provide higher voltage and current stability, lower working concentration, more concentrated stock solutions, and lower current per unit voltage, resulting in less heat generation.

pK-matched running buffers for gel electrophoresis

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&#x2032; 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&#x2032; end for the rare allele but has a mismatch at or near its 3&#x2032; terminus for the wild type allele. PAP is inhibited by a mismatch in the 3&#x2032; specific subsequence as far as 16 nucleotides away from the 3&#x2032; terminus. PAP can greatly increase the specificity of detection of an extremely rare mutant allele in the presence of the wild type allele. Specificity results from both pyrophosphorolysis and polymerization since significant nonspecific amplification requires the combination of mismatch pyrophosphorolysis and misincorporation by the DNA polymerase, an extremely rare event. Using genetically engineered DNA polymerases greatly improves the efficiency of PAP.

Serial coupling of restriction cleavage and extension for nucleic acid amplification

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&#x2032; 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&#x2032; end for the rare allele but has a mismatch at or rear its 3&#x2032; terminus for the wild-type allele. PAP is inhibited by a mismatch in the 3&#x2032; specific sequence as far as 16 nucleotides away from the 3&#x2032; terminus. PAP can greatly increase the specificity of detection of an extremely rare mutant allele in the presence of the wild-type allele. Specificity results from both pyrophosphorolysis and polymerization since significant nonspecific amplification requires the combination of mismatch pyrophosphorolysis and misincorporation by the DNA polymerase, an extremely rare event. Using genetically engineered DNA polymerases greatly improves the efficiency of PAP.

pK-matched buffers, each containing two effective buffering components: one weak base and one weak acid which have similar pK at 25&deg; 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&trade; 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. In comparison to TAE and TBE, pK-matched buffers provide higher voltage and current stability, lower working concentration, more concentrated stock solutions, and lower current per unit voltage, resulting in less heat generation.

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&prime;-deoxynucleotide at its 3&prime; 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&prime; end for the rare allele but has a mismatch at or near its 3&prime; terminus for the wild type allele. PAP is inhibited by a mismatch in the 3&prime; specific subsequence as far as 16 nucleotides away from the 3&prime; terminus. PAP can greatly increase the specificity of detection of an extremely rare mutant allele in the presence of the wild type allele. Specificity results from both pyrophosphorolysis and polymerization since significant nonspecific amplification requires the combination of mismatch pyrophosphorolysis and misincorporation by the DNA polymerase, an extremely rare event. Using genetically engineered DNA polymerases greatly improves the efficiency of PAP.

Pyrophosphorolysis activated polymerization (PAP): application to allele-specific amplification and nucleic acid sequence determination

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 dnF , reactions and electrophoretic separation using the two ddNTPs are conducted separately. In an alternative embodiment, designated dnF , 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.

Growing community of inventors

Duarte, CA, United States of America

Steve S Sommer